STEM CELLS AND USES THEREOF

FIELD OF THE DISCLOSURE

The present disclosure generally relates to methods for preparing human mesenchymal stem cells (hMSCs) that express high CD 10 phenotypes. The present disclosure further relates to methods of treating local inflammation, fibrosis, and/or musculoskeletal pain using hMSCs that express high CD 10 phenotypes.

BACKGROUND OF THE DISCLOSURE

Synovium and infrapatellar fat pad (IFP) as a single anatomical and functional unit play crucial roles in the pathogenesis and progression of Osteoarthritis (OA). They serve as sites for immune infiltration undergoing inflammation and fibrosis, and as source of joint destructive molecules (e.g., matrix metallopeptidases. Mesenchymal Stem Cell (MSC)-based therapy has gained attention as a potential therapeutic alternative in OA, given their immunomodulatory and trophic effects involving anti-inflammatory and anti-fibrotic actions, all critical for OA progression.

"Traditional" MSC processing, including fetal bovine serum (FBS) supplementation to the expanding media has been the standard of practice worldwide. However, numerous studies have raised safety concerns regarding the use of FBS-containing media during the manufacturing of MSC preparations for clinical applications/trials, most of them related to prion exposure risk, toxicological risk and immunological risk due to the presence of animal derivative. Serial MSC expansion in these conditions to obtain clinically-relevant therapeutic cell numbers may result in detrimental effects on cell performance (e.g., compromised proliferation and/or accelerated senescence), or even untoward consequences to specific cell attributes (e.g., phenotypic display and functional outcomes). These variable responses to processing steps, combined with the inherent inter-donor variability negatively affect the standardization and reproducibility of their therapeutic potential.

Thus, there is a need in this art for alternative methods for producing clinically relevant amounts of MSCs that do not involve FBS or other animal-derived media, supplements, or components, wherein the MSCs produced thereby can be more safely used for treating appropriate diseases and disorders in humans and other animals. SUMMARY OF THE DISCLOSURE

This invention provides methods for producing clinically relevant amounts of MSCs that do not involve FBS or other animal-derived media, supplements, or components, wherein the MSCs produced thereby can be more safely used for treating appropriate diseases and disorders in humans and other animals.

Provided herein is a method of preparing human mesenchymal stem cells (hMSCs) that express CD 10 at a level equal or greater than 70% positivity overall, the method comprising: culturing hMSCs in a xeno-free formulation; and thereby obtaining hMSCs having CD 10 expression at a level equal or greater than 70% positivity overall.

Further provided herein is a method of treating local inflammation and/or fibrosis in a subject in need thereof comprising administering to the subject an effective amount of human mesenchymal stem cells (hMSCs).

Also provided herein is a method of treating musculoskeletal pain in a subject in need thereof comprising administering to the subject an effective amount of human mesenchymal stem cells (hMSCs).

Also provided herein is a method of repairing altered blood vessels impacting their capacity of responding with changes in vessel tone and to contribute to inflammation and fibrosis comprising administering to the subject an effective amount of human mesenchymal stem cells (hMSCs).

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES Figures 1A-1C illustrate that IFP-MSCs showed high growth kinetics and clonogenicity when expanded in either hPL or Ch-R. Figure 1A and Figure IB demonstrate that IFP-MSCs expanded in either hPL or Ch-R showed a higher growth rate until confluency as compared with FBS alone. Figure 1C demonstrates clonogenic capacity of FBS expanded IFP-MSCs (colony-forming unit fibroblasts [CFU-Fs] per 103 MSCs seeded) showed a higher trend versus the other conditions. All experiments (n = 5) were performed independently, and data are presented as scatter plots with mean. Ch-R, chemically reinforced medium; FBS, fetal bovine serum; hPL, human platelet lysate; IFP, infrapatellar fat pad; MSC, mesenchymal stem cell.

Figures 2A-2B illustrate that IFP-MSCs expanded in either hPL or Ch-R have an enhanced immunophenotypic profile. Figure 2A shows surface markers assessed in noninduced IFP-MSCs show MSC-defining markers highly expressed in all culture conditions, whereas CD 10 and CD 146 (immunomodulatory) and CXCR4 (migratory) showed increased expression only in hPL and Ch-R. Figure 2B shows proinflammatory/profibrotic ex vivo priming with TNFα, IFNγ, and CTGF (TIC) resulted in boosted expression of CD 10 and CD 146, while HLA-DR expression was induced as expected. All experiments (n = 3) were performed independently, and data are presented as scatter plots with mean. Individual donors in each MSC type are presented with distinctive shapes and color tones to allow intradonor comparisons. Ch-R, chemically reinforced medium; FBS, fetal bovine serum; hPL, human platelet lysate; IFP, infrapatellar fat pad; MSC, mesenchymal stem cell.

Figures 3A-3B show steogenic, chondrogenic, and adipogenic differentiation potential of IFP -MSC expanded in either hPL or Ch-R. Figure 3 A shows that hPL- and Ch-R- expanded IFP-MSCs showed superior qualitative differentiation capacity upon induction in vitro for bone (mineral deposition assessed by alizarin red staining), fat (lipid accumulation assessed by oil red staining), and cartilage (glycosaminoglycan production assessed by toluidine blue staining) as compared to FBS-grown cells. Figure 3B illustrates that quantitative molecular profiling showed that differentiation-related markers in cultures expanded in hPL and Ch-R were increased versus those expanded in FBS, indicating their mature status. All experiments (n = 5) were performed independently with noninduced cells, and data are presented as scatter plots with mean ± SD. *P<05. Ch-R, chemically reinforced medium; FBS, fetal bovine serum; H&E, hematoxylin and eosin; hPL, human platelet lysate; IFP, infrapatellar fat pad; MSC, mesenchymal stem cell.

Figures 4A-4B illustrate that IFP-MSCs expanded in either hPL or Ch-R had a reduced inflammatory baseline signature while strongly secreting reparative growth factors. Figure 4A illustrates transcriptional profiling of IFP-MSCs expanded in all 3 formulations showed overall low-to-negative expression levels for most inflammation-related cytokines tested (top panel). This correlates with high delta Ct values in real-time quantitative polymerase chain reaction, representing the arithmetic subtraction of the Ct value of each gene minus the Ct value of the normalizing housekeeping gene and marked white to blue in the heat map. Lower panel shows the relative fold change expression (positive = darker, negative = lighter), calculated with the FBS-grown data as reference (set up as 1). Figure 4B shows secretory profile heat maps of noninduced and TIC-primed IFPMSCs indicated high growth factor secretion for hPL- and Ch-R-expanded IFP-MSCs (top panel). Heat maps colors are assigned according to a molecule concentration relative scale from 0 to 10,000. Venn diagram shows shared proteins among all groups (significantly different from FBS alone or FBS plus priming; middle panel). The table shows the number of proteins shared by different groups and conditions (lower panel). All experiments were performed independently (n = 3). Ch-R, chemically reinforced medium; FBS, fetal bovine serum; hPL, human platelet lysate; IFP, infrapatellar fat pad; MSC, mesenchymal stem cell; TIC, TNFα, ΙFΝγ, and CTGF.

Figure 5 illustrates FP-MSCs expanded in either hPL or Ch-R showed high growth factor secretion when compared with FBS plus priming. Venn diagram analysis revealed shared proteins among the groups (significantly different from FBS plus priming), with 14 growth factors common and highly expressed in IFP-MSCs expanded in hPL and Ch-R. All experiments were performed independently (n = 2). Ch-R, chemically reinforced medium; FBS, fetal bovine serum; hPL, human platelet lysate; IFP, infrapatellar fat pad; MSC, mesenchymal stem cell.

Figures 6A-6C illustrate IFP-MSCs expanded in either hPL or Ch-R effectively degraded substance P (SP) in vitro. Figure 6A illustrates endogenous levels of SP (quantified at ~250 pg/mL and shown as dotted colored lines) and exogenously added recombinant SP (quantified at ~ 1100 pg/mL and shown as a gray dotted line) were used for reference (as bottom and top boundaries, respectively) to quantify SP degradation activity by the cells and their supernatant in the different media formulations. When compared with FBS-grown cells, hPL- and Ch-R-expanded IFP-MSCs (cell group) strongly degraded SP, an effect also present with their supernatant (less pronounced, though). A diagram on the top of each group represents the source of the samples obtained for the measurements. CM, conditioned media; Exo SP, exogenously added SP; [SP] = SP concentration. Figure 6B illustrates CD10 immunolocalization was detected in IFP-MSCs as a concentrated punctate signal (red) around cells and highly present in hPL- and Ch-R-expanded IFP-MSCs (blue nuclei). Figure 6C shows fibrinogen matrix (green) formed within hPL cultures immobilized CD 101 vesicles (red) released from IFP-MSCs (blue nuclei). All experiments were performed independently (n = 2), and data are presented as scatter plots with mean ± SD. *P < .05. Ch-R, chemically reinforced medium; FBS, fetal bovine serum; hPL, human platelet lysate; IFP, infrapatellar fat pad; MSC, mesenchymal stem cell.

Figure 7 illustrates IFP-MSCs expanded in either hPL or Ch-R effectively reduced synovitis and IFP fibrosis in vivo. Hematoxylin and eosin (H&E) staining (top 2 panels), Masson trichrome staining (middle panel), and substance P immunolocalization (lower 2 panels) in sagitally sectioned knees of representative rats for healthy control and those injected with only MIA or both MIA and IFP-MSCs with different degrees of CD10 positivity. When compared with the MIA-only group, which showed significant synovitis and IFP fibrosis with cellular infiltrates, a striking correlation was found between magnitude of CD 10 presence in IFP-MSCs and the effect of reducing those structural changes after 4 days of a single intra-articular IFP-MSC injection. Despite an overall significant reduction in synovitis and IFP fibrosis with all treatments, the immunoselected CD101 group expanded in regulatory-compliant medium showed the strongest therapeutic effect, whereas the CD 10- re- expanded and noninduced FBS group had the poorest outcomes. The other groups (noninduced and primed IFP-MSCs expanded in regulatory-compliant medium) were effective, even at a lower dose (1/lOth). SP1 fibers in areas of active inflammation and fibrosis showed dramatic reduction for the synovium and the body of the IFP in groups similarly correlated with the degree of CD10 positivity. Of note, almost no signal was detected in healthy control rats, supporting the high specificity of the SP signal. Arrows in the top panel indicate areas of synovitis. Ch-R, chemically reinforced medium; FBS, fetal bovine serum; hPL, human platelet lysate; IFP, infrapatellar fat pad; IHC, immunohistochemistry; MIA, monoiodoacetate; MSC, mesenchymal stem cell; SP, substance P.

Figure 8 illustrates CD10 expression levels in crude and CD10 immunomagnetically sorted non-induced and TIC-primed IFP-MSC. Crude naive FBS showed low and variable CD 10 levels that were significantly enriched upon culturing in regulatory-complaint media in all cell groups tested (crude and CD 10 immuno-selected without and with TIC -priming). All experiments were performed independently (n=3).

Figure 9 illustrates the results of real time quantitative PCR (RT qPCR) showing expression of inflammatory cytokine with relative fold changes calculated using the FBS-grown data as reference (set up as 1). Compared to non-induced hPL, FBS, and Ch-R IFP-MSC.

Figure 10 illustrates protein association network of non-induced and TIC-primed hPL and Ch-R expanded IFP-MSC. STRING analysis of the proteins with statistical differences between hPL/Ch-R and FBS in naive or TIC- priming conditions (total of 41) was performed using all available interaction sources and 0.4 as a confidence interaction score. K-means algorithm revealed high protein-protein interaction (PPI) enrichment (p-value <1.0e-16) with all GFs clustered into 3 groups indicated with different colors. All experiments were performed independently (n=2).

Figures 11A- 11B illustrate protein interactomes of non-induced and TIC-primed hPL and Ch-R expanded IFP-MSC. Figure 11A and 11B show biological processes and KEGG/reactome pathways analyses revealed different type and number of proteins affected in naive and TIC-primed IFPMSC. Figure 11B shows biological processes and interactome pathways analysis of the three different groups compared showed similar protein involvement except few differences. Radar graph percentages represent the number of proteins involved in a specific pathway/ function, related to the total amount of proteins differentially expressed between hPL or Ch-R and FBS. All experiments were performed independently (n=2).

Figures 12A-12C show CD 10 immunophenotype in IFP-MSC and BMMSC pre- and post-ΊΊ and TIC priming and hPL expansion. CD 10 expression assessed by flow cytometry in naive and TI/TIC-primed IFP-MSC (Figure 12A), hPL-expanded IFP-MSC (Figure 12B) and naive and TI/TIC-rimed BM-MSC (Figure 12C). Individual donors in each hMSC type are presented with distinctive shapes and color tones to allow intra-donor comparisons between naive and both primed methods, as well as between DMEM/FBS and hPL expansion of IFP-MSC.

Figure 13 illustrates CD 10 surface expression in various MSC types cultured in regulatory-complaint condition. CD10 expression in various MSC types CD90 (>95%) and CD34 (<5%) as known markers UC-MSC (human umbilical cord - Wharton’s Jelly) BM- MSC (human bone marrow) IFP-MSC (human infrapatellar fat pad). All MSC types were grown in human platelet lysate (hPL) medium which is a regulatory-complaint medium for further in vivo therapeutics. All MSC types (UC, BM, IFP) at passage 3 grown with hPL medium demonstrated MSC related phenotypic profile CD34-CD90 ⁺ CD 146 ⁺ showing also variable expression levels of ACE2 and CD142 expression. Specifically, CD10 showed higher expression levels in UC- and IFP-MSC compared to BM-MSC.

Figure 14 illustrates CD 10 mechanism of action in Renin- Angiotensin system.

Figure 15 illustrates that MSC-bound and -released CD 10 strongly degraded Angiotensin 1 in vitro. All MSC types tested efficiently degrade Angiotensin 1 (ANG1) via both the released and cell-bound CD 10 protein. Moreover, CD 10 expression was paralleled with the significant reduction in ANG1 levels exercised by all naive MSC populations, suggesting a CD 10-dependent ANG1 degradation. The abrogation of this effect after inhibiting the enzymatic activity of CD 10 with thiorphan (CD 10 inhibitor) strongly supports this statement. Importantly, CD 10 released from MSC can equally or in some cases stronger (UC MSC) degrade ANG1 compared to the CD10 cell-bound.

Figure 16 illustrates the MSC exosomal isolation and characterization strategy disclosed in particular embodiments herein. MSC exosomes were isolated by ultracentifugation and CD63-based magnetic enrichment. NanoSight analysis revealed exosomes of size <200nm that are stained strongly positive (90±10%) for the exosomal surface marker CD9 indicating their purity. Figure 17 illustrates that CD10 actions are mediated via both CD 10-bound exosomes and soluble CD 10 protein. IFP MSC grown in ‘traditional’ medium supplemented with fetal bovine serum (FBS) [DMEM/ 10%FBS] or regulatory-complaint media [(human platelet lysate (hPL) and Chemically-reinforced medium (Ch-R)] release CD 10 protein in both naive and pro-inflammatory/pro-fibrotic conditions (TNFα/IFNγ/CTGF). Produced CD10 protein is released into the culture microenvironment packaged as exosomal cargo and as soluble protein.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure generally relates to methods for preparing human mesenchymal stem cells (hMSCs) that express high CD 10 phenotypes. The present disclosure further relates to methods of treating local inflammation, fibrosis, and/or musculoskeletal pain using hMSCs that express high CD10 phenotypes.

The methods for preparing human mesenchymal stem cells described herein advantageously provide hMSCs with increased proliferative, phenotypic, differentiation, secretory and functional profiles directly related to consistently efficient Substance P (SP) degradation in vitro and in vivo, even at significantly lower cell doses. For example, the human mesenchymal stem cells described herein allow for a significantly-reduced cell dose (1/10 ^th of the injected cells) for the generation of efficient therapeutic outcomes. Translated into a clinical protocol, this would reduce the required number of IFP-MSC injected into numbers that can be generated in vitro in a shorter period of time. Taking into account the current standards of using ~20-50 x 10 ⁶ MSC, the resulting protocol would require only -2-5 x 10 ⁶ . Thus, the human mesenchymal stem cells possessing high CD 10 described herein can be used to effectively suppress inflammation and fibrosis.

As utilized in accordance with the present disclosure, unless otherwise indicated, all technical and scientific terms shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

In some embodiments, disclosed herein is a method of preparing human mesenchymal stem cells (hMSCs) that express CD 10 at a level equal or greater than 70% positivity overall, the method comprising: culturing hMSCs in a xeno-free formulation; and thereby obtaining hMSCs having CD 10 expression at a level equal or greater than 70% positivity overall.

CD 10, also known as neprilysin, is a surface neutral endopeptidase expressed in multiple cells, including MSC and specifically IFP-MSCs. The anti-inflammatory effects of CD10 have long been recognized in other systems, while its presence in IFP-MSCs seems to be critical for efficient SP degradation, both in vitro and in vivo.

In particular embodiments, the methods disclosed herein produce MSCs that express CD10 at a level equal or greater than 30%, 40%, 50%, 60%, 70%, 80% or 90% positivity overall. For example, an MSC preparation according to the present disclosure is an MSC preparation that is 70% homogeneous (i.e., 70% positivity overall) with respect to being CD 10 positive. Thus, in particular embodiments, the methods disclosed herein produce MSCs that express a high CD 10 phenotype (i.e. express CD 10 at a level equal or greater than 50%, 60%, 70%, 80% or 90% positivity overall). The high phenotypic expression of CD 10 of the MSCs disclosed herein is desired trait during cell-based product manufacturing processes. Based on the correlation between CD 10 positivity and therapeutic outcome, levels ~70% or more indicate a high likelihood of an effective therapeutic outcome.

In particular embodiments, hMSCs that express CD 10 at a high level are obtained using flow cytometry. In particular embodiments, hMSCs that express CD 10 at a level equal or greater than 70% positivity overall are obtained using flow cytometry. In particular embodiments, hMSCs that express CD 10 at a level equal or greater than 80% positivity overall are obtained using flow cytometry. In particular embodiments, hMSCs that express CD 10 at a level equal or greater than 90% positivity overall are obtained using flow cytometry.

In some embodiments, culturing of MSCs is carried out in xeno-free medium. As used herein the term "xeno-free" refers to an absence of direct or indirect exposure to nonhuman animal components. In particular embodiments, the xeno-free formulations for MSC culturing comprises human platelet lysate (hPL) or chemically-reinforced media (Ch-R). Advantages of xeno-free medium include the absence of potential contaminants and improved consistency in both performance and quality of the culture medium.

In particular embodiments, culturing hMSCs comprises culturing hMSCs at 5 % (v/v) CO2 until the hMSCs are 80% confluent; passaging the hMSCs at a 1:5 ratio to Passage 3; and detaching the hMSCs. In some embodiments, the hMSCs are cultured at 37 degrees Celsius.

Cell priming includes preparing cells for some specific function or lineage-specific differentiation, including but not limited to cell activation, molecular signaling, genetic or epigenetic modifications, and morphology/phenotype changes. In some embodiments, the hMSCs are exposed to a proinflammatory/profibrotic environment (i.e., priming). In particular embodiments, priming comprises exposing the hMSCs to TNFα, IFNγ, and/or CTGF to enhance their immunomodulatory effects. In some embodiments, the cells are primed with 15 ng/ml TNFα, 10 ng/ml IFNγ, and 10 ng/ml CTGF). In some embodiments, the cells are primed for 48 hours or 72 hours. The time difference is based on an attempt to mimic the cascade of events that lead from pure inflammation (TI) to a more fibrotic response (TIC), as a sequential tissue response (synovitis followed by IFP fibrosis).

In some embodiments, where CD 10 is present in exosomes derived from hMSCs and/or freely released by the cells to the environment.

In particular embodiments, the hMSCs are derived from postnatal adipose-, infrapatellar fat pad-, postnatal bone marrow-, postnatal endometrial-derived, perinatal origin umbilical cord-, or perinatal origin placenta tissues.

The term "subject" is intended to include human and non-human animals, particularly mammals.

In some embodiments, the methods disclosed herein relate to treating a subject for local inflammation, fibrosis and/or musculoskeletal pain. In some embodiments, inflammation and/or fibrosis is arthritis, osteoarthritis, synovitis, tendinitis/tendinopathy, other musculoskeletal inflammation, neuroinflammation, or any tissue wound and/or inflammation. In some embodiments, the musculoskeletal pain is a result of arthritis, osteoarthritis, synovitis, tendinitis/tendinopathy, other musculoskeletal inflammation, neuroinflammation, or any tissue wound and/or inflammation

The terms "treatment" or "treat" as used herein refer to therapeutic treatment. Those in need of treatment include subjects having local inflammation, fibrosis and/or musculoskeletal pain. In some embodiments, the methods disclosed herein can be used to treat local inflammation, fibrosis and/or musculoskeletal pain.

The terms "administration" or "administering" as used herein refer to providing, contacting, and/or delivering a compound or compounds by any appropriate route to achieve the desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e g., intravenous, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, via inhalation, and implants.

The terms "pharmaceutical composition" or "therapeutic composition" as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a subject. In some embodiments, the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of human mesenchymal stem cells. The terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refer to one or more formulation materials suitable for accomplishing or enhancing the delivery of the human mesenchymal stem cells of the disclosure.

When used for in vivo administration, the formulations of the disclosure should be sterile. The formulations of the disclosure may be sterilized by various sterilization methods, including, for example, sterile filtration or radiation. In one embodiment, the formulation is filter sterilized with a pre-sterilized 0.22-micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in "Remington: The Science & Practice of Pharmacy," 21st ed., Lippincott Williams & Wilkins (2005).

The formulations can be presented in unit dosage form and can be prepared by any method known in the art of pharmacy. Actual dosage levels of the active ingredients in the formulation of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject (e.g. , "a therapeutically effective amount"). Dosages can also be administered via continuous infusion (such as through a pump). The administered dose may also depend on the route of administration. For example, subcutaneous administration may require a higher dosage than intravenous administration.

Without limiting the disclosure, a number of embodiments of the disclosure are described below for purpose of illustration.

EXAMPLES

The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only, and should not be construed as limiting the scope of the invention in any way.

MATERIALS & METHODS

Isolation, Culture and Expansion of IFP-MSC and BM-MSC

IFP-MSC were isolated from IFP tissue obtained from de-identified, non-arthritic patients (n=8, two males 16 and 26 years-old and six females, 22, 26, 31, 44, 46, and 53 years-old) undergoing elective knee arthroscopy. IFP tissue (<20ml) was mechanically dissected and washed repeatedly with Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma), followed by enzymatic digestion using 235 U/ml Collagenase I (Worthington Industries, Columbus, OH) diluted in DPBS and 1% bovine serum albumin (Sigma) for 2 hours at 37°C with agitation. Cell digests were inactivated with complete media [DMEM low glucose GlutaMAX (ThermoF isher Scientific, Waltham, MA) +10% fetal bovine serum (FBS; VWR, Radnor, PA], washed and seeded at a density of lxlO ⁶ cells/175 cm ² flask in three different complete media: hPL, Chemically-defined, DMEM/ 10%FB S . Complete hPL medium was prepared by supplementing DMEM low glucose GlutaMAX with 10% hPL solution (PL Bioscience, Aachen, Germany) and 0.024 mg/ml xeno-free heparin (PL Bioscience, Aachen, Germany). Complete chemically reinforced medium was prepared by mixing Mesenchymal Stem Cell Growth Medium 2 with supplement provided according to manufacturer’s instructions (PromoCell, Heidelberg, Germany).

All MSC were cultured at 37°C 5% (v/v) CO2 until 80% confluent (denoted as P0), then passaged at a 1:5 ratio until P3 detaching them with TrypLE™ Select Enzyme IX (Gibco, ThermoFisher Scientific) and assessing cell viability with 0.4% (w/v) Trypan Blue (Invitrogen, Carlsbad, CA).

Clonogenic assay

Passage 3 IFP-MSC (n=5) were seeded in 100mm culture plates in duplicate at a density of 10 ³ cells/plate in all three culturing conditions. On day 10, colony-forming unit fibroblasts (CFU-Fs) were manually enumerated after cytochemical staining with 0.01% Crystal Violet (Sigma).

Cell growth kinetics measurement

Passage 3 IFP-MSC (n=5) were seeded in 6- well plates at a density of 10 ⁴ cells/well in all three culturing conditions. Growth curves were generated from bright field image obtained using IncuCyte® Live Cell Analysis System with IncuCyte ZOOM® software (Essen Bioscience, Ann Arbor, MI) to quantify cell confluence as a percentage for a 10-day period.

Proinflammatory/Profibrotic Priming

With TNFα, IFNγ, and CTGF Passage 3 IFP-MSC (n=3) expanded in all three culturing conditions were subsequently primed with TIC inflammatory/ fibrotic cocktail (15 ng/ml TNFα, 10 ng/ml IFNγ, 10 ng/ml CTGF) for 72h. Non-induced and TIC-induced cultures were evaluated for their phenotypic profiles by flow cytometric analysis.

Immunophenotype Flow cytometric analysis was performed on P3 naive (n=3) and TIC-primed IFP-MSC expanded in all three culturing conditions. 2.0 x 10 ⁵ cells were labelled with monoclonal antibodies specific for: CD 10, CD44, CD56, CD73, CD90, CD 105 (Biolegend, San Diego, CA), CD 146, LepR (Miltenyi Biotec, Auburn, CA), CD 166, CD271, NG2, HLA-DR (BD Biosciences, San Jose, CA), CD200, CXCR4 (Invitrogen) and the corresponding isotype controls. All samples included a Ghost Red Viability Dye (Tonbo Biosciences, San Diego, CA). Data were acquired using a Cytoflex S (Beckman Coulter, Brea, CA) and analysed using Kaluza analysis software (Beckman Coulter). Quantitative real-time PCR (qPCR)

RNA extraction was performed using the RNeasy Mini Kit (Qiagen, Frederick, MD) according to manufacturer’s instructions. Total RNA (lpg) was used for reverse transcription with Superscript™ VILO™ cDNA synthesis kit (Invitrogen).

10 ng cDNA was analyzed by qPCR using QuantiFast SYBR Green qPCR kit (Qiagen) and a StepOne Real-time thermocycler (Applied Biosystems, Foster City, CA). For each target, human transcript primers were selected using PrimerQuest (Integrated DNA Technologies, San Jose, CA) (Table 1). All samples (n=3) were analyzed in triplicate. Mean values were normalized to GAPDH, expression levels were calculated using the method and represented as the relative fold change of the primed cohort to the naive (=1).

A pre-designed 28 gene Taqman low density cytokine array (TLDA, Applied Biosystems) was performed (n=3) using 1000 ng cDNA per IFP-MSC sample and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC). Data analysis was performed using DataAssist software v2.0 (Applied Biosystems, LLC). Mean values were normalized to GAPDH, expression levels were calculated using the method. Values were represented in a heatmap of transcript expression levels (with 34 cycles cut-off point) using Pearson’s correlation distance method and complete linkage clustering method between the different samples. Also, values were represented in a separate fold change heatmap as the relative fold change of the hPL or Chemically-reinforced medium to DMEM/10%FBS medium (reference sample) expanded IFP-MSC sample/X reference sample).

Trilineage differentiation

Tripotentiality was evaluated in IFP-MSC (n=5) expanded in all three culturing conditions. Chondrogenic differentiation (0.25x10 ⁶ IFP-MSC/pellet) was induced for 21 days with serum- free MesenCult-ACF differentiation medium (STEMCELL Technologies Inc, Vancouver, Canada). Harvested pellets were cryosectioned and 6- μm frozen sections stained with 1 % toluidine blue (Sigma) for semi-quantitative assessment of chondrogenic differentiation. Osteogenic differentiation (5000 IFP-MSC/cm ² ) was induced for 21 days with StemPro Osteogenesis differentiation kit (ThermoFisher Scientific) and semi-quantitative assessment of mineralization was performed using 1% Alizarin Red S (Sigma). Adipogenic differentiation (40000 IFP-MSC/cm ² ) was induced for 15 days with StemPro Adipogenesis kit (ThermoFisher Scientific) and semi-quantitative assessment of lipid accumulation within the cell cytoplasm was performed using 0.5% Oil Red (Sigma). Quantitative real-time PCR (qPCR) (n=3) to evaluate transcript expression for all three differentiation lineages tested is described above.

Secretome analysis

Protein array of 41 growth factors (GFs) (RayBio ^® C-Series, RayBiotech, Peachtree Comers, GA) was used to determine secreted levels obtained from IFP-MSC expanded in all three culturing conditions. For each population, 1 mL of conditioned media obtained from 2 donors, was prepared and used for each assay following the manufacturer’s instructions. Data shown represent 40 sec exposure in FluorChem E chemiluminescence imaging system (ProteinSimple, San Jose, CA). Results were generated by quantifying the mean spot pixel density of each array using protein array analyzer plugin using Image J software (Fiji/ImageJ, NIH website). The signal intensities were normalized with the background whereas separate signal intensity results represent the average pixel density of two spots per protein. The signal intensity for each protein spot is proportional to the relative concentration of the antigen in the sample. Pathway analysis

Putative interactomes were generated by Search Tool for Retrieval of Interacting Genes/Proteins (STRING 11.0; available from: http://string-db.org) database using interaction data from experiments, databases, neighborhood in genome, gene fusions, cooccurrence across genomes, co-expression and text-mining. An interaction confidence score of 0.4 was imposed to ensure high interaction probability. K-means clustering algorithm was used to organize proteins into 3 separate clusters per condition tested, discriminated by colors. Venn diagrams were used to demonstrate all possible relations between naive and/or TIC-primed IFP-MSC cultured in all three different conditions for the significantly (p<0.05) altered proteins. Functional enrichments related to biological process, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and reactome pathways were presented in radar graphs for all conditions tested.

Substance P in vitro assay

Parameter Substance P competitive immunoassay (R&D Systems, MN) was used to quantify the levels (in pg/ml) of endogenous and exogenously-added SP to culture-expanded IFP-MSC in all three culturing conditions ( 10 ⁵ /well, 12-well; n=2 per culturing condition), following manufacturer’s instructions. SP was then quantified in centrifuged (1500 rpm; 5 minutes) conditioned media (in technical triplicates run in duplicates within the membrane) obtained from IFP-MSC cultures: i) in baseline cultures (i.e., endogenous MSC-derived SP); ii) after exogenous addition of substance P (834 pg/ml) for 35 minutes to the cell-free supernatant (i.e., supernatant group); and iii) after addition of SP (834 pg/ml) for 35 minutes to the cells in fresh medium (i.e., cells group). SP final levels were determined by subtracting measured optical densities of individual wells at 450nm and 540nm (SpectraMax M5 spectrophotometer, Molecular Devices, San Jose, CA), and converted into concentrations using the reference standard curve run with the assay, and contrasted to samples with only exogenously-added SP to the medium (i.e., no cells and no supernatant).

CD10 immunolocalization

IFP-MSC groups were fixed with 4% paraformaldehyde, washed with PBS, followed by incubation with tris buffered saline (TBS; Sigma- Aldrich) containing 0.05% Triton X-100 solution (Sigma- Aldrich) for 30 minutes. Groups were then incubated with blocking solution composed of TBS with 10% normal goat serum (NGS) for 1 hour. Goat anti-human CD 10 polyclonal antibody (R&D Systems) was prepared in TBS containing 1% NGS and added to samples for 1 -hour incubation at room temperature with gentle agitation. Samples were washed with TBS and incubated for 1 hour with secondary antibody containing AlexaFluor594 conjugated rabbit anti-goat IgG antibody combined with DAPI (Thermo Fisher Scientific) at room temperature with gentle agitation in the dark. TBS was used to wash cells, and microscope images were acquired using Leica DMi8 microscope with Leica X software (Leica).

CD10 immunomagnetic separation

IFP-MSC were immunomagnetically separated based on CD 10 expression and further expanded in vitro. Briefly, IFP-MSC were suspended in staining buffer containing PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA and then incubated with biotinylated anti-human CD10 (Miltenyi Biotech, Inc., Auburn, CA) at RT for 20 minutes. Invitrogen™ CELLection Dynabeads™ Biotin Binder Kit (Thermo Fisher Scientific) were used according to manufacturer’s instructions for magnet-activated cell sorting resulting in the POS and NEG subpopulations. POS and NEG BM-MSC were directly plated in culture to obtain relevant numbers for the in vivo study yielding the CD10 ^bright and CD10 ^dim IFP-MSC populations.

Mono-iodoacetate model of acute synovial/IFP inflammation

Sixteen (#16) 10- week old Sprague Dawley rats (8 males and 8 females; mean weight 250 g and 200 g, respectively) were used. Acute synovial/IFP inflammation was generated by intra-articular injection of 1 mg of mono-iodoacetate (MIA) in 50 μΐ of saline in the right knee. Briefly, under isoflurane inhalation anesthesia rat knees were flexed 90° and MIA was injected into the medial side of the joint with a 27G needle using the patellar ligament and articular line as anatomical references. Three (3) days later, a single intra-articular injection of 500,000 cells for groups: naive crude, naive CD10 ⁺ (bright and dim), and 500,000 or

50,000 cells for group: TIC-induced IFP-MSC in 50 μl of Euro-Collins solution (MediaTech) was performed (similar injection technique), having as control: 1) rats receiving MIA but not IFP-MSC (Only MIA group); and 2) healthy rats receiving only IFP-MSC (Only IFP-MSC group). Animals were sacrificed 4 days after IFP-MSC injection (d7 total).

Substance P immunolocalization

Rat knee joints were harvested by cutting the femur and tibia/fibula 1 cm above and below the joint line, muscles were removed and joints were fixed with 10% neutral buffered formalin (Sigma- Aldrich) for 14 days at room temperature. Knee joints were decalcified, cut at sagittal plane in half, embedded in paraffin and serial 4 μm sections were obtained. Hematoxylin and Eosin (H&E) staining was performed to evaluate the structure and morphology of knee joints. Substance P and anti-human Mitochondria immunolocalization were determined by immunofluorescence and immunohistochemistry staining, respectively.

For anti-substance P immunofluorescence staining, sections were incubated with lx citrate buffer solution at 60 °C overnight for antigen retrieval, permeabilized with lx PBS + 0.2% Triton X-100 for 20 minutes at room temperature, and incubated with blocking buffer (lx PBS + 0.1% Triton X-100 with 10% rabbit serum) for 1 hour at room temperature. In between different treatments sections were washed with lx PBS. Rabbit anti-rat substance P polyclonal antibody (Millipore) was prepared in blocking buffer (1 : 100) and sections were incubated at 4 °C overnight. Sections were washed with lx PBS + 0.01% Triton X-100 and incubated for 1 hour with secondary antibody containing Alexa Fluor594 conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific) at room temperature. Controls were incubated with secondary antibody only. All sections were rinsed with lx PBS, mounted in prolong gold antifade reagent with DAPI (Invitrogen), and microscope images were acquired using Leica DMi8 microscope with Leica X software (Leica).

MSC-derived Exosomes isolation and characterization

Conditioned media from IFP-MSC groups cultured for two days in exosome-depleted media (DMEM/ 10%FB S or DMEM/ 10%hPL or chemically-reinforced) were filtered through a 0.22 μm filter to remove debris and large vesicles. Subsequently collected and differentially centrifuged for 2,000xg for 10 min, 10,000xg for 30 min, and ultracentrifuged for 120,000xg for 16hr using a sucrose gradient to increase purity. After ultracentrifugation two fractions were collected: i) the supernatant and ii) the exosomal pellet. The supernatant was concentrated at 4,000xg for 30 min using Amicon Ultra- 15 centrifugal filter devices (Millipore) and then stored at -20°C until further experimentation . Pre-enriched exosomal pellets were washed and incubated overnight at 4°C with the Dynabeads®-based Exosome- Human CD63 Isolation/Detection Reagent (Thermo Fisher Scientific). Using a magnetic separator, exosome preparations were further purified.

Exosomes collected from each group were assessed for multimodal parameters for biophysical and biochemical characterization: quantity and size determination through nanoparticle tracking analysis (NTA) (NanoSight NS300, Malver). Exosomal identity profiling was complemented by staining with CD9 antibody (Invitrogen) to validate their presence in CD63 ⁺ -gated particles by flow cytometry (CytoFLEX, Beckman Coulter). Data were acquired using a Cytoflex S (Beckman Coulter, Brea, CA) and analysed using Kaluza analysis software (Beckman Coulter).

CDlO/neprilysin in vitro quantification assay

Neprilysin SimpleStep ELISA kit (Abeam, MA, USA) was used to quantify CD10 protein levels (pg/ml) in both supernatant (soluble protein) and exosomes (exosome-bound protein) collected after ultracentrifugation of MSC conditioned media (DMEM/ 10%FB S or DMEM/ 10%hPL or chemically-reinforced, n=3 per cohort), following manufacturer’s instructions. Prior in vitro assessment collected exosomes were lysed using RIP A buffer ((Thermo Fisher Scientific). CD10 protein levels were quantified in centrifuged (2,000g; 10 minutes) conditioned media (run in duplicates) obtained from MSC cultures in all conditions. Levels were determined by measuring the fluorescence (450nm) of individual wells in endpoint mode (SpectraMax M5 spectrophotometer, Molecular Devices, San Jose, CA, USA). CD 10 protein levels were normalized to total protein per cohort.

Angiotensin I degradation in vitro assay

Angiotensin I (Angl) competitive immunoassay (Abeam, MA, USA) was used to quantify the levels (in pg/ml) of endogenous and exogenously-added Angl to culture- expanded MSC or human umbilical vein endothelial cells (HUVEC) (10 ⁵ /well, 12-well; n=2 per culturing condition), following manufacturer’s instructions. Angl was then quantified in centrifuged (1500 rpm; 5 minutes) conditioned media (in technical triplicates run in duplicates within the membrane) obtained from the cultures: i) in baseline cultures (i.e., endogenous cell-derived Angl); ii) after exogenous addition of Angl (2500 pg/ml) for 20 minutes to the cell-free supernatant (i.e., supernatant group); and iii) after addition of Angl (2500 pg/ml) for 20 minutes to the cells in fresh medium (i.e., cells group). Angl final levels were determined by subtracting measured optical densities of individual wells at 450nm and 580nm (SpectraMax M5 spectrophotometer, Molecular Devices, San Jose, CA), and converted into concentrations using the reference standard curve run with the assay, and contrasted to samples with only exogenously-added Angl to the medium (i.e., no cells and no supernatant).

Statistical analysis

Normal distribution of values was assessed by the Kolmogorov-Smimov normality test. Statistical analysis was performed using paired and unpaired Student’s /-test for normally distributed data and Wilcoxon (for paired data) or Mann Whitney (for unpaired data) test in presence of a non-normal distribution; one-way ANOVA was used for multiple comparisons. All tests were performed with GraphPad Prism v7.03 (GraphPad Software, San Diego, CA). Level of significance was set at p < 0.05. Data used for the statistical analyses is indicated in the figure legends, overall corresponding to three independent experiments from different MSC donors (n=3), unless specified.

Example 1: IFP-MSCs Show Superior Growth Kinetics When Expanded in Either hPL or Ch-Rvs FBS Growth kinetics and clonogenic potential of IFP-MSCs expanded in either hPL or Ch- R were analyzed and compared with IFP-MSCs expanded in FBS alone. All 3 culture conditions demonstrated variable growth kinetics for 8 days. In detail, hPL-expanded IFP- MSCs showed an increased growth rate, reaching 85% confluency as compared with the FBS cultures, which had only 62% confluency on day 8. Ch-R medium showed the most potent growth rate versus the other 2 culturing conditions, entering confluency (>85%) only 4 days after seeding in vitro (Figures 1A and IB). However, clonogenic capacity showed a higher trend in FBS expanded IFP-MSCs (mean ± SD, 296 ± 77 CFU-Fs) as compared with hPL and Ch-R (252 ± 43 and 233 ± 45 CFU-Fs, respectively) (Figure 1C).

Example 2: IFP-MSCs Expanded in Either hPL or Ch-R Have Privileged

Immunophenotvpic and Molecular Profiles

In all 3 culture conditions without TIC priming (i.e., noninduced), the common MSC- defining markers (CD44, CD73, CD90, CD105, CD 166) showed a similar expression pattern (>90% positivity), while LepR and CD56 were absent, and CD271 and CD200 had low to negative expression. NG2 showed high expression (approximately 90%) only in Ch-R (Figure 2A). Most important, markers related to MSC functionality toward an immunomodulatory and antifibrotic phenotype were significantly increased in noninduced IFPMSCs by solely culturing in either hPL or Ch-R. In hPL expanded IFP-MSCs, the mean expression of CD146 (44.1% ± 18.7%), CD10 (88.1% ± 6.2%), and CXCR4 (57.7% ± 17.01%) was 8-, 7-, and 5-fold enriched as compared with FBS medium cultures. Proinflammatory/ profibrotic ex vivo priming with TIC for 72 hours resulted in CD 146 and CD10 expression enrichment in all 3 culturing conditions (Figure 2B). IFP-MSCs expanded in hPL with TIC priming increased CD 10 and CD 146 expression by 1- and 1.6-fold, respectively. Even though CD10 expression increased by 1.8-fold in cultures with FBS plus TIC priming, these levels were still lower than in IFP-MSCs expanded in hPL without TIC priming (74.8 ± 3.6% vs 88.9 ± 4.1%). CD90 had stable expression (>90%) whereas, HLA- DR expression was sharply increased in all cultures with TIC priming. Example 3: IFP-MSCs Expanded in Either hPL or Ch-R Can Effectively Differentiate

Toward Bone, Fat, and Cartilage In Vitro

The tripotential capacity of IFP-MSCs to undergo osteogenic, chondrogenic, or adipogenic differentiation was comparable in FBS, hPL, and Ch-R. However, qualitative assessments showed that hPL- and Ch-R-expanded IFP-MSCs deposited higher levels of minerals on the monolayer surface in osteogenesis, had higher lipid vacuole accumulation within the cytoplasm in adipogenesis, and displayed stronger cartilage-specific metachromasia for glycosaminoglycans produced in chondrogenesis as compared with FBS- expanded IFP-MSCs (Figure 3A). Quantitatively at the molecular level, hPL- and Ch-R- expanded IFP-MSCs showed significantly (P < .05) higher expression levels for the osteogenic gene OMD, adipogenic gene FABP4, and chondrogenic genes ACAN and COMP as compared with FBS expanded IFP-MSCs (Figure 3B), indicating their increased maturity during the different differentiation schemes. Example 4: IFP-MSCs Expanded in Either hPL or Ch-R Have a Reduced Baseline

Inflammatory Transcrmtome vs FBS

To establish a baseline inflammation-related molecular signature for IFP-MSCs expanded in either hPL or Ch-R (noninduced), a multiplex transcriptional assessment was performed. IFP-MSCs grown in all 3 conditions showed an overall low-to-negative expression level for most inflammation-related cytokines tested (Figure 4A). Furthermore, when compared with FBS-grown cells, hPL- and Ch-R-expanded cells had reduced expression of most cytokines, with IL-18 significantly downregulated in both versus IL-Ιβ and IL-12a in only Ch-R (Figure 4B; Figure 9). IL-8 was the only cytokine with significantly (P <05) higher expression levels in hPL- and Ch-R-expanded IFP-MSCs as compared with the FBS reference sample, with 6.8- and 8.6-fold expression levels, respectively. Other molecules, such as IL- 6, TNF- α, and IL-1 α, showed divergent changes with hPL and Ch-R.

Example 5: IFP-MSCs Expanded in Either hPL or Ch-R Strongly Secrete Reparative

Factors Without Priming

Noninduced hPL-expanded IFP-MSCs showed overall higher secretion of GFs when compared with FBS medium (Figure 4B). Of the 41 GFs analyzed, IFP-MSCs expanded in hPL and Ch-R without priming secreted 29 and 24 growth factors, respectively, at significantly (P<0.05) higher levels compared with FBS. Upon TIC priming, hPL- and Ch- R- expanded IFP-MSCs showed increased secretion of various proteins. However, the overall number of proteins secreted was reduced, as TIC priming also affected the secretion of FBS-expanded IFP-MSCs. After TIC priming, hPL and Ch-R-expanded IFP-MSCs secreted 16 and 18 GFs, respectively, at significantly (P <05) higher amounts. Simultaneous Venn diagram representation of all 4 secretory profiles (noninduced and primed hPL and noninduced and primed Ch-R), when compared with their FBS counterparts (noninduced and primed), revealed a core of 7 GFs (EGF R, HGF, IGFBP-2, M-CSF R, PDGF-AA, SCF R, YEGF) that are commonly increased in those 4 groups. Interestingly, after priming, 13 of 16 for hPL and 14 of 18 for Ch-R were shared with the secretory profiles in their noninduced state.

In protein association network analysis, GFs appeared interconnected at least through 1 association, while K-means clustering networks showed high protein-protein interaction enrichment (P <.0000000000000001 ) and an average local clustering coefficient >0.7, indicating that the proteins used were at least partially biologically connected (Figure 10). In noninduced IFP-MSCs, hPL and Ch-R secretory profiles showed similar biological process involvement. Priming boosted the 5 of 6 biological processes of Ch-R-expanded IFP-MSCs versus only 3 of 6 in hPL-expanded IFP-MSCs (Figure 11 A, left radar chart). The involvement in those biological processes was reflected in specific signaling pathways presented in the KEGG reactome (Figure 11 A, right radar chart). In noninduced IFP-MSCs, hPL IFP-MSCs showed higher enrichment of the cytokine-cytokine receptor interaction and Jak-STAT (2 of 6) pathways, whereas Ch-R IFPMSCs showed higher enrichment of the MAPK, PI3K-Akt, Ras, and Rapl (4 of 6) pathways (Figure 11B).

According to the KEGG database, the Jak-STAT pathway, which is highly enriched in hPL IFP-MSCs, is a principal downstream mechanism for an array of cytokines and GFs and is directly involved in cytokine-cytokine receptor interaction signaling (hsa:04060). Interestingly, in Ch-R IFPMSCs — except the MAPK, PI3K-Akt, and Ras pathways, which are involved in downstream signaling upon cytokine and GF activation — the Rapl pathway, which is involved in cell adhesion, cell-cell junction formation, and cell polarity, is also highly enriched. Most important, priming resulted in further-boosted protein involvement in all signaling pathways for both hPL- and Ch-R-primed IFP-MSCs.

Example 6: IFP-MSCs Expanded in Either hPL or Ch-R Secrete More Factors vs

FBS Plus Priming

To assess the molecular similarities between the regulatory-compliant formulations and FBS plus priming, direct molecular comparisons were made between these groups. Secretory profile analysis revealed that noninduced hPL- and Ch-R-expanded IFP-MSCs shared with FBS plus priming 17 and 31 proteins, respectively. Importantly, except the common proteins shared, noninduced hPL IFP-MSCs showed a boosted secretory profile with 22 additional GFs highly secreted. In total, 14 proteins (AR, FGF-4, G-CSF, GDNF, GM-CSF, HB-EGF, IGFBP- 4, IGF-I SR, IGF-II, NT-4, PDGFRβ, SCF, TGF-α, TGFβ3) were commonly and highly expressed in IFP-MSCs expanded in hPL and Ch-R (Figure 5). Overall, biological processes and interactome pathways analysis of the 3 groups compared (common noninduced hPL vs FBS plus priming, common noninduced Ch-R vs FBS plus priming, common noninduced hPL vs noninduced Ch-R) showed similar protein involvement except few differences (Figure 11B). Interestingly, group 1 showed a higher number of proteins involved in the "regulation of signaling receptor activity" process, whereas group 2 had a higher number of proteins involved in the "positive regulation of cell migration" process (Figure 11 B, left radar chart). However, when compared with group 1 , group 2 showed higher protein involvement in almost all KEGG reactome pathways tested except PI3K-Akt and Jak-STAT pathways (Figure 11B, right radar chart).

Example 7: IFP-MSCs Expanded in Either hPL or Ch-R Show Increased

Functionality In Vitro

Upon exogenous addition of SP (834 pg/mL) in culture, the overall SP levels were significantly (P < 0.05) decreased by the cell group and the supernatant group in all 3 conditions tested (Figure 6A). As compared with FBS, hPL and more so Ch-R induced a reduction of SP, statistically significant in the cells group but not in the supernatant group (for Ch-R). CD 10 immunolocalization was detected in IFP-MSCs as a concentrated punctate signal around cells (Figure 6B), highly present in hPL- and Ch-R-expanded IFP-MSCs directly related to the SP degradation pattern observed after exogenous addition of SP in cultures. Importantly, 3 -dimensional reconstruction of fibrinogen matrix formed in hPL cultures revealed that CD 10 ⁺ vesicles released from IFP-MSCs were immobilized within the 3-dimensional matrix (Figure 6C). This effect can be correlated with the limited capacity of hPL supernatants to degrade SP: an effect that was strongly induced by hPL-expanded IFP- MSCs.

Example 8: IFP-MSCs Expanded in Either hPL or Ch-R Effectively Reverse Synovitis and IFP Fibrosis and Degrade SP In Vivo

A rat model of induced acute synovitis and IFP fibrosis was used to confirm in vivo the degradation of SP and to test the capacity of IFP-MSCs with different levels of CD 10 positivity to reverse synovial and IFP inflammation and fibrosis. In addition to signs of synovitis and early fibrotic changes of the IFP, we confirmed the hyperinnervation by SP- positive sensory fibers 7 days after the intra-articular injection of monoiodoacetate, as compared with healthy knees without inflammatory induction (Figure 7).

When compared with untreated animals, all animals that received IFP-MSCs showed a significant reduction in synovitis and IFP fibrosis 4 days after their administration (Figure 7, marked with arrows and asterisks, respectively). However, differences can be appreciated in the degree of inflammation and fibrosis reversal and SP degradation among animals that received IFP-MSCs with varying CD 10 positivity, underscoring its relevance for an efficient therapeutic effect. Of note, and as support of the inductive CD 10 enrichment with hPL and Ch-R, these formulations rapidly (1 week) turned the low CD 10 positivity of immunomagnetically sorted CD 10- IFP-MSCs (13 ± 14.8) into intermediate/high levels (74.6 ± 7.67) (Figure 8). SP presence was also significantly diminished in rats after 4 days of single intra-articular injection of IFP-MSCs, being more pronounced in peripheral areas of the IFP (close to the synovium), whereas inner parts (IFP body) showed some remaining SP- positive fibers (Figure 7). This SP degradation was uniform across animals that received IFP-MSCs with high levels of CD 10, while the ones that received IFP-MSCs with lower CD10 positivity (IFP-MSCs expanded in FBS) clearly showed suboptimal SP degradation.

Of note, CD10 ⁺ -selected IFP-MSCs showed the strongest reduction of synovitis and IFP fibrosis and SP degradation, while a reduction in the cell dose in 1 of 10 (total of 50,000 cells) still effectively generated therapeutic efficacy, as they were high in CD 10 owing to the priming stimulation.

Example 9: MSC-bound and -released CD10 strongly degraded Angiotensin 1 in vitro

All MSC types tested efficiently degrade Angiotensin 1 (ANG1) via both the released and cell-bound CD10 protein (Figure 15). Moreover, CD10 expression was paralleled with the significant reduction in ANG1 levels exercised by all naive MSC populations, suggesting a CD 10-dependent ANG1 degradation. The abrogation of this effect after inhibiting the enzymatic activity of CD 10 with thiorphan (CD 10 inhibitor) strongly supports this statement. Importantly, CD 10 released from MSC can equally or in some cases stronger (UC MSC) degrade ANG1 compared to the CD 10 cell-bound.

Example 10: MSC exosomal isolation and characterization strategy

MSC-derived exosomes were isolated by ultracentifugation and CD63-based magnetic enrichment. NanoSight analysis revealed exosomes of size <200nm that are stained strongly positive (90±10%) for the exosomal surface marker CD9 indicating their purity (Figure 16).

Example 11: CD10 actions are mediated via both CDlO-bound exosomes and soluble CD10 protein

IFP MSC grown in ‘traditional’ medium supplemented with fetal bovine serum (FB S)[DMEM/ 10%FBS] or regulatory-complaint media [(human platelet lysate (hPL) and Chemically-reinforced medium (Ch-R)] release CD10 protein in both naive and pro- inflammatory/pro- fibrotic conditions (TNFα/IFNγ/CTGF). Produced CD 10 protein is released into the culture microenvironment packaged as exosomal cargo and as soluble protein (Figure 17).

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. Citation or identification of any reference in any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.