ORGANOIDS DERIVED FROM DERMAL PAPILLA AND EPITHELIAL STEM CELLS AND PRODUCTION AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Application No. 63/396,370, filed August 9, 2022, which is incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

[0002] The present disclosure provides hair follicle organoids comprising induced dermal papilla (DP) cells and induced epithelial cells, methods of producing or growing hair follicle organoids, and methods for screening a compound for hair growth-modulating activity using the hair follicle organoids. In some embodiments, the induced DP cells and/or induced epithelial cells are derived from human induced pluripotent stem cells (iPSCs).

INTRODUCTION AND SUMMARY

[0003] Hair follicles (HF) are characterized by the ability to regenerate themselves through repetitive cycles of self-renewing stem cells. However, some follicles undergo premature degeneration leading to hair loss. Why and how this happens is unclear. Regardless of the etiology, hair loss provokes anxiety and distress more profound than its objective severity would appear to justify. Currently, there is no definitive treatment for hair loss. Both locally applied (e.g., ROGAINE® (minoxidil)) and systemic (e.g., PROPECIA® (finasteride)) drugs achieve only limited success in hair regeneration. See Price, V., N. Engl. J. Med., 1999. 341 : 964-973. Moreover, these two options are fraught with cost, side effects, and accelerated hair loss when the medications are stopped after prolonged use. Irwig, M.S., J Clin Psychiatry, 2012. 73: 1220-1223. Other hair restoration procedures involve the excision of existing HFs and re-implantation of these follicles into thinning areas. However, the number of follicles that can be harvested and re-implanted is limited, and restoration is likely temporary due to the progressive nature of many hair loss conditions. Zhang et al., J Tissue Eng, 2014. 5: 2041731414556850. Given the limited efficacy of strategies currently available in hair loss management, better solutions are urgently needed.

[0004] Dermal Papilla (DP) cells play a key role in forming and regulating hair structure during embryonic development as well as the adult hair cycle. The literature has explored one cellular approach to de novo folliculogenesis via native adult human DP cell isolation. Jahoda, et al. Nature, 1984. 311(5986):560-2. Hence, DP cells have been a fundamental target in cellular hair loss solutions. Cultured DP cells have demonstrated regenerative potential at early passages (Jahoda et al., Nature, 1984. 311(5986):560-2); however, in order to maintain these properties, aggregation of DP cells (Higgins et al., Proc Natl Acad Sci USA, 2013. 110(49): 19679-88; Ohyama, M., et al., J Cell Sci, 2012. 125(Pt 17): 4114-25) or culturing in the presence of other cells from the HF niche (Lichti et al., Nat Protoc, 2008. 3(5):799-810; Zheng et al., J Invest Dermatol, 2005. 124(5): 867-876; Toyoshima et al., Nat Commun, 2012. 3:784) appears generally necessary. More recently, an attempt to reconstitute HFs from human DP and keratinocytes (KC) within human skin constructs (HSCs) employing 3D-printed molds and recapitulating the physiological organization of cells in the hair microenvironment was reported. Abaci et al., Nat Commun, 2018. 9(1): 5301. However, due to their loss of regenerative capabilities in vitro and the existing limitations of harvesting a sufficient amount from donor skin, primary DP cells are unlikely to be a viable solution in the near term for treating alopecia.

[0005] Alternatively, an autologous stem cell-based approach would eliminate the need for extensive cell sourcing and can be fine-tuned to derive folliculogenic cell types. Lee et al. described the spontaneous formation of HFs within skin organoids using mouse and human pluripotent stem cells. Lee et al., Cell Rep, 2018. 22(l):242-254; Lee et al., Nature, 2020. 582(7812):399-404. In these studies, embryonic stem cells (ESCs) or iPSCs were aggregated and cultured in a chemically defined medium to recapitulate key steps of integumentary development. While Lee et al. demonstrated the potential for stem cell therapy to treat fullskin wounds and bums, a stem cell -based treatment of alopecia would likely require the development of discrete transplantable follicular units supported by folliculogenic cells. An early proof of concept for an alternative method that could generate hair-inducing DP cells using human embryonic stem cells (hESC) has been described. Gnedeva et al., PLoS One, 2015. 10(l):e0116892. Similarly, Veraitch et al. aimed to induce DP cells from iPSCs by an intermediate step of LNGFR(+)THY-1(+) mesenchymal cells. Veraitch et al., Sci Rep, 2017. 7:42777. Nonetheless, there is a need for further improvements and higher efficiency to offer a viable iPSC approach for HF restoration.

[0006] Hair follicles (HF) are complex, well -organized structures, often referred to as miniorgans, formed during embryonic development through communication between follicular epithelial cells and underlying DP cells. To create a new HF, DP cells interact with epithelial cells responsible for forming the multiple concentric layers that surround the hair shaft. See Millar, S.E., J Invest Dermatol, 2002. 118(2):216-25; Schneider et al., Curr Biol, 2009. 19(3):R132-42. During the natural hair follicle life cycle, these epithelial layers undergo degeneration and regrowth stages, fueled by epithelial stem cells located in the bulge area, located in the upper-middle region of each HF, at the base of the non-cycling portion. Muller- Rover et al., J Invest Dermatol, 2001. 117(1):3-15; Cotsarelis et al., Cell, 1990. 61(7):1329- 37.

[0007] The present disclosure provides, in some embodiments, methods for HF generation in vitro and in vivo, which can be applied in treatments for common hair loss disorders that severely impact the quality of life of many people. Furthermore, the hair follicle organoids described herein represent a possible platform for drug screening and personalized medicine. [0008] Certain methods described herein comprise generating DP cells and epithelial cells in vitro and creating hair follicle-like organoids suitable for skin (e.g., subcutaneous) implantation. Combining iPSC-derived DP with epithelial cells (e.g., in a 3D co-culture system) supported generation of HF organoids that resemble the primordial shape of human follicular units and gave rise to hair when transplanted under the skin of immunodeficient mice. Such approaches can provide one or more of the following advantages to the HF restoration field. First, patient-specific iPSCs represent a virtually unlimited source to generate DP cells and keratinocytes for transplantation purposes to treat alopecia and related disorders. Second, cell organization occurring within the 3D HF structures can be observed in a time-resolved manner, indicating cell functionality at early stages. Third, the molecular mechanisms involved in organoid development can be manipulated, e.g., as in a platform for automated, high-throughput screening (HTS) of compounds with potential hair growthmodulating activity. Lastly, the development of HFs ex vivo that can mimic the properties of healthy or diseased tissues represents a model that can be used to significantly improve the current knowledge of hair loss conditions, e.g., when combined with cutting-edge technology such as RNA-sequencing. Indeed, although often seen as medically irrelevant, the consequences of hair loss for men and women, especially youth, are psychologically debilitating and require better solutions.

[0009] A method for hair loss treatment was described in 2015 using human embryonic stem cells (hESCs) to generate NC cells and then hair-inducing DP -like cells in culture that were able to create HFs when transplanted under the skin of immune-deficient nude mice. See Gnedeva, et al., PLoS One (2015) 10(1): eOl 16892. Herein, methods comprising differentiation ofNC cells (e.g., IPSC-derived NC cells (“iPSC-NC cells”)) into DP-like cells are described, which could be achieved through the activation and potentiation of WNT/p- catenin pathway mediated by R-spondin 1 in combination with WNTlOb. Protein and gene analysis revealed a gradual acquisition of a mesenchymal phenotype during the differentiation process of the iPSC-NC cells that culminated in a cell population whose identity could be defined as primordial DP cells, sharing key markers with both human skin fibroblasts and human HF-derived DP cells. A precise identity of these iPSC-DP cells was delineated by comparing our results to the data previously published in the field. The iPSC- DP cells were shown to be capable of generating de novo HF structures when combined with embryonic mouse keratinocytes or primary human keratinocytes. The ability to take advantage of patient-specific iPSCs to generate a virtually unlimited source of DP cells and keratinocytes for transplantation purposes offers a new clinical approach to alopecia related disorders.

[0010] Accordingly, the following exemplary embodiments are provided.

Embodiment 1. A hair follicle organoid comprising:

(i) induced dermal papilla (DP) cells; and

(ii) induced epithelial cells; wherein the hair follicle organoid has a spheroidal or elongated shape.

Embodiment 2. The hair follicle organoid of embodiment 1, wherein the induced DP cells are derived from human induced pluripotent stem cells (iPSCs).

Embodiment 3. The hair follicle organoid of any one of the preceding embodiments, wherein the induced DP cells are derived from human induced neural crest cells, or are derived from human iPSCs via an induced neural crest intermediate.

Embodiment 4. The hair follicle organoid of embodiment 3, wherein the induced DP cells were derived from human induced neural crest cells or from human iPSCs via an induced neural crest intermediate using a medium comprising WNT-lOb and R- spondin 1 is used to induce DP cells.

Embodiment 5. The hair follicle organoid of any one of the preceding embodiments, wherein the induced epithelial cells are derived from human iPSCs.

Embodiment 6. The hair follicle organoid of any one of the preceding embodiments, wherein the induced DP cells were derived from naturally occurring cell(s) and comprise at least one epigenetic or genetic difference relative to the naturally occurring cell(s) from which they were derived.

Embodiment 7. The hair follicle organoid of any one of the preceding embodiments, wherein the induced DP cells are derived from an adult human subject or a fetal human subject. Embodiment 8. The hair follicle organoid of any one of the preceding embodiments, wherein the induced epithelial cells are derived from an adult human subject or a fetal human subject.

Embodiment 9. The hair follicle organoid of any one of the preceding embodiments, wherein the induced DP cells are derived from one or more cells obtained from a subject and comprise at least one epigenetic or genetic difference relative to naturally occurring DP cells of the subject.

Embodiment 10. The hair follicle organoid of any one of the preceding embodiments, wherein the induced epithelial cells comprise at least one epigenetic or genetic difference relative to the naturally occurring cell(s) from which they were derived.

Embodiment 11. The hair follicle organoid of any one of the preceding embodiments, wherein the induced epithelial cells are derived from one or more cells obtained from a subject and comprise at least one epigenetic or genetic difference relative to naturally occurring epithelial cells of the subject.

Embodiment 12. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid lacks a stem cell niche.

Embodiment 13. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid lacks a bulge region.

Embodiment 14. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid lacks a sebaceous gland.

Embodiment 15. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid lacks an arrector pili muscle.

Embodiment 16. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid comprises a dermal compartment and an adjacent cone of cells, wherein the cone of cells comprises transit-amplifying cells.

Embodiment 17. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid improves skin innervation and vasculature.

Embodiment 18. The hair follicle organoid of any one of the preceding embodiments, wherein induced DP cells occupy a hair bulb, and epithelial cells extend above the hair bulb. Embodiment 19. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid comprises epithelial cells wrapped within a cluster comprising induced DP cells, or DP cells wrapped within a cluster comprising epithelial cells.

Embodiment 20. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid comprises matrix produced by the epithelial cells.

Embodiment 21. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid comprises matrix produced by the dermal papilla cells.

Embodiment 22. The hair follicle organoid of the immediately preceding embodiment, wherein the matrix imparts an elongated shape to the hair follicle organoid.

Embodiment 23. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid comprises keratinocytes.

Embodiment 24. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid comprises an inner root sheath, an outer root sheath, a bulge, a companion layer, and/or a matrix.

Embodiment 25. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid comprises a hair fiber.

Embodiment 26. The hair follicle organoid of any one of the preceding embodiments, wherein the hair follicle organoid comprises about 2000 to 6000 induced DP cells and about 2000 to 6000 induced epithelial cells or about 1500 to 15000 induced DP cells and about 1500 to 15000 induced epithelial cells.

Embodiment 27. A method of producing an elongated hair follicle organoid, comprising: co-culturing induced DP cells and induced epithelial stem cells (EpSCs) in a first cell culture medium that contains basement membrane matrix, wherein the induced DP cells and induced EpSCs form an aggregate; and culturing the aggregate in a second cell culture medium comprising basement membrane matrix; wherein: the EpSCs differentiate into keratinocytes, and the induced DP cells and keratinocytes form an elongated hair follicle organoid.

Embodiment 28. A method of producing an elongated hair follicle organoid, comprising: co-culturing induced DP cells and induced epithelial stem cells (EpSCs) in a first cell culture medium, wherein the induced DP cells and induced EpSCs form an aggregate; transferring the aggregate onto a support matrix or into a suspension comprising basement membrane matrix; culturing the aggregate in a second cell culture medium comprising one or more growth factors comprising one or more of R-Spondin 1, Noggin, epidermal growth factor (EGF), one or more Wnt proteins, CHIR, Rock inhibitor, bone morphogenetic proteins (BMP), fibroblast growth factor (FGF) proteins, and Shh (sonic hedgehog) activators, wherein: the EpSCs differentiate into keratinocytes, and the induced DP cells and keratinocytes form an elongated hair follicle organoid.

Embodiment 29. A method of producing an elongated hair follicle organoid, comprising: co-culturing induced DP cells and induced epithelial stem cells (EpSCs) in a cell culture medium that contains basement membrane matrix, wherein the induced DP cells and induced EpSCs form an aggregate; culturing the aggregate in a second cell culture medium comprising one or more growth factors comprising one or more of R-Spondin 1, Noggin, epidermal growth factor (EGF), one or more Wnt proteins, CHIR, Rock inhibitor, bone morphogenetic proteins (BMP), fibroblast growth factor (FGF) proteins, and Shh (sonic hedgehog) activators, wherein: the EpSCs differentiate into keratinocytes, and the induced DP cells and keratinocytes form an elongated hair follicle organoid.

Embodiment 30. The method of any one of embodiments 28 to 29, wherein the one or more growth factors comprise R-Spondin 1, Noggin, and EGF.

Embodiment 31. The method of the immediately preceding embodiment, wherein the R- Spondin 1 is human R-Spondin 1. Embodiment 32. The method of any one of embodiments 28-31, wherein the Noggin is human Noggin.

Embodiment 33. The method of any one of embodiments 28-32, wherein the EGF is human EGF.

Embodiment 34. The method of any one of embodiments 28-33, wherein the Wnt protein is Wnt 10b.

Embodiment 35. The method any one of embodiments 28-34, wherein the BMP protein is BMP4.

Embodiment 36. The method of any one of embodiments 28-35, wherein the FGF protein is FGF10 and/or FGF20.

Embodiment 37. The method of any one of embodiments 28-36, wherein the Shh activator is SAG (signaling agonist).

Embodiment 38. The method of any one of embodiments 27-37, wherein the induced DP cells are derived from human iPSCs.

Embodiment 39. The method of any one of embodiments 27-38, wherein the induced DP cells are derived from human induced neural crest cells, or are derived from human iPSCs via an induced neural crest intermediate.

Embodiment 40. The method of the immediately preceding embodiment, wherein the induced DP cells are derived from human induced neural crest cells or from human iPSCs via an induced neural crest intermediate using a medium comprising WNT-lOb and R-spondin 1 to induce DP cells.

Embodiment 41. The method of any one of embodiments 27-40, wherein the keratinocytes are derived from human iPSCs.

Embodiment 42. The method of any one of embodiments 27-41, wherein the first cell culture medium comprises a medium suitable for culturing amniotic fluid cells.

Embodiment 43. The method of embodiment 42, wherein the medium suitable for culturing amniotic fluid cells comprises fetal bovine serum (FBS).

Embodiment 44. The method of any one of embodiments 27-43, wherein the first cell culture medium comprises a keratinocyte growth medium. Embodiment 45. The method of embodiment 44, wherein the keratinocyte growth medium comprises epidermal growth factor (EGF), insulin, hydrocortisone, cholera toxin (CT), epinephrine, and transferrin.

Embodiment 46. The method of embodiment 45, wherein the keratinocyte growth medium further comprises a growth-promoting agent, optionally wherein the growthpromoting agent comprises bovine pituitary extract.

Embodiment 47. The method of any one of embodiments 27-41, wherein the first cell culture medium comprises a mixture of a keratinocyte growth medium and a medium suitable for culturing amniotic fluid cells.

Embodiment 48. The method of embodiment 47, wherein the keratinocyte growth medium and a medium suitable for culturing amniotic fluid cells are present in the mixture at a ratio of about 1 : 1 by volume.

Embodiment 49. The method of any one of embodiments 27-48, wherein the support matrix comprises proteinaceous gel and/or basement membrane matrix.

Embodiment 50. The method of any one of embodiments 27-49, wherein the support matrix or basement membrane matrix comprises protein secreted by cells of mesenchymal origin.

Embodiment 51. The method of embodiment 50, wherein the cells of mesenchymal origin are Engelbreth-Holm-Swarm mouse sarcoma cells.

Embodiment 52. The method of any one of embodiments 27-51, wherein the support matrix or basement membrane matrix comprises laminin 1, laminin 9, laminin 11, collagen, fibronectin, hyaluronic acid, one or more proteoglycans, and/or one or more inert polymers.

Embodiment 53. The method of any one of embodiments 27-52, wherein the aggregates are cultured for about 4-45 or 4-90 days to form the elongated hair follicle organoid.

Embodiment 54. The method of any one of embodiments 27-53, wherein elongated hair follicle organoid is capable of being maintained in culture for at least about 1, 2, 3, or 4 months.

Embodiment 55. The method of any one of embodiments 27-54, wherein at least about 25%, 50%, 75% or 100% of the second cell culture medium is exchanged at least about once every three to seven days (e.g., about once every 3, 4, 5, 6, or 7 days), optionally wherein at least about 25%, 50%, 75% or 100% of the second cell culture medium is exchanged once every two to seven days (e.g., once every 2-3, 3-4, 4-5, 5- 6, or 6-7 days).

Embodiment 56. The method of any one of embodiments 27-55, wherein the basement membrane matrix is present in the suspension at about 2-4% by weight, about 2.5- 3.5% by weight, or about 3% by weight.

Embodiment 57. A hair follicle organoid produced by the method of any one of the preceding embodiments.

Embodiment 58. A method of growing a hair follicle, comprising implanting the organoid of any one of embodiments 1-24 and 57 into dermal tissue.

Embodiment 59. The method of embodiment 58, further comprising piercing the dermal tissue with a needle prior to implanting the organoid.

Embodiment 60. A method of treating hair loss in a subject, comprising implanting the organoid of any one of embodiments 1-24 and 57 into dermal tissue of the subject.

Embodiment 61. A method of screening a compound for hair growth -modulating activity, comprising: contacting a hair follicle organoid according to any one of embodiments 1-24 and 57 or the embodiment 60 with the compound; and detecting an effect or absence thereof on the hair follicle organoid.

Embodiment 62. The method of embodiment 61, wherein a plurality of compounds are screened by contacting a plurality of hair follicle organoids with a member of the plurality of compounds and detecting a plurality of effects or absences thereof on the hair follicle organoids.

Embodiment 63. The method of embodiment 61 or 62, wherein the effect or absence thereof comprises an increase, decrease, or absence of change in production of hair shaft by the hair follicle organoid.

Embodiment 64. The method of embodiments 61 or 62, wherein the effect or absence thereof comprises an increase, decrease, or absence of change in growth of the hair follicle organoid. Embodiment 65. The method of embodiments 61 or 62, wherein the effect or absence thereof comprises a change in or morphology of the hair follicle organoid or absence thereof.

Embodiment 66. The method of embodiments 61 or 62, wherein the effect or absence thereof comprises a change in or morphology of the matrix of the hair follicle organoid or absence thereof.

Embodiment 67. A method of producing DP cells from human induced neural crest cells or from human iPSCs via an induced neural crest intermediate, comprising culturing human induced neural crest cells or an induced neural crest intermediate in a medium comprising WNT-lOb and R-spondin 1 to obtain induced DP cells.

Embodiment 68. The method of embodiment 67, wherein the medium comprises about O.Olpg/ml Wnt-lOb and about 0.01 pg/ml R-Spondin 1.

Embodiment 69. The method of embodiment 68, wherein the medium further comprises AmnioMax™-!! complete medium.

Embodiment 70. The method of embodiment 67 or 68, wherein the medium comprises a mixture of a keratinocyte growth medium and a medium suitable for culturing dermal cells.

Embodiment 71. The method of any one of embodiments 67-70, wherein the culturing step is conducted for about 1-20, 2-15, 5-12, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

Embodiment 72. The method of embodiment 71, wherein the culturing step is conducted for about 12 days.

Embodiment 73. The method of any one of embodiments 67-72, wherein the culturing step further comprises changing the medium every about 1, 2, or 3 days.

Embodiment 74. The method of any one of embodiments 67-72, wherein the culturing step further comprises changing the medium about every other day.

Embodiment 75. The method of any one of embodiments 67-74, wherein the culturing step further comprises passaging the cells at about day 3, 4, or 5, optionally with an enzymatic solution (e.g., 0.25% trypsin-EDTA solution, or TrypLE Select™). Embodiment 76. The method of any one of embodiments 67-75, further comprising coculturing the induced DP cells with induced epithelial stem cells (EpSCs) to form an elongated hair follicle organoid.

Embodiment 77. A hair follicle organoid produced by the method of embodiment 76.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGs. 1A-1D show induction of dermal papilla cells from iPSC-derived neural crest cells obtained with Wnt-lOb and R-spondin 1. FIG. 1A shows an overview of the protocol to differentiate DP cells from iPSCs through NC intermediate step. FIG. IB shows differential expression of iPSC-DP at day 12 compared to original iPSC measured by RT-PCR (reverse transcription polymerase chain reaction). Error bars represent the Bonferroni -adjusted 95% confidence interval of the mean of 8 independent experiments. In this way, a gene can be seen as statistically significant if the confidence interval doesn’t contain the null hypothesis (log2 fold-change = 0), while maintaining a family-wide error rate of 5%. FIG. 1C shows a bright-field image of iPSC-derived DP cells. Scale bar 100 pm. FIG. ID shows immunostaining for DP markers and enzymatic detection for alkaline phosphatase (dark) in DP cells after 12 days of differentiation of iPSC-NC. DAPI (4',6-diamidino-2-phenylindole, a blue-fluorescent DNA stain) staining was used for nuclei detection.

[0012] FIGs. 2A-2F show molecular characterization of NC cells obtained with neurospheres protocol and growth factors effects on DP cells derivation. FIG. 2A shows an inverted microscope images of embryoid bodies (EBs) formed in AggreWell™, cultured in suspension in medium, and attached on MATRIGEL® to form neural rosettes. The insert highlights migrating cells from the rosettes. FIG. 2B shows immunofluorescence images taken at the end of the treatment for NC markers as indicated in each box. White dashed circle indicates neural rosettes (SOX2+/SOX10-), which generate migrating NC cells (SOX2- /SOX10+). DAPI was used to stain nuclei. FIG. 2C shows RT-PCR analysis of NC markers (PAX7, PAX3, AP2A, SOX10, and SOX9) and pluripotency markers (OCT4 and Nanog) at the end of the protocol. The bar graph shows the Log of relative expression of NC gene expression, normalized to iPSCs. Error bars represent the SD of three independent experiments. FIG. 2D shows quantification of versican (left bars) and alkaline phosphatase (right bars) level in NC-DP cells, at the end of the differentiation protocol, calculated as fold change in comparison to NC cells when treated with a different combination of growth factors. Versican (a large extracellular matrix proteoglycan) and alkaline phosphatase expression values were normalized on DAPI staining. FIG. 2E shows a heatmap displaying relative expression (z-score) of DP genes, assessed by RT-PCR analysis, in NC-DP cells upon treatment with the best combination of defined factors. FIG. 2F shows immunostaining for Versican and enzymatic detection for alkaline phosphatase (dark) in DP cells derived from three different iPSC lines after 12 days of differentiation using WNTlOb+R-spondinl. Each number in the DP-iPSC represents a different donor. NC cells used as a negative control. DAPI staining used for nuclei detection.

[0013] FIGs. 3A-3G show Bulk RNA sequencing showing mesenchymal features acquired by iPSC-DP cells during the differentiation process. FIG. 3 A shows a Principal Component Analysis (PCA) of iPSC, NC cells, iPSC-DP cells, native human DP (hDP) and human foreskin fibroblasts (HFF). iPSC-DP day 8 and hDP samples were also cultured in 3D as spheroids and then submitted for analysis (spheroids). At each time point, at least three replicates were analyzed for iPSC-DP, thirteen biological replicates for the native hDP and four different samples for the HFF. FIG. 3B shows a Venn diagram of differentially expressed genes (DEGs, logFC>l, adjusted p-value <0.05) in native hDP and iPSC-DP cells at different stages of cell differentiation compared to HFF. FIGs. 3C-3F show a dot plot quantifying the most enriched gene sets in hDP and iPSC-DP at different time points during the differentiation process compared to HFF based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. FIG. 3G shows a heatmap quantifying the expression level of genes selected to be representative of each stage of the DP differentiation process from iPSC to NC, to DP and compared with control cells hDP and HFF.

[0014] FIGs. 4A-4E show WGCNA highlights signaling pathways and expression patterns associated with the three populations of mesenchymal cells. FIG. 4A shows an Eigengene pattern across samples. Each line represents the trend of the module in each cell group. Similar trends are arranged under the same pattern. The stars in pattern 4 (ME9) and 7 (ME6) indicate the “positive” trends, which included all patterns where the eigengene levels in iPSC-DP were comparable to hDP but differs from HFF (either up or down-regulated). The star in pattern 6 for ME10 denotes the “negative” pattern, where the eigengene levels in iPSC-DP were equal to HFF but different than hDP (either up or down-regulated). Lastly, the stars in patterns 3 (ME5), 5 (ME7), and 7 (ME8) point to the “off’ pattern, where the eigengene levels in iPSC-DP were different than both hDP and HFF (either up or down- regulated). FIG. 4B shows KEGG signaling pathways associated with each WGCNA (weighted gene co-expression network analysis) module. The asterisk indicates a significant association. FIGs. 4C-4E each show a bar plot to show the expression trend of the most prominent hub genes extracted from the representative module of each analyzed mesenchymal population, as indicated above each panel (FIG. 4C: HFF; FIG. 4D: iPSC-DP; and FIG. 4E: hDP).

[0015] FIGs 5A-5D show RNA single-cell sequencing determines two major trajectories in the transition from NC cells to DP cells. FIG. 5A shows a UMAP (Uniform Manifold Approximation and Projection) plot of clustering of iPSC-DP cells from the NC cell state to the end of the differentiation. Sorted cell identities are color-coded by cell type. The circles identify the clusters per time point during the differentiation process. FIG. 5B shows a dot plot of the expression of dermal cells genes, signatures DP markers, ECM network, focal adhesion pathways, and cell cycle terms in each identified cluster. Gene expression frequency is indicated by dot size and expression level is indicated by color intensity. FIG. 5C shows biological processes associated with each cluster of day 8 computed using Gene Ontology (GO) database. FIG. 5D shows the expression pattern of ITGA1 (Integrin Subunit Alpha 1) overlapped with the UMAP plot showing high expression of this gene in cluster 5 (circle), among the clusters representing days 6 and 8 (box).

[0016] FIGs. 6A-6C show nascent DP cells follow two major trajectories during the differentiation process from NC cells. FIG. 6A shows an RNA Velocity analysis overlayed on UMAP projection of clustered iPSC-DP cells from day 0 to day 8. Clusters are colored- coded and labeled. The arrows indicate the transition from one state to the future one in a temporal sequence. FIG. 6B shows Violin plots representing the expression levels of some mesenchymal markers acquired during the transition from NC cells to DP-like cells. FIG. 6C shows mapping of representative genes uniquely expressed in clusters 7, 1, 3, and 6 connected by a collateral track in the pseudo-time progression.

[0017] FIGs. 7A-7D show Chimeric HF generated in vivo using the patch assay with cell suspension and organoids. FIG. 7A shows a representative picture of an Athymic Nude- Foxnlnu mouse 30 days after transplantation. Indicated by arrows are the sites of transplantation with different cell combinations. iPSC-DP 1 and iPSC-DP2 indicate two different iPSC lines differentiated into DP cells, aggregated in spheroids for 72 hours, and transplanted in combination with embryonic mouse keratinocytes (mKC). The negative control is made of embryonic mKC transplanted alone. The positive control represents embryonic mKCs transplanted in combination with mouse embryonic dermal cells (mDC). FIG. 7B shows quantification of newly formed hair shafts modeled using a generalized linear model with Poisson error distribution. Tukey’s honestly significant difference test (Tukey’s HSD) was used for pairwise post-hoc testing. In each pairwise comparison, the null hypothesis stated there is no difference between group means. FIG. 7C shows stereomicroscope images of transplanted sites on the day of sacrifice (day 30) showing hair shafts underneath the skin. FIG. 7D shows immunohistochemical images of generated HFs after transplantation of embryonic mKCs combined with human iPSC-DP cells or embryonic mDCs. A human-specific antibody (inner drop shape) recognizes only human cells, while K14 (light shading) stained keratinocytes. DAPI (light areas between inner and outer drop shapes) was used to identify nuclei. All the images are z-stack projections of 40X confocal images taken with an Apotome microscope (Zeiss).

[0018] FIGs. 8A-8C show HF generated in vivo using the slurry assay with cell suspension and organoids. FIG. 8A shows a graphical schematic of slurry preparation in which dermal and epithelial cells are combined in a basement membrane droplet and placed on a membrane, which is then transplanted into full thickness skin excisions on the back of Nu/Nu mice and representative images of a mouse 2-weeks post transplantation after bandage removal. Images highlight skin healing and hair follicle production after ten weeks. FIG. 8B shows hematoxylin and eosin staining of skin with distinct follicles when sectioned longitudinally or vertically, a plot of follicle density vs. skin thickness, and follicle density observed for the indicated conditions. Hair follicle density strongly correlates to skin thickness, with organoids generated from iDP-iEP cells resulting in greater levels of folliculogenesis than background cell type alone (gestational week 19 fetal human dermal cells combined with HFK cells in slurry format). Follicle density was calculated by taking longitudinal sections of transplanted skin and manually counting follicles, divided by the graft area (outlined in longitudinal cross section). FIG. 8C shows stereoscopic images of transplanted sites on the day of sacrifice showing hair shafts growing directionally out of the skin with hair bulbs visible from the underside of the skin.

[0019] FIG. 9 shows the fifty top hub genes extracted for each of the 13 modules (MEI to ME13) identified by the WGCNA (weighted gene co-expression network analysis), where “ME” refers to module eigengene.

[0020] FIGs. 10A-10C show iPSC-derived DP and epithelial cells spontaneously aggregating and assembling into HF -like structures. FIG. 10A shows a graphical schematic of in vivo mouse HF morphogenesis (Figure modified from Saxena et al., Exp Dermatol, 2019. 28(4):332-344). FIG. 10B shows a graphical representation of the stages observed during organoid development in vitro highlighting the similarities with the HF morphogenesis in murine models. FIG. 10C shows representative images of the key steps of organoid development obtained combining iPSC-DP cells with human keratinocytes. Shading in FIG. IOC shows the iPSC-DP, engineered via CRISPR-Cas9, with the fluorescent proteins mCherry. “Epi” refers to epithelial skin layer, “Der” refers to dermal skin layer, “pre-Pc” refers to placode precursor; “PC” refers to placode, “pre-DC” refers to dermal condensate precursor; “DC” refers to dermal condensate; “IRS” refers to inner root sheet, “TAC” refers to Transit Amplifying Cells, “HFSC” refers to HF stem cell; “Mx” refers to Matrix, and “Fb” refers to fibroblasts.

[0021] FIGs. 11A-11C show structural changes of HF organoids during their in vitro formation. In particular, FIGs. 11 A-l 1C show immunostaining at key stages in HF organoids using iPSC-DP cells, employing antibodies against epithelial markers, mesenchymal markers, and proliferative cells marker. Scale bars 25 pm. Representative slices of organoids formed with the cell combination indicated over each quadrant at very early stages (day 3 FIGs. 11 A- 1 IB) or later time point (day 9, FIG. 11C). H&E staining was used for tissue morphology and immunohistochemistry performed with specific HF markers (in white). When indicated, EpSC are green due the presence of endogenous GFP and iPSC-DP in red, due the presence of endogenous mCherry. Antibodies highlight epidermal layer (K14+, K19+, P-Cad+, P- cat+), dermal compartment (Vim+, Lef-1+) and proliferative cells (Ki67+). Dashed boxes are a magnified portion of the organoid. The white arrows indicate the jail breaking point.

[0022] FIGs. 12A-12D show the structural similarity between organoids derived from iPSC- DP cells and human native HF cells. FIG. 12A shows representative images of the key steps of organoid development obtained combining human hair follicle-derived keratinocyte cells (HF-KC) with iPSC-DP cells (upper panel) or human HF-derived dermal papilla cells (hDP) (lower panel). FIG. 12B shows confocal images of a representative organoid at the end of the elongation process. FIG. 12C shows representative images of immunocytochemistry performed on HF sections (upper panel) or human organoids sections (lower panel).

Antibody combinations are indicated above each image. Scale bar 100 pm. FIG. 12D shows a measurement system to evaluate organoid formation and elongation by plotting the number of organoids that meet the criteria of round, polarized or elongated, where the measurements were obtained with Imaged software. Each dot represents a single organoid.

[0023] FIGs. 13A-13D show a chimeric HF generated in vivo upon organoids transplantation. FIG. 13 A shows representative pictures of organoid transplantation onto an Athymic Nude-Foxnlnu mouse the day of surgery. FIG. 13B shows stereomicroscope images of transplanted organoids on the day of sacrifice (day 30) showing newly generated hair shafts. FIGs. 13C and 13D show immunohistochemistry on sections of the grafted site, employing H&E staining (FIG. 13C) and DAPI staining (FIG. 13D) to show hair shafts generated by an individual organoid.

[0024] FIG. 14 shows a graphical summary of multiple applications of the HF organoids from the bench to the clinic in the attempt to provide a better solution for the treatment of alopecia and related disorders.

[0025] FIGs. 15A-15E show induction of organoid formation upon 3D culture of iPSC- derived DP in combination with murine keratinocytes. FIG. 15A shows time-lapse representative brightfield images of iPSC-DP derived organoids formed in a 384-well plate platform. FIG. 15B shows separation of dermal papilla cells and mouse epithelial cells in organoids at day 6 and day 9 in vitro. iPSC-DP are shown in red, engineered via CRISPR- Cas9 with the fluorescent protein mCherry. Scale bar 100 pm. FIG. 15C shows whole mount immunostaining with antibodies for epidermal layer (K14), dermal compartment (Versican) and Nuclei (DAPI). FIG. 15D shows representative brightfield images of organoids obtained by combining hDP or fibroblasts (hFB) with mouse Keratinocytes (mKC) at key days during the developmental process. FIG. 15E shows quantification of organoid elongation for the conditions in FIG. 15D.

[0026] FIGs. 16A-16C show morphological features upon 3D culture of iPSC-derived DP or human HF derived DP (hDP) in combination with human hair-follicle derived keratinocytes. FIG. 16A shows multiple replicates of organoid images representing the maximum projection of the bright field and the fluorescent channels, following accumulation of non-toxic dye within the organoids as indicated above each box. Scale bar 200 pm. FIG. 16B shows plots showing the distribution of three metrics of interest (length, volume, surface) among organoids created in a 384-well plate. FIG. 16C shows a graphical representation of the measurement of some geometrical features.

[0027] FIG. 17 shows combinations of growth factors used in the initial screening. For the first 5 days of the DP differentiation, the cells were treated with the indicated growth factors in the first line, which were then withdrawn and replaced with BMP6 and FGF20 for the remaining 7 days. R = R-spondin.

[0028] FIG. 18 shows original and segmented images demonstrating the elongation score calculation, as explained in Table 8.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0029] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with such embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.

[0030] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of nucleic acids, reference to “a cell” includes a plurality of cells, and the like.

[0031] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0032] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0033] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference contradicts any explicit content of this specification, including definitions, this specification controls.

I. Definitions

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All references cited herein are incorporated by reference in their entirety as though fully set forth. In case of any contradiction or conflict between material incorporated by reference and the expressly described content provided herein, the expressly described content controls. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.

[0035] As used herein, an “induced epithelial cell” or “induced EP cell” refers an epithelial cell derived from a non-epithelial cell by artificially inducing expression of certain genes. In some embodiments, the induced epithelial cells are derived from human induced pluripotent stem cells (iPSCs). In some embodiments, the induced epithelial cells are derived from an adult human subject or a fetal human subject, e.g., via an iPSC intermediate. In some embodiments, the induced epithelial cells comprise at least one epigenetic or genetic difference relative to the naturally occurring cell(s) from which they were derived. In some embodiments, the induced epithelial cells are derived from one or more cells obtained from a subject and comprise at least one epigenetic or genetic difference relative to naturally occurring epithelial cells of the subject. Induced epithelial cells include epithelial cells at various stages of differentiation, including epithelial stem cells and keratinocytes, such as inner root sheath keratinocytes and outer root sheath keratinocytes.

[0036] As used herein, an “epithelial stem cell (EpSC)” refers to a multipotent cell which has the potential to become committed to multiple cell lineages, including cell lineages resulting in epithelial cells. In some embodiments, the EpSCs have increased expression of markers including, but not limited to, TP63 (tumor protein p63), KRT5 (keratin 5), KRT14, and KRT15, e.g., relative to iPSCs.

[0037] A “pluripotent stem cell” as used herein refers to a stem cell that can be cultured in vitro and has the ability to differentiate into any of the three germ layers (ectoderm, mesoderm, endoderm). The pluripotent stem cell may be a non-genetically modified pluripotent stem cell or a genetically modified pluripotent stem cell. The pluripotent stem cells may be mammal-derived pluripotent stem cells, such as from rodents and primates, for example where the primates are humans.

[0038] As used herein, the term “induced pluripotent stem cells” or “iPSCs”, also commonly abbreviated as “iPS cells”, refers to pluripotent stem cells artificially derived from a non- pluripotent cell, such as an adult somatic cell, by artificially inducing expression of certain genes. In some embodiments, human induced pluripotent stem cells (hiPSCs) are utilized. [0039] As used herein, “Dermal Papilla cell” or “DP cell” refers to a specialized mesenchymal cell that regulates HF development, growth, and cycling when present in the dermal papilla of a HF, and is also a reservoir of multi-potent DP stem cells.

[0040] As used herein, an “induced Dermal Papilla cell” or “induced DP cell” refers an DP cell derived from a non-DP cell by artificially inducing expression of certain genes. In some embodiments, induced DP cells are derived from human induced pluripotent stem cells (iPSCs). In some embodiments, induced DP cells are derived from human induced neural crest (NC) cells, or are derived from human iPSCs via an induced neural crest intermediate. In some embodiments, induced DP cells are derived from naturally occurring cell(s) and comprise at least one epigenetic or genetic difference relative to the naturally occurring DP cell(s) from which they were derived. In some embodiments, the induced DP cells are derived from one or more cells obtained from a subject and comprise at least one epigenetic or genetic difference relative to naturally occurring DP cells of the subject. In some embodiments, induced DP cells are derived from an adult human subject or a fetal human subject.

[0041] “WNT-lOb” is a protein that in humans is encoded by the WNT10B gene.

[0042] “R-spondin 1” is a secreted protein that in humans is encoded by the Rspol gene, found on chromosome 1.

[0043] “BMP inhibitor” as used herein is a compound or composition that targets bone morphogenetic protein (BMP) receptors or BMP signaling activity, including signaling initiated by BMP4. Exemplary BMP receptor inhibitors include, but are not limited to, DMH1, LDN-212854, ML347, dorsomorphin, LDN-193189, and LDN-213117.

[0044] As used herein, the term “growth factor” means a substance capable of stimulating cellular processes including but not limited to growth, proliferation, morphogenesis or differentiation. “Epidermal growth factor” or “EGF” is a cell signaling molecule involved in diverse cellular functions, including cell proliferation, differentiation, motility, and survival, and in tissue development. EGF is an example of a mitogenic growth factor.

[0045] As used herein, a “ROCK inhibitor” (also referred to as a “Rho-kinase inhibitor” or “rho-associated protein kinase inhibitor”) is a compound that targets rho kinase (ROCK) and inhibits the ROCK pathway. Exemplary ROCK inhibitors include, but are not limited to, AT- 13148, BA-1049, BA-210, p-Elemene, Chroman 1, DJ4, GSK-576371, GSK429286A, H- 1152, Y-27632, HA-1077 (fasudil), hydroxyfasudil, ibuprofen, LX-7101, netarsudil, RKI- 1447, ripasudil, TCS-7001, thiazovivin, verosudil (AR-12286), Y-27632, Y-30141, Y-33075, and Y-39983. In some embodiments, the ROCK inhibitor is Y-27632.

[0046] As used herein, “keratinocytes” (also referred to as corneocytes) refers to epidermal cells that produce keratin. Keratinocytes include outer root sheath keratinocytes and inner root sheath keratinocytes.

[0047] As used herein, “serum-free medium” is one that contains no animal or human serum of any type. Serum-free media may be preferred to minimize possible xeno-contamination of the stem cells. Meanwhile, a “serum replacement-free medium” is one that has not been supplemented with any commercial serum replacement formulation.

[0048] As used herein, “feeder free” refers to a minimal use of animal-derived cells and proteins.

[0049] The skin is composed of three layers: a) the epidermis, b) the dermis, and c) the hypodermis. The “epidermis” is the outermost layer of skin and comprises the “interfollicular epidermis” (IFE), which is a stratified squamous epithelium of the skin, and HFs. The “dermis” is the inner layer of the skin and is composed of a network of collagenous extracellular material, fibroblasts, blood vessels, nerves, sebaceous glands, and elastic fibers. The “hypodermis” is the layer underneath the dermis and mainly comprises adipose tissue and sweat glands.

[0050] The “hair follicle” or “hair follicle mini-organ” is a peg of tissue that comprises a highly organized system of recognizably different layers arranged in concentric series. Active hair follicles extend down through the dermis, the hypodermis (a loose layer of connective tissue), and into the fat or adipose layer (Ross M H, Histology: A text and atlas, 3rd Ed., Williams and Wilkins, 1995: Chapter 14; Burkitt et al, Wheater's Functional Histology, 3rd Ed., Churchill Livingstone, 1996: Chapter 9).

[0051] At the base of an active HF lies a “hair bulb.” The hair bulb comprises a body of dermal cells or “dermal compartment”, also known as “dermal papilla” cells or “DP” cells, contained in an inverted cup of epidermal cells known as the epidermal “matrix.” Irrespective of follicle type, the germinative epidermal cells at the very base of this epidermal “matrix” produce the hair fiber, together with several supportive epidermal layers. In some embodiments, the HF organoid comprises a dermal compartment and an adjacent cone of cells, wherein the cone of cells comprises transit-amplifying cells. In some embodiments, the induced DP cells occupy a hair bulb, and epithelial cells extend above the hair bulb. In some embodiments, the HF organoid improves skin vasculature and innervation. [0052] “Organoid”, as used herein, refers to a three-dimensional cell cluster or aggregate derived through culture of stem cells that has structural similarity to an organ, or part of an organ, and possesses cell types relevant to that particular organ. Organoids include miniaturized and/or simplified versions of an organ and usually replicate much of the complexity of an organ, or at least express selected aspects of it, such as producing only types of cells relevant to that particular organ. HF organoids are described herein.

[0053] As used herein, a “bulge region” refers to a region in a hair follicle with increased thickness (e.g., cross-sectional diameter) relative to other parts of the follicle that is ordinarily located between the opening of the sebaceous gland and the attachment site of the arrector pili muscle. The bulge region is associated with the presence of hair follicle stem cells and/or epidermal stem cells. A bulge region may or may not be present in an organoid.

[0054] As used herein, a “fibroblast” is a cell in connective tissue which produces an extracellular matrix rich in collagen and other fibers.

[0055] As used herein, “skin graft” or “replacement skin graft” refers to a patch of skin for transplantation, implantation, or other attachment, to a subject. Methods of generating skin grafts from EpSC are generally known. For example, Zang et al., Nat Commun. 5:3071 (2014), discloses generation of skin grafts from EpSCs. As another example, Morris et al., Nat Biotechnol. 22(4):411-7 (2004), and Ito et al., Nature 447(7142):316-20 (2007), discloses generation of skin grafts from naturally occurring bulge EpSC.

[0056] As used herein, a “cellular scaffold” or “tissue scaffold” refers to a material designed to cause desirable cellular interactions to contribute to the formation of new functional tissues for implantation or other medical purposes. In some embodiments, the cellular scaffold is in the form of a hydrogel, a fiber network (e.g., via electrospun mat), or a photoinitiated 3D structure. In some embodiments, the cellular scaffold comprises a biodegradable polymer. In some embodiments, the cellular scaffold is in the form of a hydrogel.

[0057] As used herein, “hydrogel” refers to a gel in which the liquid component is water. Examples of hydrogels include, but are not limited to, gelatin-based hydrogels, collagen- based hydrogels, hyaluronic-based hydrogels, polynucleotide-based hydrogels, polypeptide- based hydrogels, polysaccharide-based hydrogels, poly(ethylene glycol) (PEG)-based hydrogels, and polyacrylate-based hydrogels. See, e.g., El-Sherbiny et al., Glob Cardiol Sci Pract. 2013(3):316-342. In some embodiments, the cellular scaffold includes a cell reservoir and a guide attached to the cell reservoir comprising one or more first biodegradable polymers. First biodegradable polymers include, but are not limited to, poly(glycolic acid) (PGA), poly(lactic-co-glycolic) acid (PLGA), poly(lactic acid) (PLA), polycaprolactone (PCL), polyethylene glycol, poly(butylene succinate) (PBS), polyphosphazenes, polyanhydrides, polyphosphoesters, polyurethanes, polycarbonates, and combinations thereof.

[0058] The terms “or a combination thereof’ and “or combinations thereof’ as used herein refers to any and all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0059] “ Or” is used in the inclusive sense, i.e., equivalent to “and/or,” unless the context requires otherwise.

[0060] “About” indicates a degree of variation that does not substantially affect the properties of the described subject matter, e.g., within 10%, 5%, 2%, or 1%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed considering the number of reported significant digits and by applying ordinary rounding techniques.

II. Hair Follicle Organoids

[0061] Hair follicle (HF) organoids, methods of use thereof, and methods of producing or growing HF organoids are provided herein.

[0062] In some embodiments, the HF organoid comprises: (i) induced dermal papilla (DP) cells; and (ii) induced epithelial cells, such as those described herein or those produced by any of the methods described herein; wherein the HF organoid has a spheroidal or elongated shape.

[0063] In some embodiments, an HF organoid is considered elongated when the roundness score is below 0.6. In some embodiments, the roundness score is calculated using the formula: roundness = (4K X area)/(convex perimeter) ² which generates a value from 1 to 0. Ranges of values may then be used to assess the development of the organoids as follows: organoids are considered round if the roundness score is between 1 and 0.8, polarized if the roundness score is between 0.8 and 0.6, and elongated when the roundness score is below 0.6. In some embodiments, an HF organoid is considered elongated when the elongation score is greater than 0.4. In some embodiments, the elongation score is calculated using the formula: 1 -(roundness = (4K X area)/(convex perimeter) ² ) which generates a value from 0 to 1. Ranges of values may then be used to assess the development of the organoids as follows: organoids are considered round if the elongation score is between 0 and 0.2, polarized if the elongation score is between 0.2 and 0.4, and elongated when the elongation score is greater than 0.4. See also, Example 5, Table 8, and FIG. 18.

[0064] In some embodiments, the induced DP cells are derived from human induced pluripotent stem cells (iPSCs). In some embodiments, the induced DP cells are derived from human induced neural crest cells, or are derived from human iPSCs via an induced neural crest intermediate. In some embodiments, the induced DP cells were derived from human induced neural crest cells or from human iPSCs via an induced neural crest intermediate using a medium comprising WNT-lOb and R-spondin 1 is used to induce DP cells. In some embodiments, the medium comprises about O.Olpg/ml Wnt-lOb and about 0.01 pg/ml R- Spondin 1. In some embodiments, the medium comprising WNT-lOb and R-spondin 1 further comprises AmnioMax™-II complete medium (GIBCO™, a fully-supplemented medium for short-term culture of human amniotic fluid cells containing fetal bovine serum (FBS), gentamicin, and L-glutamine to maximize cell attachment and growth, proteins (e.g., one or more of insulin, fibronectin, mediator of RNA polymerase II RNA polymerase transcription subunit 24, antithrombin-III, methyltransferase-like protein 7B, Beta-actin-like protein 2, heterochromatin protein 1 -binding protein 3 (HP1BP3), hepatocyte growth factor activator (HGFAC), ferritin heavy chain, pre-rRNA processing protein FTSJ3, chitinase domaincontaining protein 1, unconventional myosin-lc, torsin-lA-interacting protein 1, 60S ribosomal protein L6, myosin-9, acetyl-CoA carboxylase 1, filamin-A, ceruloplasmin, hemopexin, hornerin, desmoglein-2, SUN domain-containing protein 2, coagulation factor XIII A chain, complement factor H, and/or fibroblast growth factor receptor 1) and an enhanced buffering system for greater pH stability).

[0065] In some embodiments, the induced epithelial cells are derived from human iPSCs. In some embodiments, the induced DP cells were derived from naturally occurring cell(s) and comprise at least one epigenetic or genetic difference relative to the naturally occurring cell(s) from which they were derived. In some embodiments, the induced DP cells are derived from an adult human subject or a fetal human subject. In some embodiments, the induced epithelial cells are derived from an adult human subject or a fetal human subject. In some embodiments, the induced DP cells are derived from one or more cells obtained from a subject and comprise at least one epigenetic or genetic difference relative to naturally occurring DP cells of the subject.

[0066] In some embodiments, the induced epithelial cells comprise at least one epigenetic or genetic difference relative to the naturally occurring cell(s) from which they were derived. [0067] In some embodiments, the induced epithelial cells are derived from one or more cells obtained from a subject and comprise at least one epigenetic or genetic difference relative to naturally occurring epithelial cells of the subject.

[0068] In some embodiments, the HF organoid lacks a stem cell niche, a bulge, a sebaceous gland, and/or an arrector pili muscle.

[0069] In some embodiments, the HF organoid comprises a dermal compartment and an adjacent cone of cells, wherein the cone of cells comprises transit-amplifying cells.

[0070] In some embodiments, the induced DP cells occupy a hair bulb, and epithelial cells extend above the hair bulb.

[0071] In some embodiments, the HF organoid comprises: a) epithelial cells wrapped within a cluster comprising induced DP cells; or b) DP cells wrapped within a cluster comprising epithelial cells.

[0072] In some embodiments, the HF organoid comprises matrix produced by the epithelial cells. In some embodiments, the matrix imparts an elongated shape to the HF organoid.

[0073] In some embodiments, the HF organoid comprises keratinocytes.

[0074] In some embodiments, the HF organoid comprises an inner root sheath (IRS), an outer root sheath (ORS), a bulge, a companion layer, and/or a matrix. The bulge is a region of the ORS surrounding the hair shaft (HS) generally located around the attachment site of an arrector pili muscle, if present. The bulge generally houses several types of stem cells. The companion layer, much like the HS and the IRS, is derived from the hair matrix. The companion layer is generally situated between the IRS and the ORS. The matrix is the part of the HF organoid where matrix keratinocytes proliferate to form the HS of growing hair.

[0075] The lowermost dermal sheath or IRS is contiguous with the papilla basal stalk, from where the sheath curves externally around all of the hair matrix epidermal layers as a thin covering of tissue. The lowermost portion of the dermal sheath or IRS then continues as a sleeve or tube for the length of the follicle (Ross MH, Histology: A text and atlas, 3rd Ed., Williams and Wilkins, 1995: Chapter 14; Burkitt HG, et al, Wheater's Functional Histology, 3rd Ed., Churchill Livingstone, 1996: Chapter 9). [0076] In some embodiments, the HF organoid comprises a hair fiber.

In some embodiments, the HF organoid comprises about 1,000 to about 15,000, about 1,000 to about 10,000, about 2,000 to 6,000, or about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, or 15,000 induced DP cells, and about 1,000 to 15,000, about 2,000 to 6,000, or about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000 or 15,000 induced epithelial cells. In some embodiments the HF organoid comprises induced DP cells and induced epithelial (EP) cells at a ratio of about 1 : 1 DP:EP (e.g., 6,000 DP cells and 6,000 EP cells) or about 1 :2 DP:EP.

III. Exemplary Methods

[0077] The HF organoids of the present disclosure, generated by combining specialized epithelial and dermal papilla cells, are a promising therapy to treat baldness as well as a powerful tool in drug discovery and disease modeling in the hair loss field (FIG. 13). They also can provide more physiologically relevant and realistic 3D models of cellular interactions than a monolayer having a single type of cell. These 3D models can be used to study how cells interact with each other and to gain insight into cell signaling and pathway acti vati on/inhibiti on .

[0078] Moreover, HF organoids can be employed in precision medicine, particularly for the development of individualized treatments, as they can be derived from patient’s somatic cells, which are used to generate induced pluripotent stem cells (iPSC). Indeed, a library of hundreds of molecules can be tested in patient-derived organoids to identify the best therapies in vitro, which is considered predictively useful for efficacy in the clinic. This bio-engineered approach allows one to recapitulate native tissue architecture, cellular composition, and function, in order to achieve a higher level of fidelity to the in vivo condition in a high- throughput system, leading to an increase in the accuracy of pharmacological ‘hits’ during screening. The foundation for the organoid library is based on patients’ iPSC, which are used to derive dermal papilla cells (DP) and epithelial stem cells (EpSC), according to methods described herein. Once the two cell types are generated and processed for quality attributes (gene and protein analysis), they are combined 1 : 1, distributed into 3D platforms, and fed with commercially available media to induce organoid formation. This culture system requires minimal tissue handling, and allows for real-time analysis structural parameters, thus enabling high-content screening of mature HF organoids. In addition, this organoid system is compatible with both real-time imaging and terminal analysis methods. This includes interfacing with high content brightfield imaging to track changes in tissue structure, array methodologies for measuring molecule signals, and systems for gene profile analysis. Through the use of robotics, liquid handling, and data analysis systems, the compounds screening could be fully automated to identify candidate molecules for the appropriate biological responses.

A. Methods for Producing Induced DP Cells

[0079] In some embodiments described herein, a method of producing DP cells from human induced neural crest cells or from human iPSCs via an induced neural crest intermediate, comprises culturing human induced neural crest cells or an induced neural crest intermediate in a medium comprising WNT-lOb and R-spondin 1 to obtain induced DP cells. The methods may comprise any of the features described below and elsewhere herein relevant to producing DP cells.

[0080] In some embodiments, the medium comprises about O.Olpg/ml Wnt-lOb. In some embodiments, the medium comprises about 0.01 pg/ml R-Spondin 1. In some embodiments, the medium comprises about O.Ol pg/ml Wnt-lOb and about 0.01 pg/ml R-Spondin 1. Wnt- lOb and R-Spondin 1 concentrations may range from 10 ng/ml to 500 ng/ml, for example, in an AmnioMax™-II complete medium. Considering the role of the Wnt pathway in the differentiation process of iPSC-NC into DP cells together with the results described herein, one would expect other known WNT activators, such as CHIR99021 (an aminopyrimidine derivative that is a potent glycogen synthase kinase (GSK) 3 inhibitor; also referred to herein as “CHIR”), BIO (6-bromoindirubin-3 '-oxime) or BML-284 hydrochloride, to lead to a similar outcome.

[0081] In some embodiments, the medium further comprises AmnioMax™-II complete medium. Different media (e.g., DMEM/F12 with or without GlutaMAX™, basic DMEM medium, or similar) may be combined with AmnioMax™-II complete medium to change its concentration. In some embodiments, the possible concentration range of AmnioMax™-II complete medium could be from 25% to 75%, such as a 50:50, 25:75, or 75:25 ratio.

[0082] In some embodiments, the culturing step is conducted for about 1-20, 2-15, 5-12, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the culturing step is conducted for about 12 days.

[0083] In some embodiments, the culturing step further comprises changing the medium about every 1, 2, or 3 days.

[0084] In some embodiments, the culturing step further comprises changing the medium every other day. [0085] In some embodiments, the culturing step further comprises passaging the cells at about day 3, 4, or 5, optionally under conditions that promote detachment of adherent cells, e.g., with a recombinant enzyme solution (e.g., a protease solution such as rProtease-EDTA solution), such as a trypsin-EDTA solution. The concentration of trypsin-EDTA can be adjusted, for example, by diluting it in PBS (and equivalent solution) or base culture media. In some embodiments, the concentration range of trypsin-EDTA is from 0.05% to 0.25%. In some embodiments, the concentration range of trypsin-EDTA is 0.125%. In some embodiments, conditions that promote detachment of adherent cells are provided by using one or more alternatives to a recombinant enzyme solution for cell detachment, such as: Trypsin-EDTA (0.25%) (ThermoFisher), ACCUTASE™, Accumax®, EDTA only (though some cell types may require more potency than EDTA alone), and/or citrate saline (135 mM potassium chloride and 15 mM sodium citrate) with or without 10 mM EDTA. See, e.g., Barber, L., “Detaching Adherent Cells Using Citric Saline,” available at medicine.uiowa.edu/flowcytometry/protocolssample-prep/staini ng-protocols/detaching- adherent-cells-using-citric-saline, accessed July 2022.

[0086] In some embodiments, the method further comprises producing an elongated hair follicle organoid from the induced DP cells, e.g., in combination with induced epithelial stem cells (EpSCs). For example, in one embodiment, a flask is coated with a surface coating solution comprising a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., a MATRIGEL® surface coating solution; see, e.g., Kretzschmar et al., Dev Cell 38(6):590-600 (2016); Sato et al., Nature 459(7244):262-5 (2009)).

[0087] The surface coating solution comprising a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., a MATRIGEL® surface coating solution) and DMEM/F12+GlutaMAX™ (200 mM L-alanyl- L-glutamine dipeptide in 0.85% NaCl), and then neural progenitor cell (NPC) complete media of Table 1 is added to the flask, wherein the NPC Basal Medium is as described in Table 2 below. The basic amino acid L-glutamine may also be used in the alternative to GlutaMAX™, though GlutaMAX™ minimizes toxic ammonia build-up, which can improve cell health.

[0088] In some embodiments, the method further comprises co-culturing the induced DP cells with induced epithelial stem cells (EpSCs) to form an elongated hair follicle organoid. In some embodiments, the hair follicle organoid is elongated when the roundness score is below 0.6. In some embodiments, the hair follicle organoid is elongated when the elongation score is greater than 0.4. The roundness or elongation score may be determined as described in the Examples.

Table 1: NPC Complete Medium

Table 2: NPC Basal Medium

[0089] In some embodiments, the iPSC-NC cells are seeded into the coated flasks. After 24 hours, the media is changed by aspiration of the media and adding pre-warmed NPC Complete Medium. The iPSC-NC are allowed to recover for a minimum of 48 hours before beginning differentiation.

[0090] In some embodiments, cells are seeded for iPSC-DP differentiation at Day 0 (DO). The NPC Complete Medium is aspirated from the flasks, the flasks are rinsed with PBS and the PBS aspirated. A suitable cell dissociation reagent (e.g., ACCUTASE®) is added and the flasks are incubated, e.g., for 4 min at 37°C. The flasks are rinsed with PBS, and the cell suspension is put in a conical tube and spun down, e.g., at 300 x g for 3 minutes. The supernatant is aspirated and resuspended in the iPSC-DP Complete Medium of Table 3 below. Cells are seeded into a new flask with iPSC-DP Complete Medium and placed into a humidified 5% CO2 and 37°C incubator.

Table 3: iPSC-DP Complete Medium R-spondin 1, lOug/mL (working stock) lOOpL 0.01 pg/mL

[0091] In some embodiments, differentiated iPSC-DP cells are fed (e.g., on Day 2 and Day 6) with 100% media change with the iPSC-DP Complete Medium and returned to a humidified 5% CO2 and 37°C incubator.

[0092] In some embodiments, the medium in each flask is aspirated out on Day 4, the flask is rinsed with PBS and the PBS aspirated. A recombinant enzyme (e.g., Trypsin 0.25% and PBS with EDTA) is added to each flask and incubated at 37°C for 5 min, or until all cells are lifted (e.g., incubate another 3 min for 8 min total). Flasks are rinsed with PBS and iPSC-DP are collected into a tube pre-filled with FBS, and spun down at 300 x g for 3 min. The supernatant is aspirated and iPSC-DP are resuspended in the iPSC-DP Complete Medium. [0093] In some embodiments, iPSC-DP cells are collected on Day 8, e.g., by aspirating media from each flask, rinsing with PBS, aspirating the PBS, adding a recombinant enzyme solution (e.g., Trypsin 0.25% and PBS with EDTA) to each flask and incubating at 37°C for 5 min or until all cells are lifted (e.g., incubate another 3 min for 8 min total). Flasks are rinsed again with additional PBS, iPSC-DP are collected into a tube, and spun down at 300 x g for 3 min. The supernatant is aspirated and iPSC-DP are resuspended in an organoid media for organoid formation or iPSC-DP Complete Medium for use for other purposes.

[0094] In some embodiments, cells are counted (e.g., using a Cellometer® Auto 2000 (Nexcelom Bioscience) using the Trypan Blue Assay) to confirm total cell count of approximately 0.5e6 and viability > 80% throughout this process.

[0095] In some embodiments, after about 12 days of culture in presence of WNT-lOb and R- spondin 1, the iPSC-DP cells express known markers such as one or more or each of Versican, Vimentin, Alkaline phosphatase, Lamin A/C and WNT -pathways effector like B- catenin and LEF1 (see FIG. ID) with an immunofluorescence assay. In some embodiments, the iPSC-DP cells show a loss of expression of one or more pluripotency markers (e.g., OCT4 and/or NANOG), e.g., a complete loss of the one or more pluripotency markers.

B. Methods for Producing Hair Follicle Organoids

[0096] In some embodiments described herein, a method of producing an elongated hair follicle organoid comprises: co-culturing induced DP cells and induced epithelial stem cells (EpSCs) in a first cell culture medium that contains basement membrane matrix, wherein the induced DP cells and induced EpSCs form an aggregate; and culturing the aggregate in a second cell culture medium comprising basement membrane matrix; wherein: the EpSCs differentiate into keratinocytes, and the induced DP cells and keratinocytes form an elongated hair follicle organoid. In such embodiments, the same type of medium (e.g., any of the first cell culture media discussed elsewhere herein) can be used as the first and second cell culture media. Regardless, the culture medium is replaced or changed between the steps of co-culturing induced DP cells and induced epithelial stem cells (EpSCs) in a first cell culture medium and culturing the aggregate in a second cell culture medium. For example, most or all of the first cell culture medium can be removed and the second cell culture medium (which may be, but is not necessarily, identical to the first cell culture medium) can then be added to the aggregate. [0097] In some embodiments described herein, a method of producing an elongated hair follicle (HF) organoid comprises: co-culturing induced DP cells and induced epithelial stem cells (EpSCs) in a first cell culture medium, wherein the induced DP cells and induced EpSCs form an aggregate; transferring the aggregate onto a support matrix or into a suspension comprising basement membrane matrix; culturing the aggregate in a second cell culture medium comprising one or more growth factors comprising one or more of R-Spondin 1, Noggin, epidermal growth factor (EGF), one or more Wnt proteins, CHIR, Rock inhibitor, bone morphogenetic proteins (BMP), fibroblast growth factor (FGF) proteins, and Shh (sonic hedgehog) activators, wherein: the EpSCs differentiate into keratinocytes, and the induced DP cells and keratinocytes form an elongated HF organoid.

[0098] In some embodiments described herein, a method of producing an elongated hair follicle (HF) organoid comprises: co-culturing induced DP cells and induced epithelial stem cells (EpSCs) in a suspension containing basement membrane matrix, wherein the induced DP cells and induced EpSCs form an aggregate; culturing the aggregate in a second cell culture medium comprising one or more growth factors comprising one or more of R-Spondin 1, Noggin, epidermal growth factor (EGF), one or more Wnt proteins, CHIR, Rock inhibitor, bone morphogenetic proteins (BMP), fibroblast growth factor (FGF) proteins, and Shh (sonic hedgehog) activators, wherein: the EpSCs differentiate into keratinocytes, and the induced DP cells and keratinocytes form an elongated HF organoid.

[0099] In some embodiments, the one or more growth factors comprise R-Spondin 1, Noggin, and EGF. In some embodiments, the R-Spondin 1 is human R-Spondin 1. In some embodiments, the Noggin is human Noggin. In some embodiments, the EGF is human EGF. In some embodiments, the Wnt protein is WntlOb. In some embodiments, the BMP protein is BMP4. In some embodiments, the FGF protein is FGF10 and/or FGF20. In some embodiments, the Shh activator is SAG (signaling agonist).

[0100] In some embodiments, the induced DP cells are derived from human iPSCs. In some embodiments, the induced DP cells are derived from human induced neural crest cells, or are derived from human iPSCs via an induced neural crest intermediate. In some embodiments, a medium comprising WNT-lOb and R-spondin 1 is used to induce DP cells from human induced neural crest cells or from human iPSCs via an induced neural crest intermediate.

[0101] In some embodiments, the medium comprises about 0.01 pg/ml Wnt-lOb and about 0.01 pg/ml R-Spondin 1. In some embodiments, the medium comprising WNT-lOb and R- spondin 1 further comprises AmnioMax™-II complete medium.

[0102] In some embodiments, the keratinocytes are derived from human iPSCs.

[0103] In some embodiments, the first cell culture medium comprises a medium suitable for culturing amniotic fluid cells. In some embodiments, the medium suitable for culturing amniotic fluid cells may contain serum. In some embodiments, the first cell culture medium comprises a medium suitable for culturing dermal cells. In some embodiments, the medium suitable for culturing dermal cells may contain serum. Serum obtained from any appropriate source may be used, including fetal bovine serum (FBS), bovine calf serum (BCS), goat serum, or human serum. In some embodiments, human serum is used. In some embodiments, the medium suitable for culturing amniotic fluid cells comprises fetal bovine (FBS). Serum may be used at between about 1% and about 30% by volume of the medium, according to conventional techniques.

[0104] In other embodiments, the medium suitable for culturing amniotic fluid cells may contain a serum replacement. Various serum replacement formulations are commercially available and are known to the skilled person. Where a serum replacement is used, it may be used at between about 1% and about 30% by volume of the medium, according to conventional techniques. [0105] In other embodiments, the medium suitable for culturing amniotic fluid cells may be a serum-free medium and/or a serum replacement-free medium. Serum-free media may be preferred to avoid possible xeno-contamination of the stem cells.

[0106] In some embodiments, induced epithelial stem cells (EpSCs) are generated from hiPSCs by induced differentiation using a serum-free media with retinoic acid (RA), BMP4, EGF, and Rho-Kinase inhibitor (Y27632). In some embodiments, the induced EpSCs express EpSC markers, such as one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or each of CD59, CD99, KRT15, KRT19, DKK3, FZD1, FZD2, CTNNB1, TGFB2, FST, DCN, SOX9, NFATC1, ITGA6, ITGB1, LEF1, TCF4, TP63, and LGR6, e g., one, two, three, or each of CD59, CD99, LEF1, and TP63.

[0107] The medium suitable for culturing amniotic fluid cells may also include a feeder support system supplemented with fetal bovine serum (FBS) or serum replacer, or the medium may be a feeder-free system optionally supplemented with defined culture media, such as mTeSR™l and/or TeSR™8.

[0108] In some embodiments, the first cell culture medium comprises a keratinocyte growth medium. In some embodiments, the keratinocyte growth medium comprises epidermal growth factor (EGF), insulin, hydrocortisone, cholera toxin, epinephrine, and transferrin. In some embodiments, wherein the keratinocyte growth medium further comprises a growthpromoting agent, optionally wherein the growth-promoting agent comprises bovine pituitary extract. In some embodiments, the growth-promoting agent is one or more of a lipid supplement (e.g., comprising linoleic acid and vitamin E (see Sawada et al., Investigative Opthalmology & Visual Sci 143: 137 (2002), or a combination of prolactin and prostaglandin El. See Hammond et al., Proc Natl Acad Sci USA 81 :5435-5439 (1984).

[0109] In some embodiments, the first cell culture medium comprises a mixture of a keratinocyte growth medium and a medium suitable for culturing amniotic fluid cells in the presence of support matrix. In some embodiments, the keratinocyte growth medium and a medium suitable for culturing amniotic fluid cells are present in the mixture at a ratio of about 1 : 1 by volume.

[0110] In some embodiments, the support matrix comprises proteinaceous gel and/or basement membrane matrix. In some embodiments, the support matrix or basement membrane matrix comprises protein secreted by cells of mesenchymal origin. In some embodiments, the cells of mesenchymal origin are Engelbreth-Holm-Swarm mouse sarcoma cells. In some embodiments, the support matrix or basement membrane matrix comprises laminin 1, laminin 9, laminin 11, collagen, fibronectin, hyaluronic acid, one or more protecoglycans, and/or one or more inert polymers.

[OHl] In some embodiments, the aggregates are cultured for about 3-90 days, about 3-60, about 4-45, or about 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 days to form the elongated HF organoid.

[0112] In some embodiments, elongated HF organoid is capable of being maintained in culture for at least about 1 to 6 months, 1 to 4 months, or about 1, 2, 3, 4, 5, or 6 months. [0113] In some embodiments, at least about 25%, 50%, 75%, or 100% of the second cell culture medium is exchanged at least about once every 1, 2, 3, 4, 5, 6, or 7 days, optionally wherein at least about 25%, 50%, 75% or 100% of the second cell culture medium is exchanged once every 3-7 or 2-3 days.

[0114] In some embodiments, the basement membrane matrix is present in the suspension at about 2-4% by weight, about 2.5-3.5% by weight, or about 3% by weight.

[0115] In some embodiments described herein, a method of producing a HF organoid comprises: combining dermal cells, such as neonatal dermal cells (e.g., neonatal dermal cells) with induced epithelial stem cells (EpSCs), wherein the dermal cells and induced EpSCs form an aggregate; and implanting the aggregate into dermal tissue of a subject (e.g., by subcutaneous injection), wherein: the induced EpSCs are generated from hiPSCs; the EpSCs differentiate into keratinocytes; and the dermal cells and keratinocytes form HF organoids in the dermal tissue. In some embodiments the dermal cells are DP cells. The keratinocytes may comprise, e.g., inner root sheath keratinocytes and/or outer root sheath keratinocytes.

[0116] In some embodiments, an HF organoid is disclosed, which is produced by any of the above methods.

[0117] In some embodiments, a method of growing a HF comprises implanting the HF organoid into dermal tissue. In some embodiment, the method further comprises piercing the dermal tissue with a needle prior to implanting the organoid.

C. Methods for Treating Hair Loss

[0118] In some embodiments, a method of treating hair loss in a subject comprises implanting the HF organoid into dermal tissue of the subject. [0119] In some embodiments described herein, a method of treating hair loss in a subject comprises: combining dermal cells, such as neonatal dermal cells (e.g., neonatal dermal cells) with induced epithelial stem cells (EpSCs) as described herein, wherein the dermal cells and induced EpSCs form an aggregate; and implanting the aggregate into dermal tissue of a subject (e.g., by subcutaneous injection), wherein: the induced EpSCs are generated from hiPSCs; the EpSCs differentiate into keratinocytes; and the dermal cells and induced keratinocytes form HF organoids in the dermal tissue. [0120] In some embodiments the dermal cells are DP cells. The EpSCs may be produced according to any of the methods described herein.

D. Screening Methods

[0121] In some embodiments, a method of screening a compound for hair growth-modulating activity, comprises: contacting a HF organoid with the compound; and detecting an effect or absence thereof on the HF organoid.

[0122] In some embodiments, a plurality of compounds is screened by contacting a plurality of HF organoids with a member of the plurality of compounds and detecting a plurality of effects or absences thereof on the HF organoids.

[0123] In some embodiments, the effect or absence thereof comprises an increase, decrease, or absence of change in production of hair shaft by the HF organoid. In some embodiments, the effect or absence thereof comprises an increase, decrease, or absence of change in growth of the HF organoid. In some embodiments, the effect or absence thereof comprises a change in or morphology of the HF organoid or absence thereof, such as a change in the roundness and/or elongation of the organoid. In some embodiments, the effect or absence thereof comprises a change in or morphology of the matrix of the hair follicle organoid.

[0124] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the invention. It is therefore contemplated that the disclosure shall also cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0125] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the methods, systems, computer readable media, and/or component features, steps, elements, or other aspects thereof can be used in various combinations.

[0126] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number, if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant, unless otherwise indicated. To the extent any material incorporated by reference is inconsistent with the express content of this disclosure, the express content controls.

EXAMPLES

[0127] The following examples are meant to be illustrative and can be used to further understand embodiments of the present disclosure and should not be construed as limiting the scope of the present teachings in any way. [0128] Materials and Methods

[0129] Generation of NC cells from iPSCs

[0130] To generate NC cells from human iPSC, a neurosphere-based migration protocol was used as previously described using the AggreWell™ system (StemCell Technologies) to create neurospheres. See Cimadamore et al., Stem Cells (2009) 27(8): 1772-81; Curchoe et al., PLoS One (2010) 5(11): el3890. In brief, stem cells were detached as single cells using ACCUTASE™ (a natural enzyme mixture with proteolytic and collagenolytic enzyme activity). According to the manufacturer's instructions, cells were counted to calculate the necessary number of cells dependent on the size of spheres and number of wells according to the vendor table and resuspended in the AggreWell ™ media. The cells were transferred to the AggreWell™ plate, centrifuged for 5 minutes at 300 g, and incubated for 24 hours. Neuralization was initiated by carefully moving the spheres to low attachment polypropylene dishes (CORNING®) in serum-free chemically defined, NPC complete medium composed of NPC base medium (1 : 1 ratio of DMEM/F12 glutaMAX™ (Gibco) and neurobasal medium supplemented with 0.05X B27 without vitamin A (Gibco), 10% BIT 9500 (StemCell Technologies), 5 pg/ml insulin (Sigma-Aldrich), 20 ng/ml bFGF (Chemicon), 20 ng/ml EGF (Sigma-Aldrich), and 5 mM nicotinamide (Sigma-Aldrich). Half of the medium was changed every day for 5 days. Then, the neurospheres were plated on MATRIGEL®-coated culture dishes and allowed to migrate for 4-6 days in the same NPC complete medium. The medium was changed every other day. The stem cell neuralization was carried out under 5% CO2, 3% O2 condition.

[0131] Generation of DP cells in vitro from NC cells

[0132] To generate DP cells from iPSC-NC cells, the protocol described in Gnedeva et al., PLoS One (2015) 10(1): eOl 16892, was optimized to activate known molecular pathways in defined culture conditions. NC cells were detached using ACCUTASE™ and dissociated to single-cell suspensions. Cells were centrifuged at 300 g for 5 minutes, resuspended in AmnioMax™-II Complete medium (Gibco), and seeded on cell-treated plastic without any coating solution to have 30-40% confluency the day after plating them. Cells were grown with AmnioMax™-II Complete medium supplemented with 10 ng/ml of recombinant human Wnt-lOb Protein (R&D systems, cat: 7196-WN-010/CF) and 10 ng/ml of recombinant human R-Spondin 1 Protein (R&D systems, cat: 4645-RS-025/CF). [0133] The medium with the addition of the two growth factors was changed every other day, and cells were passaged at day 4 with a recombinant enzyme solution (e.g., Trypsin 0.25% and PBS with EDTA). The treatment was carried on for 8 days.

[0134] Recombinant human FGF-20 (R&D systems, cat: 2547-FG-025/CF) and recombinant human BMP-6 (R&D systems, cat: 507-BP-020/CF) were tested at the concentration of 10 ng/ml during the developmental phase of the differentiation protocol. After 12 days, cells were analyzed to evaluate the expression of known DP markers using immunofluorescence staining and qPCR as described below.

[0135] Immunocy tochemi stry

[0136] Cells were cultured and imaged in 96 well plates. Briefly, media was aspirated, and wells rinsed once with PBS for 2 minutes. Cells were fixed with 4% formaldehyde for 10 minutes at room temperature, followed by two washes with PBS. Cells were blocked and permeabilized with PBSAT (0.5% Triton X-100 and 2% BSA diluted in PBS) for 1 hour and incubated overnight with the following primary antibodies: goat anti-versican (1 :200; R&D), goat anti-SOX2 (1 :500, R&D), rabbit anti-SOX9 (1 : 100, Millipore), rabbit anti-SOXlO (1 : 1000, R&D), rabbit anti-p75 (1 :200, Abeam), goat anti-Nestin (1 :500, Santa Cruz Biotechnology). The cells were washed three times with PBSAT and then incubated with the corresponding secondary antibody in blocking solution for 1 hour at room temperature. After washing 3 times with PBSAT, nuclei were stained with DAPI (diluted 1 :000 in PBSAT) for 10 minutes and washed twice with PBS and kept in PBS at 4°C until imaged. Images were acquired using Zeiss LSM 800 confocal microscope or EVOS M7000 Imaging System.

[0137] RT-PCR

[0138] Total RNA was extracted using the RNeasy® kit (Qiagen), and 1 pg of total RNA was reverse transcribed using the QuantiTect® Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions to make cDNA. RNA concentrations were measured using a NanoDrop spectrophotometer (NanoDrop Technologies) or Qubit™ (ThermoFisher), and samples were stored at -80°C. qPCR was performed with the SYBR™ Green master mix (Invitrogen) according to the manufacturer's recommendation. For q-PCR, GAPDH (glyceraldehyde-3 -phosphate dehydrogenase) expression level was used for normalization, and the data were analyzed using the AACT method. q-PCR was performed by the LightCycler® 480 II instrument (Roche) using the following conditions: initiation at 95°C for 10 minutes followed by 50 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. GraphPad Prism was used to prepare graphs and perform statistical analysis.

[0139] Bulk RNA-sequencing

[0140] For bulk-sequencing, RNA was extracted from each sample and quantified with Qubit™. cDNA libraries were generated by using NEBNext® Ultra™ II Directional RNA Library Prep Kit (Illumina®) according to the manufacturer’s protocol. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The libraries were ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The libraries were checked with Qubit™ and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms, according to each effective library concentration and data amount. For the analysis, reads were first analyzed by fastQC vO.11.9 (Andrews, S., FastQC: A Quality Control Tool for High Throughput Sequence Data. Available online at: www.bioinformatics.babraham.ac.uk/projects/fastqc/) and then trimmed for low-quality bases with Trimmomatic v0.39 (Bolger et al., Bioinformatics (2014) 30( 15):2114-2120). Clean reads were then aligned to the human reference genome (GRCh38) with Ensembl gene annotation using STAR v2.7.3a (Dobin et al. Bioinformatics (2013) 29(1): 15-21). Transcripts per million (TPM) estimates were calculated using RSEM vl.3.3 (Li et al., BMC Bioinformatics (2011) 12:323) and were used for visualization and dimensionality reduction. All differential expression analyses were performed on raw abundance estimates via edgeR v3.34.1 (Robinson et al., Bioinformatics (2010) 26(1): 139-140). Differentially expressed genes were defined as having logFC > 1 and false discovery rate (FDR) < 0.05. KEGG pathway analysis was performed using enrichR v3.0 (Kuleshov et al., Nucleic Acids Res (2016) 44(Wl):W90-7) and the significance was determined by FDR < 0.05.

Single Cell Library Preparation and Sequencing

[0141] Libraries for single cell analysis were prepared using 10X Genomics droplet-based single-cell RNA-seq, the Chromium Single Cell 3’ Library, and Gel Bead Kit v2 (Cat. #120267) following the manufacturer’s protocol. Briefly, we targeted 1000 single cells per time point. To compensate for cell loss and count errors, 2000 iPSC-DP cells were collected every 2 days (starting from day 0 when they are still NC cells, up to day 8) and methanol fixed according to lOx protocol recommendation. Cells were kept at -80°C until all sample collection was completed. When ready, cells were re-hydrated and loaded onto the Chromium. Libraries were quantified using by a Bioanalyzer. DNA libraries were sequenced using a NextSeq 500/550 Mid Output Kit v2.5 (150 Cycles), suitable for 130 million reads (-25000 reads/cell). For the analysis, 10X Genomics Cell Ranger v6.1.1 (Zheng et al., Nat Commun (2017) 8: 14049) was used to align raw sequencing reads to the human reference genome (GRCh38) and quantify the gene expression in each cell. Single cell analysis was carried out using Seurat v4.0.4 (Hao et al., Cell (2021) 184(13):3573-3587.e29). Low quality cells were filtered out by the following criteria: less than 1000 or more than 5000 unique genes expressed or more than 5% of reads mapping to mitochondria genome. A total of 7352 cells was used for downstream analysis. Top 2000 highly variable genes were determined by the variance stabilizing transformation (vst) method. Clustering was performed based on 20 principal components (PCs). Differentially expressed genes between cell types were determined using a Wilcoxon test with logFC > 1 and FDR < 0.05. To explore dynamic patterns of transcriptional changes during cell differentiation across different days, RNA velocity was examined using Velocyto vO.17.17 (generating spliced vs unspliced reads) (La Manno et al., Nature (2018) 560(7719): 494-498) and scVelo vO.2.4 (computing velocity) (Bergen et al., Nature Biotechnology (2020) 38: 1408-1414).

Example 1: Activation and potentiation of WNT/p-catenin signaling pathway support the differentiation of DP cells from NC cells

[0142] Although DP cells have been previously generated from human iPSC, see Gnedeva, et al., PLoS One (2015) 10(1): eOl 16892, a modified approach was developed in an effort to improve performance. The importance of engaging NC cells is supported by rich literature that emphasizes the key contribution of these cells to cranial and facial fibroblast and DP cell development. See Driskell et al., Journal of Cell Science (2011) 124(8): 1179-1182; Fernandes et al., Nat Cell Biol, (2004) 6(11): 1082-93; Nagoshi et al., Cell Stem Cell (2008) 2(4):392- 403; and Wong et al., J Cell Biol (2006) 175(6): 1005-15. Herein, known growth factors were tested with NC cells being differentiated into DP cells (FIG. 1 A). Among the numerous candidates available, WNTlOb, BMP6, FGF20 and R-spondin 1 were selected. For background regarding these candidates, see Mok et al., BioRxiv, (2018) at 414839; Chen et al., Development (2012) 139(8): 1522-33; Millar, S.E., J Invest Dermatol (2002) 118(2):216- 25; Sennett et al., Semin Cell Dev Biol (2012) 23(8): 917-27; Zhang et al., Development (2008) 135(12): 2161-72; Rabbani et al., Cell (2011) 145(6): 941-955; See Schuijers et al., Cell Stem Cell (2015) 16(2): 158-70; Huh et al., Genes Dev (2013) 27(4): 450-8; Rendl et al., Genes Dev (2008) 22(4): 543 -57. Several growth factor combinations, timing, and concentrations were tested to develop this protocol (range concentration from 10 ng/ml to 100 ng/ml in AmnioMax™ medium), and the resulting cell populations were characterized to evaluate the maximal differential capabilities of NC cells to DP cells (FIGs. 2A-2F). Our initial growth factor screen (see the culture conditions discussed in detail below) relied on the common DP cell markers Versican and Alkaline phosphatase to determine the degree of DP cell differentiation and enrichment after 12 days of culture (FIGs. 2D and 2E). See Rendl et al., Genes Dev (2008) 22(4): 543-57; Greco et al., Cell Stem Cell (2009) 4(2): 155-69; Kishimoto et al., Proc Natl Acad Sci USA (1999) 96(13): 7336-41. Growth factors tested are shown in FIG. 17. For the first 5 days of the DP differentiation, the cells were treated with the indicated growth factors in the first line, which were then withdrawn and substituted with BMP6 and FGF20 for the remaining 7 days. Altogether, the results demonstrated that the activation of the WNT/p-catenin pathway by WNT-lOb and its potentiation mediated by R- spondin 1 was the best performing combination in generating DP-like cells from iPSC-NC cells and its effect was robust and reproducible across three different iPSC lines, labeled as iPSC-61, iPSC-101 and iPSC-126 (FIGs. 2D-2F).

[0143] Therefore, the combination of WNT-lOb and R-spondin 1 was selected for an additional protocol to carry out the differentiation of iPSC-derived NC cells into DP, whose nature and profile were studied on multiple levels as herein described.

[0144] For example, in one embodiment, a flask was coated with a MATRIGEL® surface coating solution comprising MATRIGEL® and DMEM/F12+GlutaMAX™, and then neural progenitor cell (NPC) complete media of Table 4 was added to the flask, wherein the NPC Basal Medium was as described in Table 5 below.

Table 4: NPC Complete Medium

Table 5: NPC Basal Medium

[0145] iPSC-NC cells were seeded into the MATRIGEL® coated flasks. After 24 hours, the media was changed by aspiration of the media and adding pre-warmed NPC Complete Medium. The iPSC-NC was allowed to recover for a minimum of 48 hours before beginning differentiation.

[0146] Cells were seeded for iPSC-DP differentiation at Day 0 (DO). The NPC Complete Medium was aspirated from the flasks, the flasks were rinsed with PBS and the PBS aspirated. ACCUTASE® was added and the flasks were incubated for 4 min at 37°C. The flasks were rinsed with PBS, and the cell suspension was put in a conical tube and spun down at 300 x g for 3 minutes. The supernatant was aspirated and resuspended in the iPSC-DP Complete Medium of Table 6 below. Cells were seeded into a new flask with iPSC-DP Complete Medium and placed into a humidified 5% CO2 and 37°C incubator.

Table 6: iPSC-DP Complete Medium

[0147] Differentiated iPSC-DP cells were fed on Day 2 and Day 6 with 100% media change with the iPSC-DP Complete Medium and returned to a humidified 5% CO2 and 37°C incubator.

[0148] On Day 4 the medium in each flask was aspirated out, the flask was rinsed with PBS and the PBS aspirated. A recombinant enzyme solution (e.g, Trypsin 0.25% and PBS with EDTA or TrypLE) was added to each flask and incubated at 37°C for 5 min, or until all cells were lifted (e.g., incubate another 3 min for 8 min total). Flasks were rinsed with PBS and iPSC-DP were collected into a tube, and spun down at 300 x g for 3 min. The supernatant was aspirated and iPSC-DP were resuspended in the iPSC-DP Complete Medium.

[0149] On Day 8, iPSC-DP cells were collected by aspirating media from each flask, rinsing with PBS, aspirating the PBS, adding a recombinant enzyme solution (e.g, Trypsin 0.25% and PBS with EDTA or TrypLE) to each flask and incubating at 37°C for 5 min or until all cells were lifted (e.g., incubate another 3 min for 8 min total). Flasks were rinsed again with additional PBS, iPSC-DP were collected into a tube, and spun down at 300 x g for 3 min. The supernatant was aspirated and iPSC-DP were resuspended in an organoid media for organoid formation or iPSC-DP Complete Medium for use for other purposes.

[0150] Cells were counted (e.g., using a Cellometer® Auto 2000 (Nexcelom Bioscience) using the Trypan Blue Assay) to confirm total cell count of approximately 0.5e6 and viability > 80% throughout this process.

[0151] Immunofluorescence assay showed that after 12 days of culture in presence of WNT - 10b and R-spondin 1, the iPSC-DP cells expressed known markers such as Versican, Vimentin, Alkaline phosphatase, Lamin A/C and WNT-pathways effector like B-catenin and LEF1 (FIG. ID). Along with protein expression, the iPSC-DP cells' genetic profile was characterized to evaluate each pathway's contribution to the differentiation process, as shown in FIG. IB, confirming the identity of these newly differentiated cells and the complete loss of pluripotency markers (OCT4, NANOG).

[0152] Considering the role of the Wnt pathway in the differentiation process of iPSC-NC into DP cells, one would expect other known WNT activators, such as CHIR, BIO or BML- 284 hydrochloride, to lead to a similar outcome.

Example 2: Bulk RNA-seq shows the acquisition of mesenchymal phenotype of the iPSC- DP cells during their differentiation process

[0153] To define the transcription profile of viable and functional iPSC-DP cells, closely mimicking the native cells, bulk RNA sequencing was performed at multiple time points during the cell differentiation process from NC cells (namely at days 4, 8 and 12) and compared with human native DP cells (hDP) and human foreskin fibroblasts (HFF), cultured in vitro for multiple passages. Thirteen patient-derived DP cells were used as the positive reference population, while four primary fibroblast lineages were considered the negative reference, as they are the most similar cell type to DP cells and yet not as specialized as the HF cells counterpart. Additionally, nine day 8 DP samples and 4 hDP samples were cultured in 3D (as spheroids), as dermal papilla identity is known to be impacted by culture conditions. See Andi et al., Cell and Tissue Research (2023) 391 :221-233. The transcriptomes of each sample were compared by principal component analysis (PCA) (FIG. 3A). The plot showed a great degree of reproducibility within all the replicates in each population, as well as some segregation of each cluster in discrete areas of the plot. Interestingly, the nascent iPSC-DP cells moved along an iPSC-to-DP trajectory with clear separation from the original iPSC and the intermediate NC cells populations. hDP and HFF were positioned in proximity in the geometrical space due to the nature of the DP compartment, which can be defined as a cluster of active specialized fibroblasts derived from dermal mesenchyme. Driskell et al., Journal of Cell Science (2011) 124(8): 1179-1182. Spherizing DP of hDP cells does not drastically alter cell identity. Nonetheless, the PCI and PC2 values for the iDP cells were consistently distinct from those of native hDP and HFF, showing that the gene expression pattern and therefore the epigenetic state of the iPSC-DP cells is not identical to HFF or hDP. Because of the unique capability of DP cells to govern HF morphogenesis and life cycle, the dissimilarities between native DP cells and the HFF were explored to define a list of key markers for each one of these mesenchymal populations that could be useful to determine the identity of the iPSC-derived DP cells. Therefore, the differentially expressed genes (DEGs) between HFF and hDP, as well as iPSC-DP, were analyzed. As shown in the Venn diagram analysis (FIG. 3B), a total of 1375 genes were differentially expressed between hDP and HFF, while within the iPSC-DP population, cells at day 4 had the highest number of unique genes when compared to HFF (1816), suggesting that the initial cell state had less similarity with the mesenchymal phenotype and is gradually acquired throughout the differentiation protocol. This trend was confirmed by the enrichment analysis using the KEGG database (FIGs. 3C-3F) that identified several biological processes and pathways differentially regulated in DP cells, both native and iPSC-derived, when compared to HFF. The differences included, but were not limited to p53, HIPPO, PI3K, RAP and RAS signaling pathways and were less pronounced at day 12, suggesting that a longer differentiation in vitro might generate fibroblast-like cells rather than DP cells. For each of these signaling pathways, the expression was higher in the hDP or iDP (at any day) when compared to HFF. Some hypotheses as to why these pathways are differentially expressed include: 1) Rapl signaling has been implicated in cell-to-cell interaction and in ECM binding via integrin- and cadherin- mediated adhesion events. In the dataset, the Rapl pathway was highly expressed at the early stages of the iDP differentiation process (day 4) when the transition process from NC cells to mesenchymal cells occurs and the cells are transferred from a MATRIGEL®-coated petri dish to an uncoated surface, stimulating the expression of integrin proteins. See, e.g., Boettner et al., Current Opinion in Cell Biology (2009) 21(5):684-693; 2) RAS pathway is involved in cell cycle regulation, wound healing, tissue repair, integrin signaling and cell migration. In addition, there are supportive data reporting that EGFR-Ras-Raf signal pathway plays crucial roles in hair follicle development and regeneration. See, e.g., Dorna et al., Int. J. Mol. Sci. (2013) 14(10): 19361-19384; and 3) PI3K-AKT is a pathway known to contribute to the secretion of the Wnt family proteins and has been previously shown to be enriched in dermal papilla cells. See e.g., Jin et al., Stem Cell Rep (2021) 16: 1568-1583; Yang et al., Genomics (2022) 114: 110316. In the present context, many of the genes identified under this pathway are related to focal adhesion formation and cell-matrix interaction via collagen, PDGFR protein, laminin and integrins. This is consistent with the changing nature of the nascent iDP at early stages.

[0154] In addition to the unique gene set that characterized each population, 465 genes were found to be shared between all the iPSC-DP samples and the hDP cells (FIG. 3B). Among this list of genes, genes were filtered for those that have been previously described as part of the DP core signature, both in human adult and neonatal mouse DP cells, either via bulk sequencing or microarray analysis in existing publications in the field. See Higgins et al., Proc Natl Acad Sci USA (2013) 110(49): 19679-88; Ohyama et al., J Cell Sci (2012) 125(Pt 17): 4114-25, and Sennett, et al., Semin Cell Dev Biol (2012) 23(8): 917-27. As shown in the heatmap (FIG. 3G), many of these genes displayed similar level of expression in iPSC-DP and hDP such as SDC1, RGS5, FZD2, HEY1, BMP2, BMP4, SOX9, EDNRA, and WNT5B that are highly expressed in the iPSC-DP cells, reinforcing the discovery that iPSC-derived cells acquire a DP-like profile following our protocol described above.

[0155] Moreover, a weighted gene co-expression network analysis (WGCNA) was performed to identify eigengenes, key modules, and possible targets to modulate in iPSC- derived cells to augment their folliculogenic profile. The analysis was carried out on the same data set as the bulk sequencing samples. A total of 13 module eigengenes (MEs) were identified. Fifty top hub genes were extracted for each ME and listed in FIG. 8. Each module represents a network of genes that can be up or down-regulated in each of the samples analyzed, namely iPSC, NC, IDP-day4-8-12, DP, and HFF. To understand if the genes in each module are up or downregulated in the iDP, it is helpful to look at the pattern in FIG. 4 A. The genes that are likely to be upregulated in the iDP relative to hDP belong to MEI 1, ME5 and ME7. Conversely, the genes that are likely to be downregulated in iDP relative to hDP belong to ME10, ME13, and ME3. For example, Table 7 provides a list of genes that are upregulated or downregulated in iDP relative to hDP and shows the log of fold change in natural-log transformed transcripts per million between hDP and iDP (“logFC”)and a p value (as false discovery rate, “FDR”) of this difference for the 50 top hub genes. Only the genes with a significant fold change are listed. The cutoff for the gene selection was a False discovery rate (FDR) <0.05. TABLE 7

Gene logFC FDR module Gene logFC FDR module DNMT3B 2.3790126 0.0000000 MEI KIAA0895 1.2972724 0.0010091 ME8 PHC1 0.6175530 0.0002770 MEI NRIP1 0.3901036 0.0373529 ME8 CTSV 2.1037413 0.0000137 MEI DZIP1 0.5138024 0.0001133 ME8 LAPTM4B 0.5202978 0.0004899 MEI BCORL1 0.5042448 0.0406270 ME8 ZNF121 0.7766349 0.0000291 MEI SOX6 2.8994079 0.0000000 ME8 ZNF589 0.6479651 0.0042718 MEI APLP1 1.2583615 0.0012307 ME8 MY05C 1.7131105 0.0018208 MEI GPRASP1 -0.5520251 0.0472948 ME8 KIF1A 6.7612305 0.0000000 MEI MECOM 2.5491022 0.0000000 ME8 SLC7A3 5.1263546 0.0000326 MEI SENP7 -0.6149767 0.0187927 ME8 RNF125 1.7060178 0.0002219 MEI KLHL8 0.5210955 0.0052357 ME8 ZNF649 0.6109168 0.0027399 MEI DDAH2 0.5190650 0.0221113 ME8 PARG 0.2561957 0.0366063 MEI AURKB 0.8855726 0.0346117 ME9 JPH3 5.5859094 0.0000006 MEI LMNB1 0.9397724 0.0263765 ME9 TEX 15 5.5674469 0.0000000 MEI ORC6 1.0147336 0.0131308 ME9 PLA2G3 4.6720091 0.0000177 MEI HAGLR -11.984499 0.0000000 ME10 H00K1 6.5487807 0.0000000 MEI COL10A1 -8.7892273 0.0000000 ME10

GLDC 2.0600262 0.0217737 MEI RARB -5.7716851 0.0000000 ME10 ZNF721 0.4279478 0.0238028 MEI MILR1 -9.5330101 0.0000000 ME10 MPP6 0.9360237 0.0000001 MEI ALDH3A1 -6.7177566 0.0000000 ME10 BEND3 0.7631810 0.0032666 MEI ROBO2 -9.9951879 0.0000000 ME10 MAP3K21 1.6461107 0.0076734 MEI AGTR1 -6.4882192 0.0000000 ME10 PGBD5 2.5180691 0.0008179 MEI IL12RB1 -8.8522027 0.0000000 ME10 ANXA2 -0.3447018 0.0333807 ME2 CAMK2G -1.8304644 0.0000000 ME10 MYOF -1.2100525 0.0000000 ME2 LGR5 -6.0308462 0.0000000 ME10 FAM114A1 -0.5567565 0.0070591 ME2 HECW2 -3.5729250 0.0000000 ME10 ITGB1 -0.5297783 0.0005313 ME2 CA5B -2.6697976 0.0000000 ME10 SMPD1 -0.9331866 0.0000011 ME2 GPR65 -4.0557289 0.0000000 ME10 ANXA6 -0.6419818 0.0003161 ME2 RAET1E -5.6966589 0.0000003 ME10 TMEM263 -0.5482018 0.0007687 ME2 CMKLR1 -3.4746178 0.0000371 ME10 COL5A2 -0.6454486 0.0194121 ME2 LINC01117 -6.3546468 0.0000000 ME10 EFEMP2 -1.1068067 0.0000000 ME2 IL17RE -2.6463322 0.0000000 ME10 PRSS23 -0.6480790 0.0261835 ME2 SLC7A4 -7.3973101 0.0000000 ME10 PALLD -0.5167559 0.0201127 ME2 HOXB4 -7.1875503 0.0000160 ME10 GSN -0.6193805 0.0027638 ME2 EVI2A -4.6004865 0.0000000 ME10

GSTK1 -0.5476042 0.0001379 ME2 EVI2B -5.4024424 0.0000009 ME10 PMM1 -0.4430124 0.0056788 ME2 HOXD1 -8.4648511 0.0000000 ME10 TRADD -0.4413892 0.0375911 ME2 HHIPL2 -6.9458159 0.0000000 ME10 AHNAK -1.1602881 0.0000000 ME2 FAM225B -6.2291495 0.0000324 ME10 ALPK1 -4.2351509 0.0000000 ME3 LTC4S -4.8905884 0.0000000 ME10 MR1 -5.3195759 0.0000000 ME3 HOXB7 -8.8196743 0.0000000 ME10 LAMA2 -4.1255162 0.0000000 ME3 ZIC4 -9.9159978 0.0000000 ME10 TMEM119 -6.4801441 0.0000000 ME3 GPR85 -4.2394502 0.0000000 ME10 CYBRD1 -2.9633626 0.0000000 ME3 HOXB3 -7.2269301 0.0000000 ME10 STXBP5 -1.8371725 0.0000000 ME3 RETREG1 -4.5669813 0.0000000 ME10 TPP1 -1.3286656 0.0000000 ME3 WNT16 -3.9309523 0.0001222 ME10 GPNMB -6.3587899 0.0000000 ME3 MGARP -4.5001413 0.0000000 ME10 COL6A3 -6.6682208 0.0000000 ME3 PCNX1 -1.2653990 0.0000000 ME10 PLSCR4 -4.6704916 0.0000000 ME3 0AS2 -5.4190409 0.0000067 ME10

AP0L6 -3.1055170 0.0000000 ME3 TMEM155 -6.2316984 0.0000000 ME10

ABHD14B -1.9069916 0.0000000 ME3 STPG1 -0.9983934 0.0000001 ME10

KCNE4 -7.1274922 0.0000000 ME3 NDP -5.9317717 0.0000003 ME10

TNFRSF14 -3.5149826 0.0000000 ME3 RANBP3L -9.0716879 0.0000000 ME10

CALHM2 -3.7099265 0.0000000 ME3 MTUS2 -7.1124703 0.0000001 ME10

BMPER -6.4801824 0.0000000 ME3 TFAP2C -3.2557464 0.0000000 ME10

SH0X2 -9.7318725 0.0000000 ME3 NRN1 -3.9265678 0.0000766 ME10

FBLN5 -5.1053198 0.0000000 ME3 ALDH3A2 -2.3193660 0.0000000 ME10

CD68 -4.7205457 0.0000000 ME3 ACVR1C -5.5772750 0.0000000 ME10

FLT3LG -3.6242036 0.0000000 ME3 MBOAT1 -2.6826172 0.0000000 ME10

CACNA1C -5.4769764 0.0000000 ME3 LYPD6B -4.8121438 0.0000066 ME10

VSTM4 -4.1398312 0.0000000 ME3 ALX4 -12.447348 0.0000000 ME10

ALDH3B1 -2.9581031 0.0000000 ME3 SLC5A3 -3.1565261 0.0000000 ME10

METRNL -3.1823262 0.0000000 ME3 IFI44 -4.3575220 0.0000000 ME10

CALC0C02 -1.2624530 0.0000000 ME3 ABCC12 -4.6003189 0.0001965 ME10

ADAM33 -5.7926616 0.0000000 ME3 MRPL12 0.6105783 0.0197178 ME11

MMP19 -2.3887983 0.0000000 ME3 MRPL20 0.4040631 0.0461220 ME11

SECTM1 -5.0962270 0.0000000 ME3 NME1 0.8391457 0.0149980 ME11

NBR1 -1.1778199 0.0000000 ME3 PSMC4 0.6056408 0.0008587 ME11

VASN -2.5172011 0.0000000 ME3 PSMB6 0.4351081 0.0382947 ME11

PLXDC1 -4.3706015 0.0000000 ME3 NHP2 0.6105542 0.0114906 ME11

FAP -5.4298114 0.0000000 ME3 CCDC137 0.7833759 0.0012935 ME11

CASP1 -5.8742249 0.0000000 ME3 PSMA7 0.5672493 0.0076734 ME11

UBA7 -4.3087761 0.0000000 ME3 MRPL15 0.9393268 0.0000652 ME11

CSF1 -3.1292039 0.0000000 ME3 EBNA1BP2 0.6845928 0.0046207 ME11

NEK7 -1.9658340 0.0000000 ME3 ATAD3A 0.8787347 0.0000328 ME11

ATP8B2 -1.2104167 0.0000000 ME3 MRPS12 0.6864225 0.0080727 ME11

NGF -2.9774805 0.0000006 ME3 TXNDC17 0.7364932 0.0024487 ME11

DIPK1A -3.3726894 0.0000000 ME3 N0L7 0.4145842 0.0482447 ME11

AP0L3 -3.7610397 0.0000000 ME3 ADRM1 0.7390053 0.0004833 ME11

CTSO -2.3518696 0.0000000 ME3 IFRD2 0.7308684 0.0002881 ME11

CD81 -1.2832469 0.0000000 ME3 WDR46 0.4268160 0.0371462 ME11

NAALADL2 -3.8575169 0.0000000 ME3 UCHL3 0.6978031 0.0013481 ME11

FTH1 -1.9539461 0.0000000 ME3 FARSA 1.0124801 0.0000056 ME11

LINC02202 -8.5674711 0.0000000 ME3 CCDC86 1.2973809 0.0000000 ME11

PPP1R3C -3.6512493 0.0000000 ME3 TIMM13 1.1575179 0.0000027 ME11

MEG3 -16.4035659 0.0000000 ME3 CYC1 0.6037913 0.0051555 ME11

HSPB7 -7.4424456 0.0000000 ME3 EMC8 0.5255315 0.0016923 ME11

LY96 -4.1448873 0.0000000 ME3 TSTA3 0.8281653 0.0001651 ME11

EBF1 -3.2317945 0.0000000 ME3 PDF 1.0934423 0.0000073 ME11

CRB2 4.4208388 0.0016256 ME4 LSM10 0.5905967 0.0113850 ME11

FIGNL2 2.2491528 0.0428986 ME4 BUD23 0.6142878 0.0000167 ME11

TMEM170B 0.8867954 0.0025530 ME4 NOL6 0.5357571 0.0032871 ME11

PRTG 4.4753483 0.0000000 ME4 TOMM5 0.7252121 0.0006351 ME11

SLC16A14 2.0144037 0.0066847 ME4 MRPL34 0.5659158 0.0182234 ME11

C0L2A1 5.9240801 0.0000001 ME4 RABGGTB 0.8461693 0.0000000 ME11

SLITRK5 3.6914814 0.0085212 ME4 PTGES2 0.6771377 0.0011155 ME11

CCDC160 4.5760554 0.0000000 ME4 PSMB5 0.5837831 0.0042928 ME11 DIPK1B 6.2076728 0.0000000 ME5 1.0252148 0.0000000 ME11 MEX3A 3.7912147 0.0000000 ME5 0.8127778 0.0000479 ME11 PIAS4 0.9854060 0.0000000 ME5 0.5840057 0.0169798 ME11 ZBED9 6.1810705 0.0000000 ME5 0.5153950 0.0045474 ME11 HS6ST2 10.1852085 0.0000000 ME5 0.6703209 0.0140528 ME11 PTK2 1.3151391 0.0000000 ME5 0.9669301 0.0000018 ME11 PCDHA12 6.0156597 0.0000000 ME5 0.4597748 0.0072768 ME11 CAPN10 0.8490436 0.0000000 ME5 -2.0693949 0.0000000 ME13 ZBTB46 3.8917678 0.0000000 ME5 -2.0325824 0.0000000 ME13 CAMSAP 1 1.2744324 0.0000000 ME5 -2.1401156 0.0000000 ME13 FBXL19 1.2978621 0.0000000 ME5 -3.7325277 0.0000003 ME13 IGF2BP1 6.2071910 0.0000000 ME5 -1.4075934 0.0000000 ME13 CTXN1 2.9734157 0.0000000 ME5 -4.1085584 0.0000000 ME13 PLAGL2 1.6832696 0.0000000 ME5 -4.8091205 0.0000000 ME13 FKBP5 2.1853618 0.0000000 ME5 -1.4286992 0.0000000 ME13 PCDHB2 3.3869914 0.0000000 ME5 -2.0806571 0.0000000 ME13 ZBED4 1.1268935 0.0000000 ME5 -2.2699625 0.0000000 ME13 ATRNL1 4.8027929 0.0000000 ME5 -0.9421046 0.0000135 ME13 SEC14L6 6.5506233 0.0000000 ME5 -2.2975572 0.0000000 ME13 KRT8 8.2036849 0.0000000 ME5 -3.1544146 0.0000000 ME13 PCDHA4 4.0661651 0.0000000 ME5 -6.5863005 0.0000000 ME13 LOC339260 2.8909689 0.0000000 ME5 -1.6981832 0.0000000 ME13 GPRC5C 4.6397855 0.0000076 ME5 -1.4354364 0.0000008 ME13 ALPK3 7.0365014 0.0000000 ME5 -4.7231081 0.0000000 ME13 CCND2 8.8002221 0.0000000 ME5 -1.5359686 0.0000000 ME13 PLCG1 0.9947133 0.0000000 ME5 -0.6682173 0.0000005 ME13 KIF21B 5.4203620 0.0000000 ME5 -0.9588485 0.0000001 ME13 BCL9 I.4899949 0.0000000 ME5 -0.9290212 0.0000001 ME13 DSC2 I I.5830136 0.0000000 ME5 -4.5491507 0.0000000 ME13 CCNJL 2.8740339 0.0000000 ME5 -2.4206127 0.0000000 ME13 PEG 10 2.5681467 0.0000000 ME5 -1.7837461 0.0000070 ME13 C0R07 1.1510497 0.0000000 ME5 -1.6701708 0.0000007 ME13 LRRC20 2.6515120 0.0000000 ME5 -1.1966232 0.0000000 ME13 CADM4 4.7726502 0.0000000 ME5 -1.1729754 0.0000229 ME13 BRAT1 0.6066975 0.0000082 ME5 -2.8037589 0.0000502 ME13 CBARP 1.6169745 0.0000000 ME5 -1.0389763 0.0000000 ME13 MERTK 9.0058245 0.0000000 ME5 -1.3044984 0.0000803 ME13 0TUD3 1.2854724 0.0000000 ME5 -0.6155513 0.0008313 ME13

UBQLN4 0.7992297 0.0000000 ME5 -0.7827770 0.0002402 ME13 SHROOM2 2.8514140 0.0000000 ME5 -0.9469069 0.0000003 ME13 PLEKHG3 1.9058750 0.0000000 ME5 -3.3694714 0.0000000 ME13 INAVA 7.7603663 0.0000000 ME5 -0.6909703 0.0000463 ME13 MDFI 6.0212485 0.0000000 ME5 -0.5564119 0.0000505 ME13 PUDP 1.2497569 0.0000000 ME5 -0.7302590 0.0000066 ME13 SPINDOC 1.3082677 0.0000000 ME5 -1.1281088 0.0000000 ME13 SYTL1 4.1524660 0.0000000 ME5 -2.3254071 0.0000028 ME13

GAL3ST1 7.7703002 0.0000000 ME5 -0.9050113 0.0036475 ME13 ACHE 4.1588186 0.0000000 ME5 -2.6595403 0.0000018 ME13 PLCB2 3.9592521 0.0000000 ME5 ZNF454 -3.8847375 0.0000000 ME13

COMP -3.9420118 0.0104646 ME6 LHPP -1.3432093 0.0000006 ME13

HOXAIO -3.0202807 0.0472640 ME6 TRPM7 -0.5856249 0.0002706 ME13

OXTR -2.7318474 0.0000000 ME6 CDHR3 -2.2152519 0.0002971 ME13

HOXC6 -3.9192012 0.0083830 ME6 ZNF578 -5.1392134 0.0000000 ME13

AOC3 2.3629517 0.0001401 ME6 MINDY2 -0.8288741 0.0002684 ME13

CRLF1 2.1605267 0.0000380 ME6 LMO4 -1.5428564 0.0000000 ME13

PGDN -5.3809638 0.0013290 ME6 DENND1A -0.8900475 0.0000003 ME13

UBL4B -4.8325020 0.0045816 ME6 ADH5 -0.5775103 0.0007866 ME13

FOXL1 2.8671156 0.0000000 ME7

TNFRSF10D 2.5368675 0.0000000 ME7

KCNJ15 4.1105184 0.0000000 ME7

FOXC2 4.1789858 0.0000000 ME7

IL11 2.7742469 0.0000000 ME7

EDN1 2.7767355 0.0000000 ME7

CPA4 6.8407977 0.0000000 ME7

DDA1 0.7678576 0.0000385 ME7

RPS6KB2 1.0418741 0.0000000 ME7

ATP6V1F 0.7380079 0.0012980 ME7

LOC91370 5.1578970 0.0000000 ME7

SMURF2 1.3326624 0.0000000 ME7

MAPK12 0.9423114 0.0000074 ME7

LINC01638 5.2199622 0.0000000 ME7

LIF 4.4471168 0.0000000 ME7

LINC02802 3.9445372 0.0000000 ME7

GTPBP6 0.6132249 0.0000805 ME7

TM4SF1 4.8843611 0.0000000 ME7

THSD1 2.7305926 0.0000000 ME7

SVIL 2.1487554 0.0000000 ME7

ZGPAT 0.6727864 0.0016680 ME7

CYGB 2.5825431 0.0000000 ME7

TTYH3 1.0666762 0.0000000 ME7

SH3TC1 8.1408914 0.0000000 ME7

AGAP3 0.5584489 0.0001060 ME7

BCL2L1 1.3265258 0.0000000 ME7

IL32 6.2167518 0.0000000 ME7

STK25 0.6940011 0.0000000 ME7

GMPPB 0.9100871 0.0000001 ME7

ZNF276 0.8629701 0.0000003 ME7

SIX1 3.9671020 0.0000000 ME7

ROMO1 0.8879908 0.0025380 ME7

AMIGO2 3.5572884 0.0000000 ME7

ABCA12 3.4539597 0.0000001 ME7

PLA1A 9.4290105 0.0000000 ME7

HRAS 0.7083809 0.0028514 ME7

DCBLD2 2.2293755 0.0000000 ME7

SPRED3 1.8536125 0.0000000 ME7

BCAR1 0.9383424 0.0000003 ME7

ARL6IP4 0.5656486 0.0057540 ME7 CLPP 0.6319964 0.0000967 ME7 GIPC1 0.6387105 0.0005712 ME7 UBE2J2 0.8592577 0.0000018 ME7

PRKAG2 1.0432618 0.0000308 ME7 GPSM3 1.4529731 0.0000000 ME7 GDF15 3.9425230 0.0000000 ME7 PYGB 1.6862157 0.0000000 ME7 PLCH2 4.3852536 0.0000000 ME7 MCAM 1.7378911 0.0000508 ME7

MRPL41 0.7677772 0.0032565 ME7

[0156] Genes were extrapolated from these modules that were uniquely expressed in each of the mesenchymal populations analyzed (iPSC-DP, hDP and HFF) that could serve as signature markers for further identity and functionality investigations (FIGs. 4C-4E). For example, FIG. 4D shows genes expressed at an elevated level in iDP relative to hDP (e.g., EDN1, FOXC2, ITGA11, LIF, SIX2, and IGFBP1).

[0157] Surprisingly, many genes previously described in the literature to be involved in HF morphogenesis or hair growth cycle and specifically related to the DP compartment were found to be expressed in human foreskin fibroblasts only (HFF). Some examples are the HOX family, which has been proposed as a master regulator of appendages development and skin regional specificity in different organisms (see Chang et al., Proc Natl Acad Sci USA (2002) 99(20): 12877-82; Duverger et al., Birth defects research. Part C, Embryo today: reviews (2009) 87(3): 263-272); FGF9, an effector of WNT activation able to induce HF neogenesis after wounding in mice (see Gay et al., Nat Med (2013) 19(7):916-23; Kinoshita- Ise et al., Inflamm Regen (2020) 40:35); IGF1, reported to be specifically expressed in DP and previously investigated for its role in controlling the hair growth cycle as well as hair shaft differentiation. See Weger et al., J Invest Dermatol (2005) 125(5): 873-82; Tavakkol et al., J Invest Dermatol (1992) 99(3): 343-9. On the other hand, EDN1 (endothelin 1), FOXC2 (Forkhead box protein C2) and LIF (leukemia inhibitory factor) clearly showed unique expression in the iPSC-DP cells, gaining a potential role as signature genes for viable and functional iPSC-DP cells (FIG. 4D).

[0158] EDN1 is one of the three ligands of the endothelin signaling system which are implicated in the development of several Neural crest-derived tissues in vertebrate organisms. Although very little is known about the normal role of endothelin receptor type A (EDNRA) in hair follicle development, strong expression of EDN1 and its receptor EDNRA was observed in hair follicles at multiple stages of follicular development and particularly in DP cells. See Gordon et al., Am J Hum Genet (2015) 96(4):519-31. Interestingly, genetic mutations of EDNRA caused near-complete alopecia in four unrelated individuals, in addition to other craniofacial abnormalities. Id. F0XC2 is part of the Forkhead box (FOX) family of transcription factors, which are essential components in the embryonic development of several systems, including hair follicle and their stem cell niche. F0XC2 role and expression of in DP cells have not been investigated yet. However, mutations in the FOXC2 winged helix transcription factor gene have been identified in more than 170 individuals affected by distichiasis, which manifests as a double row of eyelashes arising from the meibomian glands at birth. See, e.g., Chen et al., Nucleic Acids research (2019) 47(7):3752- 3764; Kriederman et al., Hum Mol Genet (2003) 12(10): 1179-1185. LIF (leukemia inhibitory factor) is expressed in many different cell types and has pleiotropic actions. In HF, LIF is involved in the development of the follicle and partial or full knock-out of LIF gene has been demonstrated to cause a delay in the HF formation and decreased epidermis thickness. See Pichel et al., Mechanisms of Development (2003) 120(3): 349-361. IGFBP1 (insulin-like growth factor binding protein 1) is part of the insulin-like growth factors (IGFs) signaling networks, whose role in hair follicle biology has recently been recognized to stimulate hair elongation and facilitate maintenance of the hair follicle in the anagen phase. Some studies identified these proteins as being produced and released by dermal papilla cells and may modulate IGF-I action by interaction with matrix proteins. See, e.g., Batch et al., J Investigative Dermatology (1996) 106(3): 471-475. SIX2, also known as Sine Oculis Homeobox (Drosophila) Homolog, is a transcription factor that plays an important role in the development of several organs, including the kidney, skull, and stomach. There are no studies reporting the role of SIX2 in DP cells or hair follicle biology. On the other hand, studies on embryonal renal mesenchyme report its interaction with TCF7L2 and OSR1 and involvement in a canonical Wnt signaling independent manner preventing transcription of differentiation genes in mesenchymal stem cells. See, e.g., Senanayake et al., Human Pathology (2013) 44(3):336-345. Considering the crucial role that Wnt has in the functionality of dermal papilla, SIX2 could represent a novel player in the regulation of this pathways in hair follicle cells.

[0159] Lastly, we studied the eigengene pattern across cell groups to determine genes with similar expression trend to evaluate their possible link to folliculogenesis (FIG. 4A). Interestingly, three major scenarios were noticed. The first one, referred to as “positive”, included all patterns where the eigengene levels in iPSC-DP were comparable to hDP but differ from HFF (either up or down regulated). This scenario accounts for ME9 in pattern 4 and ME6 in pattern 7 (starred). The second one, referred to as “negative”, described patterns where the eigengene levels in iPSC-DP were equal to HFF but different than hDP (either up or down regulated), such as pattern 6 ME10 (starred). Lastly, the third one, referred to as “off’, comprised patterns where the eigengene levels in iPSC-DP were different than both hDP and HFF (either up or down regulated), for instance pattern 3 ME5, pattern 5 ME7, and pattern 7 ME 13 (starred).

[0160] Gene set mapping analysis revealed some noteworthy pathways activated in the modules identified above (Figure 4B). For instance, module 6 was enriched for skin development pathways, estrogen signaling, and ECM receptor interaction; module 7 was linked to TNF, Shh, PI3K-AKT and NF-kp signaling pathways and ECM receptor interaction; module 9 was associated to cell cycle and p53 signaling, and module 10 was inclusive of WNT signaling pathway and adhesion molecules previously associate to DP cells. See Rezza et al., Cell Rep, (2016) 14(12): 3001-18.

[0161] Knowing the pathways that are differently regulated between iPSC-DP and both the native hDP and the HFF, offered a great opportunity to gain control over the newly differentiated cells by intentionally modulating the targeted eigengenes using the proper set of small molecules. Ongoing screenings will continue to shed light on the pathways that characterize DP cells.

[0162] Taken together, the bulk sequencing data revealed the acquisition of a mesenchymal profile of the iPSC-DP cells upon differentiation from iPSC-NC cells, with commonalities with the two reference cell populations. The presence of both native DP markers and typical fibroblast genes in the iPSC-DP cells could be a reflection of a heterogenous population that emerged during the differentiation process, which needed to be addressed.

Example 3: Single-cell RNA-seq of iPSC-derived cells determines a gene trajectory from the NC state to the DP fate

[0163] To evaluate the purity of the iPSC-derived cell population and explore cell-to-cell transcriptomic changes that define cell heterogeneity during the differentiation process, we performed a single-cell RNA sequencing (scRNAseq) analysis on DP cells at different time points during cell development from day 0 (NC cell state) to day 8. The differentiation process was reduced from day 12 to day 8 as bulk sequencing analysis revealed little to no changes in the transcriptome profile of these two times points. In addition, there are several technical advantages related to a shorter protocol that are worth consideration especially for scalability purposes including less media and reagents consumption, faster turnaround time, and eased human resources. [0164] After the removal of cells that didn't meet high-quality criteria, the number of total cells profiled in the data set was 7352 cells.

[0165] The extrapolated cells were clustered based on cell differentiation day in a UMAP plot, which clearly showed a progressive transcription state that significantly changed from day 0 to day 2 and reached a more uniform but yet dynamic profile from day 4 throughout day 8 (FIGs. 5A and 5B). Day 2 (clusters 1 and 8) appeared to be a transient state that gave rise to four clearly distinguishable clusters by day 8. To define the transcriptomic profiles for each subpopulation, we performed a differentially expressed genes analysis (DEGs, logFOl, adjusted q-value <0.05) between clusters. The dot plot in FIG. 5B showed the expression frequency and level of five major groups of genes related to dermal cells and to key features of the DP cells such as signature genes, extracellular matrix, focal adhesion and cell cycle. This plot highlighted an enrichment of the cell cycle genes mainly in four particular clusters, namely 1, 3, 6 and 7, while ECM network and focal adhesion genes were highly expressed in clusters 2 (day 4), 4 and 5 (day 8). The enrichment analysis of the four clusters emerged at day 8, confirmed the presence of genes related to ECM matrix, tissue development and cell adhesion as well as genes related to cell cycle and chromatin organization (FIG. 5C). These findings supported the hypothesis, formulated based on bulk sequencing, of the co-existence of two subgroups of mesenchymal cells in the targeted iPSC-DP population, where clusters 4 and 5 could represent more committed cells and clusters 3 and 6 more immature or progenitor cells.

[0166] Surface markers that may be used to purify and optimize the iPSC-DP population for functionality were screened for by sorting a subcluster of cells. This screen identified ITGA1 as a best candidate to isolate cells in cluster 5 from the co-existing cell population (FIG. 5D) by using the pattern of expression among all the clusters at day 8 (highlight in the box in FIG. 5D).

[0167] To further investigate the dynamic nature of nascent DP cells during the differentiation process by performing an RNA velocity analysis.

[0168] RNA velocity estimates the future state of individual cells by calculating the ratio of unspliced and spliced mRNAs to predict lineage trajectories within scRNA-seq data. See La Manno et al., Nature (2018) 560(7719):494-498. scVelo was utilized to perform this analysis on all conditions. The velocity analysis revealed two main paths in the trajectory ordering (FIG. 6A). Specifically, the major trajectory, generated as differentiation progressed after the starting point (clusters 0 and 7), connected day 2 (cluster 8 and partially cluster 1), to day 4 (cluster 2) to day 6 and 8 (clusters 4 and 5). Interestingly, this trajectory seemed to merge into cluster 5 (part of day 6 and 8), one of the most favorable clusters in terms of expression of genes known to be activated in mesenchymal and DP cells, such as PDGF, TGFP, VEGF, IGF or encoding for extracellular matrix production such as glycoproteins, collagens, proteoglycans (FIG. 6B), confirming not only the acquirement of a mesenchymal phenotype at the end of differentiation protocol but also the presence of a more folliculogenic group of cells in the iPSC-DP population Ohyama et al., J Cell Sci (2012) 125(Pt 17): 4114-25; Rezza et al., Cell Rep (2016) 14(12): 3001-18 ; Shin et al., Dev Cell (2020) 53(2): 185-198.e7; Heldin et al., Physiol Rev (1999) 79(4): 1283-316; Kim et al., Aging (Albany NY) (2021) 13(16): 19978-19995.

[0169] However, the co-existence of subclusters of DP-like cells within the iPSC-derived population might have a supporting role for the functionality of this nascent population. Thus, the iPSC-DP were characterized not only by marker expression, but also by in vitro behavior and ultimately by in vivo transplantation to test functional capabilities.

Example 4: In vivo transplantation of iPSC-derived DP generates new HFs

[0170] To be fully functional, iPSC-DP cells should be able to demonstrate essential physiological attributes of HFs, such as hair shaft production. Therefore, different transplantation methods were employed to evaluate cell potency and characterize the emerging HFs.

[0171] First, spheroids made of iPSC-derived DP cells were combined with a suspension of embryonic mouse epithelial cells (mKC) and transplanted intradermally into immune- deficient nude mice (FIGs. 7A-7D). Positive control combinations, consisting of embryonic mKC and dermal cells (mDC), generated new hair around day 14, as previously observed in what is generally known as patch assays. See Zheng et al., J Invest Dermatol (2005) 124(5): 867-876. The dark coloration of hair shafts at the injection site of both chimeric and fully murine hair was most likely due to melanocyte progenitors' presence within epidermal preparations from E18.5 black hair-bearing C57BL/6 mice (FIGs. 7A and C). HF-like structures were observed underneath the skin 60 days post-surgery (FIG. 7C). To ensure that such growth was driven by human cells, human-specific antibodies and confocal microscopy were used. hiPSC-DP cells were noted to be incorporated into the DP of the newly formed HFs (FIG. 7D). Unfortunately, the in vivo experiments' success rate was low and highly variable, as previously reported by others, see Abaci et al., Nat Commun (2018) 9(1): 5301; Lee et al., Nature (2020) 582(7812): 399-404; Oh et al., J Invest Dermatol (2016) 136(1):34- 44, likely reflecting mouse-to-mouse variability in graft rejection and technical difficulties to inject cells into the dermis. Moreover, the majority of the HFs grew disorganized underneath the skin growing in subcutaneous cysts, adding a layer of complexity in the histological analysis of the transplanted site. Also, despite being easy to perform and compatible with large-scale screening, this procedure is only valid for evaluating the efficacy of hair formation on a short-term basis as the newly generated HFs cannot cycle normally.

[0172] In order to evaluate the folliculogenic potential of iDP aggregates and iDP-iEP organoids in a setting of all-human hair follicles, we tested the addition of iDP aggregates, fetal DP aggregates, or iDP-iEP organoids into a 3D reconstructed human skin xenograft model (FIG. 8A). In this model, a full thickness skin wound is created on the dorsal aspect of athymic nude mice and 3D construct containing only human cells is grafted in place to generate a reconstructed hair-bearing human skin xenograft with anatomically appropriate directional growth of hair follicles (FIG. 8B). Human hair shafts were observed to emerge from the human skin pad and the dermal papilla/bulb region of the hair follicles was observed from the underside of the human skin xenograft (FIG. 8C). Hair follicle density was quantified in grafts comprising the fetal human dermal + HFK baseline xenograft and also after spiking in 50 iDP aggregates or 50 iDP-iEP organoids (FIG. 8D). Increases in HF density were observed when adding in the trichogenic cells to the baseline dermal plus epidermal xenograft condition.

[0173] Subsequently, iPSC-DP cells were tested in organoids, created using the 3D coculture method, and embedded in MATRIGEL® before being transplanted into immune- deficient nude mice. See details in the Material and Method section above, FIGs. 13A-13D and Example 9 below.

Example 5: Organoid generation and analysis

[0174] The protocol described below is for an ultra-low attachment microsphere 384-well plate (CORNING® 3830 or equivalent). If differently sized well plates are used, the protocol can be modified proportionately. After harvesting and counting the appropriate cell types needed to form organoids (for example, iPSC-DP and hKCs), each cell type was resuspended at a density of 150k cells/mL in Organoid Medium, composed of 50% of GIBCO™ AMNIOMAX™-!! (ThermoFisher Scientific, catalog # 85850) and 50% of fully supplemented keratinocyte growth medium 2 (KG2) (PromoCell®, catalog # C-20211 and C- 39016) with 2% MATRIGEL® (CORNING®, cat #354230). The two cell types were then combined in a 15 mL tube at a 1 : 1 ratio. The cell suspension was dispensed into 384-well plate by using an electric repeating pipet (Integra 125). All wells were added to the plate, and the plates were then transferred to a humidified 5% CO2, 37°C incubator. After seven days, the organoid medium was changed by removing 25% of the media (20 pL) and replacing it with Organoid Medium. A multichannel system or a microfluid automated system was used to ensure that organoids were not aspirated during the media change process. Media change was performed every 7 days with 2% MATRIGEL® Organoid Medium followed by a fullplate scan with any automated imaging system (e.g., a Zeiss LSM 800 microscope, a multimode plate reader CYTATION™ 5 by BioTek, or alternative instrument). The images acquired were subjected to automated analysis to measure the roundness score (also known as elongation score) by using the GEN5™ Data Analysis Software (BioTek). The software provides cell segmentation and values for several parameters such as minimum and maximum object size, surface, perimeter, longitudinal axis length. Based on these parameters, the elongation score is calculated using the formula: 1 - (roundness = (4K X area)/(convex perimeter) ² ), which generates a value from 0 to 1. A range of values is then defined for each stage of the development of the organoids as follows: organoids are considered round if the elongation score is between 0 and 0.2, polarized if the elongation score is between 0.2 and 0.4, and elongated when the elongation score is greater than 0.4. See also, Table 8 below and FIG. 18.

Table 8: Elongation Score Calculation

Example 6: Mouse transplantation

[0175] For the animal procedures Athymic Nude-Foxnlnu mice obtained from Charles River Laboratories were used. All animal procedures were performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals and under IACUC protocol. For neonatal mouse HF progenitor cells, truncal skin was removed from El 8.5 C57BL/6 pups and rinsed in Ca2+ and Mg2+ free PBS (phosphate-buffered saline). The skin was incubated in 0.2% dispase overnight at 4°C to separate the epidermis from the dermis. The dermis was then digested with 0.2% collagenase (Sigma-Aldrich, St. Louis) at 37°C for 60 minutes, while the epidermis was digested with 0.125% trypsin/EDTA for 10 minutes at 37°C. Singlecell suspensions were strained through 40 pm filters and pelleted at 300 g. When stated, these cells were used as positive control for folliculogenesis in the organoid’s transplantation.

[0176] For the patch assay, 0.5xl0 ⁶ dermal cells (mouse dermal cells, human DP cells, or iPSC-derived DP cells) and 0.5xl0 ⁶ single epidermal cells were mixed and resuspended in a total volume of 50 pl of DMEM-F12 medium and injected (28G needle) into the hypodermis of the mouse skin, forming a bleb as previously described. After recovery from anesthesia (isofluorane), mice were kept under normal husbandry conditions. The animals were monitored on a weekly basis, and the darkening of the skin at the site of the injection could be visible in correspondence with the control condition (mouse dermal and epithelial cells) two weeks after. The skin sites that had received the injected cells referred to as “patch” were harvested usually 30-45 days later. The number of HFs formed in each patch assay was manually quantified in each stereomicroscope image.

[0177] To transplant the organoids, a MATRIGEL® (Corning®, basement-membrane matrix extracted from Engelbreth-Holm-Swarm mouse sarcomas) dome containing 200 organoids per site was created in the lab 24 hours before transplantation. Briefly, the organoids were collected into a sterile reservoir from the 384-well plate by gentle centrifugation (300g for 3 minutes). Organoids were then transferred in a 1.7 ml tube (200 organoids/tube) with the minimum volume of media possible. The organoids were allowed to settle down by gravity and were then transferred with 50 pl of medium to a slick coated side of a 1x1 cm sterile sheet (placed in a 12-well plate with sterile tweezers) to create a drop. The excess media was removed from the drop using a p200 micropipette. After most of the media was aspirated, 30 pl of pure MATRIGEL® was added on top of the organoids to create a dome. To ensure proper gelation, the plate containing drops of MATRIGEL® with embedded organoids was placed into a 37°C incubator for 10 minutes. Then ImL of fibroblast media (89% DMEM/F12, 10% FBS, 1% Antibiotic/ Antimycotic) was added dropwise to each well on top of the sheet and the plate was returned to the 37°C incubator until the transplantation. For transplantation, the mice were anesthetized by isoflurane. The back of each mouse was wiped with betadine solution followed by alcohol. An anti-inflammatory and pain relief drug (Carprofen 0.03 mg/ml) was administered before proceeding with the surgery. With surgical sterile scissors, a 2-sided flap was created on the back skin of the nude mice, big enough to accommodate the MATRIGEL® dome with organoids. With sterile tweezers, the sheet with organoids was transferred from the plate to the proximity of the flap and the MATRIGEL® dome accommodated underneath the skin flap. The mice were covered with the following bandages: Rylon-1® wound dressing, Tegaderm™, and elastic bandage (any brand). After recovery from anesthesia, mice were kept under normal husbandry conditions. The animals were monitored daily, and the darkening of the skin at the site of the injection was visible in correspondence with the control condition (mouse dermal and epithelial cells) two weeks after. The skin sites that received the injected cells referred to as “graft” were harvested usually 30-45 days later. The number of HFs formed in each patch assay was manually quantified and imaged with a stereomicroscope.

[0178] For the reconstructed human skin model, populations of human dermal and epidermal cells were mixed to generate a 3D construct which was engrafted onto a full thickness skin wound on the dorsal aspect of athymic nude mice See Wu et al., Tissue Eng Part A, (2014) 20(23-24): 3314-3321. In this model, directional growth of human hair follicles including hair shafts that emerge from the skin make it more physiologically relevant than patch assays. Dermal cells were isolated from human fetal scalp tissues (estimated gestational age 11-22 weeks). Fetal scalp skin pieces were diced into pieces and dissociated in a solution of collagenase type I (100 U/mL), dispase (0.2%) and DNAse I (0.1 mg/mL) with agitation for 2 hours at 37 degrees followed by 5 minutes dissociation with trypsin with EDTA. Cell suspensions were filtered through a 40-um strainer, washed and cultured on matrix-coated vessels (collagen I, fibronectin and laminin 521) in DMEM/F12 (3: 1) containing 0.1% penicillin/streptomycin, 40 mg/mL fungizone, 40 ng/mL FGF2, 20 ng/mL EGF, and 2% B27 supplement with 5% FBS until the first passage and then transitioned to tissue-treated plastic with AmnioMax™-II media. Human foreskin keratinocyte (HFK) cultures were established from neonatal foreskin tissue by similar methods and cultured in KG2 basal medium :DMEM/F 12 (2: 1) supplemented with FBS (5% v/v), adenine (0.18mM), hydrocortisone (0.5 ug/mL), cholera toxin (0.1 nM), EGF (10 ng/mL), and insulin (5 ug/mL) on a mitomycin C-treated 3T3 fibroblast feeder layer. To generate 3D constructs for grafting onto mice, human fetal dermal cells and human foreskin keratinocytes were mixed into a dense cell suspension at 3:2 ratio in DMEM/F12 media with MATRIGEL, placed on transwell inserts bathed from below with DMEM/F12 media containing 5% FBS and allowed to gel overnight (also referred to as a cell slurry). For conditions that tested the addition of dermal papilla aggregates (iDP or fetal DP) or iDP-iEP organoids, the aggregates and organoids were generated 1 day prior to the 3D construct and 50 aggregates or 50 organoids were added into dense suspension of fetal dermal cells and HFK prior to placing on the transwell insert. For transplantation to athymic nude mice, full thickness skin wounds were created using a 8-mm biopsy punch below nape and along midline of animal. The 3D cell constructs were placed onto the fascia sites of the animal. Suprathel, an absorbable wound/burn dressing or similar, was placed above the 3D constructs as a securing agent. Rylon, mesh adhesive fabric or similar, was the next layer added. Tegaderm, or similar, was used as a tertiary bandage material to ensure constructs remain on the wound site. Other bandage material (i.e., vetwrap, elasticon, sterile tape) was used as the last layer to ensure proper compression and reduce the risk of infection. Bandaging remained in place for 10 days and animals were sacrificed after 10 weeks for histological evaluation of graft sites.

Example 7: Histology and immunohistochemistry

[0179] After 6-10 weeks, mice were sacrificed, and grafts were removed. The tissue was subsequently fixed overnight in 4% formaldehyde in phosphate-buffered saline (PBS). The fixed tissue was frozen in N2 liquid in OCT (optimal cutting temperature) compound and stored at -80°C or processed for paraffin embedding and kept at room temperature. Serial sections (5-7 pm) were cut on a cryostat or on a microtome. Some sections were analyzed by hematoxylin/eosin staining, and the remaining sections were used for immunofluorescence analysis. For hematoxylin and eosin staining, slides were washed three times in PBS, three times in water, and then counterstained in Mayer's hematoxylin, followed by eosin. Finally, sections were dehydrated through graded ethanol solutions and xylene and mounted. Sections were scanned using an APERIO® automated system (Leica) or with a Nikon inverted microscope.

[0180] For immunofluorescence, slides were washed in PBS for 5 min after fixation and incubated with a blocking solution (5% of serum derived from the same species as the secondary antibodies diluted in PBS with 0.5% Triton X-100) for 1 hour. The slides were incubated overnight at 4°C with the specific primary antibody. The slides were washed three times with PBS-0.5% Triton X-100 and incubated with the corresponding secondary conjugated antibody in blocking solution for 1 hour at room temperature. After washing out the secondary antibody, slides were mounted with ProLong® Diamond Antifade Mountant with DAPI (for nuclei staining). Sections were imaged with a confocal Zeiss LSM 800 microscope. The same staining procedure can be used for organoid slices or whole mount staining.

Computational analysis

[0181] Computational analysis for morphological features of organoids was performed using the Harmony platform version 4.9 (Perkin Elmer) to extract different organoid metrics from the experiment. Statistical analysis

[0182] Each experiment was analyzed using an appropriate statistical method as indicated in each figure. GraphPad Prism was used unless otherwise specified.

Results

[0183] A 3D cell culture approach was employed to recapitulate HF morphogenesis and generate HF units. For background regarding organoids and 3D cell culture, see Method of the Year 2017: Organoids. Nature Methods, 2018. 15(1): 1-1; and Freedman et al., Nature Communications, 2015. 6(1): 8715; McCracken et al., Nature, 2014. 516(7531): 400-4. The system employed here relies on the co-culture of dissociated DP cells and epithelial cells, mixed to enhance continuous crosstalk between them. A static culture environment was developed where the two cell types self-assemble into organoids to induce an architecture similar to an endogenous hair follicle. In particular, the formation of organoids was initiated by combining equal numbers of epithelial and dermal cells of both human and murine origin in microsphere 384-well plates, whose specific geometry is conducive to the formation of organoids of uniform size and shape and is compatible with a high-throughput compound screening (see Material and Methods for details). C57BL/6 mouse embryonic cells were employed as a positive control due to their high proliferative and trichogenic ability as previously reported. Zheng et al., J Invest Dermatol, 2005. 124(5): 867-876. To culture the combined cell lines, a homogenous culture medium was used without intentional nutritional gradients to permit the cells to freely grow and remodel their environment without imposing guidance on spatial cell patterning. After 24 hours in culture, dermal and epithelial cells spontaneously aggregated in spheroids and engaged in a self-directed organization to form polarized clusters that further developed into more complex and organized structures that mimicked the organizational features of HFs (FIG. 10C). Brightfield and fluorescence images showed the morphological changes of these cell aggregates during culture, revealing a selforganization process through four consecutive stages, which replicate the major stages of the folliculogenesis (FIGs. 10A-10B): 1) induction phase, consisting of the thickening of epithelial cells to form a placode, 2) organogenesis stage, which takes place when the epithelial cells first send signals to dermal cells to proliferate and form dermal condensate (DC), and then directs epithelial cells to proliferate and migrate towards dermis, 3) cytodifferentiation stage, when the dermal condensate transition into a more mature dermal papilla upon being encapsulated by the follicular epithelial cells and 4) HF downgrowth, in which the growing HF has elongated past the boundary of the lower dermis, and the hair canal is morphologically visible. Saxena et al., Exp Dermatol, 2019. 28(4):332-344. Remarkably, in the area above the dermal compartment in the HF organoids, a dense cone of cells emerged, resembling the hair matrix region characterized by the presence of Transit- Amplifying Cells (TAC), likely generated by the epithelial compartment (FIGS. 9A-9C and FIG. 11C). To visualize step-by-step the temporal and spatial dynamics of these organoids, human iPSC-DP cells were engineered with the fluorescent protein mCherry via CRISPR- Cas9 (FIG. IOC, FIGs. 11A-11C, FIG. 15B). The resultant structures were highly reminiscent of a primordial HF, with the DP cells occupying the stereotypical position in the hair bulb and the epithelial cells extending above it. However, one noticeable difference between the in vitro model and the physiological morphogenesis was the distribution of dermal papilla around the epithelial cells in the first stages of organoid formation. Escaping from the dermal enveloping the epithelial cells seemed to create an opening through the external layer, defined as the jailbreaking point (see arrows in FIG. 10B and FIGs. 11A-1 IB). This portion functions as an anchor between the two cell populations and creates the foundation for the elongation of the epithelial cell. Marker analysis revealed that this segment is positive for Lefl, one of the downstream effectors of Wnt pathway (FIGs. 11 A-l IB). This unexpected phenomenon is likely related to the absence of contextual clues from the surrounding environment in the in vitro system and the different cohesive properties of mesenchymal and epithelial cells. Nevertheless, cells could reroute and follow a different morphogenetic path to acquire the same phenotype of endogenous follicles. Notably, Lei at al. in 2017 showed a comparable self-organization pattern of dissociated cells from newborn mouse skin, which aggregated to generate a cell slurry that grow hairs robustly in vivo after transplantation into nude mice. Lei et al., Proceedings of the National Academy of Sciences, 2017. 114(34):E7101-E7110. A well-organized matrix is produced during the organoid elongation to serve as a scaffold for its shaping. Meanwhile, the dermal compartment acquired a more compact and round shape, approximately 100 pm in diameter, the size of which didn't appear to change for the entire developmental process (FIGS. 15A-15C). This aligns with literature data that established that cells in mature dermal papillae are quiescent and very rarely divide. See, e.g., Moffat, G.H., J Anat, 1968. 102(Pt 3):527-40; Tobin et al., J Investig Dermatol Symp Proc, 2003. 8(l):80-6. In addition, this capability was confirmed to be specific to iPSC-DP cells, as fibroblasts in combination with mouse embryonic keratinocytes never elongated, despite being able to aggregate in spheroids (FIGS. 15D-15E). Example 8: Confirmation that human native HF cells follow the structural pattern of iPSC-DP cells

[0184] To validate the suitability of only primed HF cells to form organoids in vitro, native human HF cells obtained from donors undergoing hair transplantation were tested. Remarkably, native human DP (hDP) and human HF keratinocytes (HF-KC) followed the exact same step-wise progression, from a round organoid to an elongated structure, when coculture in our 3D system (FIG. 12A). Marker analysis and confocal microscopy revealed that organoids made of both iPSC-derived and native cells presented the same segmentation in two major compartments, defined by a clear separation between the dermal papilla (Vim+) and the epithelial cells (K14+) (FIG. 12B). A panel of essential markers, whose localization patterns have been well characterized in human and murine HFs, was further analyzed to assess which cell types were present in the iPSC-DP derived organoids and how they compare to a real mature tissue (FIG. 12C). Although these in vitro self-organized structures were not identical to actual mature HFs, they achieved such a similar phenotype and markers distribution that they can be used to corroborate the link between the identity of iPSC-derived cells and their functionality as hair forming cells. To quantitively assess the reproducibility of organoid elongation and provide an early indicator of cell functionality in vitro, an automated computational image-based analysis using Gen5™ software (BioTek) was employed that calculated the elongation of the 3D structures, assigning values between 0 and 1, where 0 represents a perfectly round object and 1 a long shaped one. Ranges of values were assigned to account for the different stages of the organoid development and defined organoids as aggregated (round) if the elongation score was between 0 and 0.2, polarized between 0.2 and 0.4 and elongated when the elongation score was greater than 0.4. By plotting time-lapse values of elongation score in a graph, the ability of iPSC-DP cells to generate elongated structure in combination with human primary keratinocytes or iPSC-EpSC (FIG. 12D) was confirmed.

[0185] Additional computational analysis could be performed to extract a multitude of morphological features such as shape, size, dimension, geometry, and orientation to further characterize the HF organoids (FIGs. 16A-16C). To get a basic idea of the follicle size, the metrics associated with the minimal box's dimensions that contain the whole structure was plotted. From FIG. 16B, the structures grew to a mean length of approximately 1.5 mm (Longest Length). The other two dimensions (Medium Length and Shortest Length) were mainly influenced by how the structure lay on the concave surface and were therefore surrogates for the height and perpendicular span of the organoid in relation to the Longest Length measurement. (FIG. 16C). All of these metrics in combination with artificial intelligence system could provide real-time in-depth knowledge on the organoids, which can be leveraged to monitor shrinkage, expansion, or structure stagnation upon treatment or perturbation of the organoid’s environment via small molecules, gene editing, or matrices addition in the near future.

Example 9: In vivo transplantation of HF organoids to generate new HFs

[0186] To be fully functional and accurately mimic the phenotype of native hair, HF organoids should be able to demonstrate essential physiological attributes, such as hair shaft production. Therefore, organoids created using the 3D co-culture method were transplanted and embedded in MATRIGEL® into immunodeficient nude mice (FIG. 13 A). Three iPSC- derived DP lines (DP61, DP101, DP 126) were tested in combination with embryonic mouse keratinocytes (mKC). The dark coloration of the hair shafts at the injection site of the chimeric hair was most likely due to melanocyte progenitors' presence within epidermal preparations, generated from black hair-bearing C57BL/6 mice. Transplanted animals were sacrificed 30 days post-surgery, and the skin was excised and examined under a dissecting microscope (FIG. 13B). HF-like structures were observed underneath the skin and quantified. Histological section of the skin at day 30 post-transplantation, stained with H&E and DAP I, showed the presence of follicular units generated by individual organoids, bearing one or two follicles each (FIGs. 13C-13D). The development of these discrete transplantable follicular units supported by iPSC-derived cells represents an innovative approach, different than anything studied before, to achieve natural-looking hair. Indeed, this procedure could pose the basis for practical clinical applications in the future and offer a major advantage over similar approaches, providing individual follicular units ready to be implanted in the desired amount on the target area on the scalp. However, due to the limitation of the mouse skin and its reduced thickness, a physiological spatial organization of the newly regenerated HF was not achieved in the murine models, as the organoids were implanted underneath the dermis and the shaft couldn’t emerge through the skin surface. Also, despite being easy to perform and compatible with large-scale screening, this procedure is only valid for evaluating the efficacy of hair formation on a short-term basis as the newly generated HFs cannot cycle normally.

[0187] Also, the in vivo experiments’ success rate was low and highly variable, as previously reported by others. See, e.g., Abaci et al., Nat Commun, 2018. 9(1): 5301 ; Lee, J., et al., Nature, 2020. 582(7812): 399-404.; Oh et al., J Invest Dermatol, 2016. 136(l):34-44.), likely reflecting mouse-to-mouse variability in graft rejection and technical difficulties to inject cells into the dermis. Nonetheless, these results demonstrate that the organoids described herein are capable of functioning as hair follicles following implantation.