BACKGROUND

[0001 ] Cultured endothelial cells (ECs) can find applications in a variety of areas in medicine and biotechnology. These applications include coating of vascular grants and stents, generation of artificial blood vessels and in vitro models for studying (e.g., blood- brain barrier, kidney filter, etc.). They can also be used for a variety of functional assays as part of drug development and testing.

[0002] It would be desirable to be able to provide large quantities of endothelial cells for various applications. Although human umbilical vascular endothelial cells (HUVECs) isolated and expanded from umbilical cords are currently being used for such purposes, such cells isolated from human organs vary from batch to batch. HUVECs are not stable and the phenotype of the cells varies from batch to batch and cell line to cell line.

[0003] In addition, HUVECs can readily de-differentiate and quite rapidly lose their phenotype, as can be observed by loss of specific EC markers. One major reason for the loss of cellular phenotypes of primary cells cultured in vitro is a lack of normal cell- matrix interactions. In vivo, most organized cells such as ECs are positioned as cell monolayers that are tightly anchored to a special matrix (e.g., basement membrane, basal lamina, etc.). The basement membrane contains several specific components such as collagen IV, proteoglycans and laminin proteins. However, this environment is difficult to replicate in a xeno-free fashion.

[0004] Endothelial cells differentiated with current methods from human pluripotent stem cells (hPSCs), either iPSCs (induced pluripotent stem cells) or hESCs (human embryonic stem cells), are not appropriate for use in regenerative medicine in patients as there is not a method for making such cells in chemically defined, fully human cell culture systems. Most current protocols to derive hESCs into ECs use embryoid bodies cultured on undefined matrices such as Matrigel® or feeder cells (e.g., fibroblasts, etc.) as well as a culture medium containing bovine serum. Such cells are not suitable for human treatments or for medical devices used in patients, as they can contain contagious organisms and can be immunogenic. This is a typical effect of using an undefined medium. The resulting cells are not of the highest clinical quality. [0005] Since stem cell derived ECs can be intended for use in humans or on medical devices intended for human therapies, it is of central importance to develop methods that contain no animal-derived materials, are chemically defined, and are reproducible. It would be desirable to develop methods that allow for the differentiation of pluripotent human stem cells under fully defined, xeno-free, and stable batch-to-batch conditions into substantially homogenous differentiated cells, particularly endothelial cells.

BRIEF DESCRIPTION

[0006] Disclosed herein in various embodiments are methods for producing endothelial cells. Generally, these methods comprise plating pluripotent human stem cells on multiple cell culture substrates that contain laminins, and then exposing the stem cells to various differentiation mediums to obtain endothelial cells.

[0007] Disclosed herein are methods for producing endothelial cells, comprising: plating pluripotent human stem cells on a first cell substrate comprising a first laminin selected from the group consisting of laminin-521 or laminin-51 1 ; culturing the cells in a mesoderm induction medium; removing the mesoderm induction medium and culturing the cells in a vascular specification medium; purifying the differentiated cells; replating the differentiated cells on a second cell substrate comprising either (a) laminin-521 , (b) laminin-51 1 , (c) a mixture of laminin-521 and laminin-421 , or (d) a mixture of laminin- 51 1 and laminin-421 ; and culturing the cells in an endothelial amplification medium to obtain the endothelial cells.

[0008] The volume ratio of (i) the laminin-521 or the laminin-51 1 to (ii) the laminin- 421 in the second cell substrate may be from about 1 :2 to about 2:1 .

[0009] Sometimes, the methods further comprise culturing the cells on the first cell substrate in a xeno-free serum-free culture medium until the cells reach from about 40% to about 50% confluency, prior to culturing the cells in the mesoderm induction medium.

[0010] The mesoderm induction medium may contain an activin, a bone morphogenic protein (BMP), and a GSK-3 inhibitor. In particular embodiments, the activin is activin A, the bone morphogenic protein is BMP4, and the GSK-3 inhibitor is CHIR99021 . The activin may be present in an amount of about 5 ng/mL to about 15 ng/mL. The bone morphogenic protein may be present in an amount of about 10 ng/mL to about 30 ng/mL. The GSK-3 inhibitor may be present in a concentration of about 3 micromolar (μΜ) to about 9 μΜ.

[0011 ] The vascular specification medium may contain a vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), a bone morphogenic protein (BMP), and a Notch signaling inhibitor. In particular embodiments, the vascular endothelial growth factor is VEGF ₁₆ 5, the bone morphogenic protein is BMP4, and the Notch signaling inhibitor is DAPT. The vascular endothelial growth factor may be present in an amount of about 25 ng/mL to about 75 ng/mL. The bFGF may be present in an amount of about 5 ng/mL to about 15 ng/mL. The bone morphogenic protein may be present in an amount of about 10 ng/mL to about 30 ng/mL. The Notch signaling inhibitor may be present in a concentration of about 5 μΜ to about 15 μΜ.

[0012] The endothelial amplification medium may contain a vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and a TGF-β inhibitor. In specific embodiments, the vascular endothelial growth factor is VEGF ₁₆ 5, and the TGF-β inhibitor is SB431542. The vascular endothelial growth factor may be present in an amount of about 25 ng/mL to about 75 ng/mL. The bFGF may be present in an amount of about 5 ng/mL to about 15 ng/mL. The TGF-β inhibitor may be present in a concentration of about 5 μΜ to about 15 μΜ.

[0013] The cells may be cultured in the mesoderm induction medium for a period of about 48 hours to about 96 hours. The cells may be cultured in the vascular specification medium for a period of about 72 hours to about 120 hours. The cells may be cultured in the endothelial amplification medium for a period of at least 96 hours.

[0014] These and other non-limiting characteristics of the disclosure are more particularly disclosed below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same. It is noted that the labels CD144 and VE-cadherin are used in different figures, but that these labels refer to the same biomarker. Similarly, the labels KDR and VEGFR2 are used in different figures, but refer to the same biomarker.

[0017] FIGs. 1 A-1 D are bright-field images of human embryonic H1 cells over 1 1 days of differentiation, cultured on a LN-521 substrate for all 1 1 days, on Days 0, 3, 7, and 1 1 (i.e. without purification). FIG. 1 A is for Day 0, FIG. 1 B is for Day 3, FIG. 1 C is for Day 7, and FIG. 1 D is for Day 1 1 . FIG. 1 E is a bright-field image of H1 cells cultured according to Protocol I (i.e. purification on Day 7, then replating on an LN-521 substrate) on Day 1 1 , for comparison with FIG. 1 D.

[0018] FIGs. 2A-2F are a set of six bar graphs of pluripotency markers and biomarkers Nanog, Mixl1 , CD34, CD31 , KDR, and CD144 expression for the cells of FIGs. 1A-1 D. The y-axis for each graph is "relative expression, normalized to GAPDH". The x-axis is Day 0, 3, 5, 7, 9, and 1 1 of differentiation for each graph. The y-axis for FIG. 2A (Nanog) runs from 0 to 0.15 at intervals of 0.05. The y-axis for FIG. 2B (Mixl1 ) runs from 0 to 0.20 at intervals of 0.05. The y-axis for FIG. 2C (CD34) runs from 0 to 1 .2 at intervals of 0.4. The y-axis for FIG. 2D (CD31 ) runs from 0 to 1 .6 at intervals of 0.4. The y-axis for FIG. 2E (KDR) runs from 0 to 4 at intervals of 1 . The y-axis for FIG. 2F (CD144) runs from 0 to 1 .5 at intervals of 0.5. In these Figures, results are reported as Means ± SEM, and each dot represents one independent differentiation batch (n=4- 7). P< 0.05.

[0019] FIGs. 3A-3F show FACS analysis for various markers on Day 0 for H1 cells plated on LN-521 substrate. For these figures, the red line is the isotype control (labeled C), and the blue line is the positive population (labeled P); the y-axis is % of max, and runs from 0 to 100 in intervals of 20; and the x-axis is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ . FIG. 3A is for CD34. FIG. 3B is for CD31 . FIG. 3C is for CD144. FIG. 3D is for KDR. FIG. 3E is for Oct3/4, and has a value of 98.9%. FIG. 3F is for TRA-1 -60, and has a value of 99.8%.

[0020] FIGs. 4A-4E show FACS analysis for various markers on Day 1 1 for H1 cells initially plated on LN-521 substrate to which Protocol I is applied and replated on LN- 521 substrate. For FIGs. 4A-4D, the red line is the isotype control (labeled C), and the blue line is the positive population (labeled P); the y-axis is % of max, and runs from 0 to 100 in intervals of 20; and the x-axis is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ . FIG. 4A is for CD34, and has a value of 96.3%. FIG. 4B is for CD31 , and has a value of 98.1 %. FIG. 4C is for CD144, and has a value of 97.1 %. FIG. 4D is for KDR, and has a value of 98.6%. FIG. 4E is for CD31 ⁺ and CD144 ⁺ within the KDR ⁺ population. 98.2% of cells in this KDR ⁺ population are also CD31 ⁺ and CD144 ⁺ . Altogether, 96.8% of the original population was triple positive for KDR, CD31 and CD144. The y-axis is for CD31 , and is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ . The x-axis is for CD144, and is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ .

[0021 ] FIGs. 5A-5E show FACS analysis for various markers on Day 1 1 for H1 cells initially plated on LN-521 substrate to which Protocol I is applied and replated on LN- 521/421 substrate. For FIGs. 5A-5D, the red line is the isotype control (labeled C), and the blue line is the positive population (labeled P); the y-axis is % of max, and runs from 0 to 100 in intervals of 20; and the x-axis is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ . FIG. 5A is for CD34, and has a value of 94.8%. FIG. 5B is for CD31 , and has a value of 97.5%. FIG. 5C is for CD144, and has a value of 96.0%. FIG. 5D is for KDR, and has a value of 98.2%. FIG. 5E is for CD31 ⁺ and CD144 ⁺ within the KDR ⁺ population. 97.8% of cells in this KDR ⁺ population are also CD31 ⁺ and CD144 ⁺ . Altogether, 96.1 % of the original population was triple positive for KDR, CD31 and CD144. The y-axis is for CD31 , and is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ . The x-axis is for CD144, and is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ .

[0022] FIGs. 6A-6E show FACS analysis for various markers on Day 15 for H1 cells initially plated on LN-521 substrate to which Protocol II is applied and replated on LN- 521 substrate. For FIGs. 6A-6D, the red line is the isotype control (labeled C), and the blue line is the positive population (labeled P); the y-axis is % of max, and runs from 0 to 100 in intervals of 20; and the x-axis is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ . FIG. 6A is for CD34, and has a value of 90.7%. FIG. 6B is for CD31 , and has a value of 91 .3%. FIG. 6C is for CD144, and has a value of 89.8%. FIG. 6D is for KDR, and has a value of 85.9%. FIG. 6E is for CD31 ⁺ and CD144 ⁺ within the KDR ⁺ population. 96.8% of cells in this KDR ⁺ population are also CD31 ⁺ and CD144 ⁺ . Altogether, 83.1 % of the original population was triple positive for KDR, CD31 and CD144. The y-axis is for CD31 , and is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ . The x-axis is for CD144, and is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ .

[0023] FIGs. 7A-7E show FACS analysis for various markers on Day 15 for H1 cells initially plated on LN-521 substrate to which Protocol II is applied and replated on LN- 521/421 substrate. For FIGs. 7A-7D, the red line is the isotype control (labeled C), and the blue line is the positive population (labeled P); the y-axis is % of max, and runs from 0 to 100 in intervals of 20; and the x-axis is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ . FIG. 7A is for CD34, and has a value of 92.3%. FIG. 7B is for CD31 , and has a value of 93.6%. FIG. 7C is for CD144, and has a value of 91 .3%. FIG. 7D is for KDR, and has a value of 85.9%. FIG. 7E is for CD31 ⁺ and CD144 ⁺ within the KDR ⁺ population. 97.7% of cells in this KDR ⁺ population are also CD31 ⁺ and CD144 ⁺ . Altogether, 84% of the original population was triple positive for KDR, CD31 and CD144. The y-axis is for CD31 , and is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ . The x-axis is for CD144, and is fluorescent intensity and is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ .

[0024] FIGs. 8A-8E show FACS analysis comparing the markers levels at different time points for different protocols. Letters A and B refer to a test where H1 cells were differentiated upon a LN-521 substrate for 1 1 days without purification. Letter A refers to the results on Day 7, and Letter B refers to the results on Day 1 1 of this data set where no purification occurred. Letter C refers to the results on Day 1 1 for cells cultured initially on LN-521 , then replated on LN-521 using Protocol I. Letter D refers to the results on Day 1 1 for cells cultured initially on LN-521 , then replated on LN-521 /421 using Protocol I. Letter E refers to the results on Day 1 5 for cells cultured initially on LN- 521 , then replated on LN-521 using Protocol II. Letter F refers to the results on Day 15 for cells cultured initially on LN-521 , then replated on LN-521/421 using Protocol II. For all figures, the y-axis is the % of positive population, and runs from 0 to 100% in intervals of 25%. FIG. 8A is for CD34 ⁺ . FIG. 8B is for CD31 ⁺ . FIG. 8C is for CD144 ⁺ . FIG. 8D is for KDR ⁺ . FIG. 8E is for KDR ⁺ CD31 ⁺ CD144 ⁺ . Results are reported as Means ± SEM, and each dot represents one independent differentiation batch (n=6-10). P< 0.05.

[0025] FIGs. 9A-9D are immunofluorescent pictures of CD31 ⁺ cells after purification, stained with anti-CD31 antibody. Green is for CD31 , and blue is for DAPI. FIG. 9A is on Day 1 1 for H1 cells initially plated on LN-521 substrate to which Protocol I is applied and replated on LN-521 substrate. FIG. 9B is on Day 1 1 for H1 cells initially plated on LN- 521 substrate to which Protocol I is applied and replated on LN-521 /421 substrate. FIG. 9C is on Day 15 for H1 cells initially plated on LN-521 substrate to which Protocol II is applied and replated on LN-521 substrate. FIG. 9D is on Day 15 for H1 cells initially plated on LN-521 substrate to which Protocol II is applied and replated on LN-521 /421 substrate.

[0026] FIGs. 10A-10D are fluorescent pictures of H1 -derived cells after purification taking up acetylated LDL, showing punctate perinuclear staining of DilAcLDL dye (red), blue=DAPI. FIG. 10A is for Day 1 1 , Protocol I, LN-521 substrate. FIG. 10B is for Day 1 1 , Protocol I, LN-521/421 substrate. FIG. 10C is for Day 15, Protocol II, LN-521 substrate. FIG. 10D is for Day 15, Protocol II, LN-521/421 substrate.

[0027] FIGs. 11 A-11 E show FACS analysis for the H1 -derived cells of FIGs. 10A- 10D. For FIGs. 11 A-11 D, the red line represents cells that were not incubated with DilAcLDL dye (labeled C), and the blue line represents cells that were incubated with DilAcLDL dye (labeled P); the y-axis is % of max, and runs from 0 to 100 in intervals of 20; and the x-axis is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ .

[0028] FIG. 11A is the results on Day 1 1 for H1 cells cultured initially on LN-521 , then replated on LN-521 using Protocol I, and has a value of 97.7%. FIG. 11 B is the results on Day 1 1 for H1 cells cultured initially on LN-521 , then replated on LN-521/421 using Protocol I, and has a value of 95.7%. FIG. 11 C is the results on Day 15 for H1 cells cultured initially on LN-521 , then replated on LN-521 using Protocol II, and has a value of 94.6%. FIG. 11 D is the results on Day 15 for H1 cells cultured initially on LN- 521 , then replated on LN-521/421 using Protocol II, and has a value of 91 .4%. For these figures, the y-axis is the % of maximum, and runs from 0 to 100% in intervals of 20%; and the x-axis is logarithmic, and is labeled as 0, 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ . [0029] FIG. 11 E compares the results of FIGs. 11 A-11 D with each other. Letter A is the results on Day 1 1 for cells cultured initially on LN-521 , then replated on LN-521 using Protocol I, and has a value of 97.7%. Letter B is the results on Day 1 1 for cells cultured initially on LN-521 , then replated on LN-521/421 using Protocol I, and has a value of 95.7%. Letter C is the results on Day 15 for cells cultured initially on LN-521 , then replated on LN-521 using Protocol II, and has a value of 94.6%. Letter D is the results on Day 15 for cells cultured initially on LN-521 , then replated on LN-521/421 using Protocol II, and has a value of 91 .4%. For these figures, the y-axis is the % of DilAcLDL ⁺ , and runs from 80% to 100% in intervals of 5%. Results are reported as Means ± SEM, and each dot represents one independent differentiation batch (n=5-6).

[0030] FIG. 12A is a picture of cells cultured on LN-521 substrate, purified on day 7, replated on LN-521/421 substrate, and allowed to grow until day 1 1 . Cells were then dissociated, plated on Matrigel at the density of 35,000 cells/cm ² , and allowed to grow for 4 days before visualization. FIG. 12B is a picture of the cells of FIG. 12A stained with Calcein-AM dye.

[0031 ] FIGs. 13A-13E show FACS analysis comparing the markers levels at different time points for different protocols using H1 cells on LN-51 1 as the first cell culture substrate and LN-51 1 or LN-51 1/421 for the second cell culture substrate. Letters A and B refer to a test where H1 cells were differentiated upon a LN-51 1 substrate for 1 1 days without purification. Letter A refers to the results on Day 7, and Letter B refers to the results on Day 1 1 of this data set where no purification occurred. Letter C refers to the results on Day 1 1 for cells cultured initially on LN-51 1 , then replated on LN-51 1 using Protocol I. Letter D refers to the results on Day 1 1 for cells cultured initially on LN-51 1 , then replated on LN-51 1/421 using Protocol I. Letter E refers to the results on Day 15 for cells cultured initially on LN-51 1 , then replated on LN- 51 1 using Protocol II. Letter F refers to the results on Day 1 5 for cells cultured initially on LN-51 1 , then replated on LN-51 1/421 using Protocol II. For all figures, the y-axis is the % of positive population, and runs from 0 to 100% in intervals of 25%. FIG. 13A is for CD34 ⁺ . FIG. 13B is for CD31 ⁺ . FIG. 13C is for CD144 ⁺ . FIG. 13D is for KDR ⁺ . FIG. 13E is for KDR ⁺ CD31 ⁺ CD144 ⁺ . Results are reported as Means ± SEM, and each dot represents one independent differentiation batch (n=3-5). P< 0.05. [0032] FIG. 14 summarizes FACS data from cells incubated with DilAcLDL dye. Letter A is the results on Day 1 1 for cells cultured initially on LN-51 1 , then replated on LN-51 1 using Protocol I. Letter B is the results on Day 1 1 for cells cultured initially on LN-51 1 , then replated on LN-51 1/421 using Protocol I. Letter C is the results on Day 15 for cells cultured initially on LN-51 1 , then replated on LN-51 1 using Protocol II. Letter D is the results on Day 15 for cells cultured initially on LN-51 1 , then replated on LN- 51 1/421 using Protocol II. For these figures, the y-axis is the % of DilAcLDL ⁺ , and runs from 80% to 100% in intervals of 5%. Results are reported as Means ± SEM, and each dot represents one independent differentiation batch (n=4-5).

[0033] FIGs. 15A-15D are bright-field images of differentiated HS1001 cells on Days 0, 3, 7, and 1 1 (i.e. without purification on an LN-521 substrate). FIG. 15A is for Day 0, FIG. 15B is for Day 3, FIG. 15C is for Day 7, and FIG. 15D is for Day 1 1 . FIG. 15E is a bright-field image of HS1001 cells cultured according to Protocol I (i.e. purification on Day 7, then replating on an LN-521 substrate) on Day 1 1 , for comparison with FIG. 15D.

[0034] FIG. 16A is a graph illustrating the total expression variabilities of the first principal component due to the separation of hESCs from other cell types and the second principal component originating from the separation of endothelial progenitor cell (EPC) samples from mature HUVECs.

[0035] FIG. 16B is a set of four heat maps exhibiting gene-level expression patterns in KEGG and GOBP pathways in hESCs, EPCs differentiated from Protocols I and II, and HUVECs, as shown on the x-axis. The first graph titled "KEGG: JAK-STAT SIGNALING PATHWAY" illustrates the expression patterns, in descending order, of ACTN4, CAPN2, CAV2, COL5A1 , GRB2, ITGA10, ITGA1 1 , ITGA2, ITGA3, JUN, LAMB1 , MET, MYL12A, MYL12B, PARVA, PDGFA, PIP5K1 C, PRKCA, PTK2, RAC2, RAP1 A, THBS1 , VWF, XIAP, ACTG1 , FLNC, PGF, VAV2, ZYX, CCND1 , CCND3, LAMA5, PARVB, PDGFB, RAPGEF1 , AKT1 , AKT3, BAD, BRAF, COL3A1 , CTNNB1 , DOCK1 , GSK3B, ITGA5, ITGB3, LAMA4, MAPK1 , MAPK3, PAK2, PDGFC, PTEN, PXN, RHOA, ROCK1 , THBS3, TLN1 , VEGFB, CDC42, BIRC2, COL5A2, IGF1 R, PDPK1 , PIK3R3, SRC, ACTB, ITGB4, ITGB8, PPP1 CB, RAC1 , VEGFA, ACTN1 , DIAPH1 , EGFR, FLNA, ITGA6, MAPK8, ELK1 , PAK4, PIK3CB, PIK3CD, PIK3R2, RAC3, THBS2, CRKL, ERBB2, RAF1 , VCL, CRK, BCL2, FYN, HRAS, ILK, ITGA8, ITGB1 , KDR, LAMB2, PDGFD, PIK3R1 , RAP1 B, ROCK2, SHC2, SOS2, VASP, COL4A1 , ARHGAP5, COL4A2, COL6A2, FLT1 , FLT4, LAMC1 , MAP2K1 , MYL5, MYLK, PIK3CA, PIK3CG, S0S1 , VEGFC, ITGA1 , EGF, ITGA4, ITGA9, PPP1 CA, VAV1 , VAV3, ITGB5, MAPK9, CCND2, ITGA7, LAMA1 , MYL9, PPP1 CC, SPP1 , TLN2, C0L6A1 , AKT2, PAK1 , FN1 , PPP1 R12A, C0L1 A2, C0L1 A1 , and LAMC2.

[0036] The second graph (top right) titled "KEGG: ECM-RECEPTOR INTERACTIONS" illustrates the expression patterns, in descending order, of HSPG2, COL5A1 , ITGA10, ITGA2, ITGA3, LAMB1 , SDC3, THBS1 , VWF, LAMA5, CD44, ITGA5, COL3A1 , ITGB3, LAMA4, THBS3, COL5A2, COL4A1 , CD47, COL4A2, LAMC1 , GP9, ITGB5, ITGB1 , ITGA8, LAMB2, ITGA1 , ITGA4, ITGA9, ITGA7, LAMA1 , SPP1 , SV2A, HMMR, COL1 A2, FN1 , COL6A1 , SDC1 , ITGA6, ITGB4, ITGB8, SDC2, SDC4, and THBS2.

[0037] The third graph (middle right) titled "GOPP: WOUND HEALING" illustrates the expression patterns, in descending order, of GNA12, MIA3, GNAQ, PLAT, ITGB3, ENTPD1 , DCBLD2, THBD, F2RL2, ADORA2A, NF1 , GP9, LMAN1 , GGCX, ITGA2, PROS1 , PABPC4, F2R, ACVRL1 , MMRN1 , VWF, TFPI, CD59, and SERPINE1 .

[0038] The fourth graph (bottom right) titled "GOPP: VASCULATURE DEVELOPMENT" illustrates the expression patterns, in descending order, of AGGF1 , NF1 , ANGPTL4, TNFSF12, AMOT, EGF, VEGFA, FOXC2, ERAP1 , IL8, FOX04, GLMN, TNNI3, SERPINF1 , C1 GALT1 , PML, CUL7, RASA1 , CCM2, HTATIP2, NPR1 , PDPN, BTG1 , ROB04, THY1 , NOTCH4, CDH13, RNH1 , EMCN, SPHK1 , ACVRL1 , STAB1 , CANX, MYH9, NCL, ATPIF1 , RHOB, EGFL7, and COL4A2.

[0039] FIGs. 16C-16E are three sets of graphic representations of expression profiles. Each of these sets has four smaller graphs arranged vertically. The top graph is hESCs, the second graph from the top is labeled "EPCs Protocol I", the third graph is labeled "EPCs Protocol II", and the bottom graph is HUVECs.

[0040] FIG. 16C is an expression profile of some major ECM genes during maturation of hESC-derived endothelial lineage. The y-axis on each graph is a numeric representation of FPKM values (Fragments Per Kilobase of Exon per Million Fragments Mapped) from 0 (bottom) to 1200 (top) with intervals of 400. From left to right, the ECM genes profiled are LAMA1 , LAMA4, LAMA5, LAMB1 , LAMB2, LAMC1 , FN1 , HSPG2, AGRN, SDC4, FBN1 , VTN, NID1 , FBLN1 , COL4A1 , COL4A2, COL18A1 , MMP1 , MMP2, and MMP14.

[0041 ] FIG. 16D is an expression profile of major receptors for ECM proteins. The y- axis on each graph is a numeric representation of FPKM values from 0 (bottom) to 1200 (top) with intervals of 400. From left to right, the ECM receptors profiled are ITGA1 , ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGA9, ITGAE, ITGAV, ITGB1 , ITGB3, ITGB4, and ITGB5.

[0042] FIG. 16E is an expression profile of some genes within the differentiated EPCs. The y-axis on each graph is a numeric representation of FPKM values from 0 (bottom) to 1200 (top) with intervals of 400. From left to right, the genes profiled are THY1 , CD34, VEGFR1 , VEGFR2, NRP1 , NRP2, NOS3, TIE2, ENG, ANKRD1 , VE- cadherin, CD31 , TIE1 , MCAM, and VWF.

[0043] FIGs. 17A-I are graphs indicating the relative mRNA expression patterns of hESC-derived EPCs at 2, 3, 4, 5, and 6 weeks after purification according to Protocols I and II. Each x-axis is broadly divided into two groups: Purification on Day 7 (Protocol I) and Purification on Day 1 1 (Protocol II) displayed left to right. Further, 10 bars are displayed in conjunction with each protocol. The leftmost five bars are expression patterns of the selected EPC cultured on LN-521 according to Protocol I. The next five bars are expression patterns of the selected EPC cultured on LN-521/421 according to Protocol I. The next five bars are expression patterns of the selected EPC cultured on LN-521 according to Protocol II. The next five bars are expression patterns of the selected EPC cultured on LN-521/421 according to Protocol II. Each set of five bars illustrates the expression of the selected EPC, from left to right, at 2 weeks, 3, weeks, 4 weeks, 5 weeks, and 6 weeks. The bar furthest right is the expression pattern of HUVECs. Results are reported as Means ± SEM from 3 to 10 independent differentiation batches.

[0044] FIG. 17A is a VEGFR2 mRNA expression profile over time. The y-axis indicates the mRNA expression pattern (relative expression, normalized to GADPH) from 0.0 (bottom) to 2.0 (top) in intervals of 0.5. [0045] FIG. 17B is a CD31 mRNA expression profile over time. The y-axis indicates the mRNA expression pattern (relative expression, normalized to GADPH) from 0.0 (bottom) to 1 .5 (top) in intervals of 0.5.

[0046] FIG. 17C is a VE-Cadherin mRNA expression profile over time. The y-axis indicates the mRNA expression pattern (relative expression, normalized to GADPH) from 0.0 (bottom) to 0.8 (top) in intervals of 0.2.

[0047] FIG. 17D is a VWF mRNA expression profile over time. The y-axis indicates the mRNA expression pattern (relative expression, normalized to GADPH) from 0.0 (bottom) to 0.6 (top) in intervals of 0.2.

[0048] FIG. 17E is a LAMA4 mRNA expression profile over time. The y-axis indicates the mRNA expression pattern (relative expression, normalized to GADPH) from 0.00 (bottom) to 0.08 (top) in intervals of 0.02.

[0049] FIG. 17F is a LAMA5 mRNA expression profile over time. The y-axis indicates the mRNA expression pattern (relative expression, normalized to GADPH) from 0.00 (bottom) to 0.08 (top) in intervals of 0.02.

[0050] FIG. 17G is a LAMB1 mRNA expression profile over time. The y-axis indicates the mRNA expression pattern (relative expression, normalized to GADPH) from 0.00 (bottom) to 0.15 (top) in intervals of 0.05.

[0051 ] FIG. 17H is a LAMB2 mRNA expression profile over time. The y-axis indicates the mRNA expression pattern (relative expression, normalized to GADPH) from 0.0 (bottom) to 0.6 (top) in intervals of 0.2.

[0052] FIG. 171 is a MMP1 mRNA expression profile over time. The y-axis indicates the mRNA expression pattern (relative expression, normalized to GADPH) from 0.0 (bottom) to 2.5 (top) in intervals of 0.5.

DETAILED DESCRIPTION

[0053] A more complete understanding of the compositions and methods disclosed herein can be obtained by reference to the accompanying drawings. These figures are merely schematic representations based on convenience and the easy of demonstrating the present disclosure, and are, therefore, not intended to define or limit the scope of the exemplary embodiments. [0054] Although specific terms are used in the following description for the sake of clarity, these terms are intended to refer only to the particular structure of the embodiments selected for illustration in the drawings, and are not intended to define or limit the scope of the disclosure. In the drawings and the following description below, it is to be understood that like numeric designations refer to components of like function.

[0055] All publications, patents, and patent applications discussed herein are hereby incorporated by reference in their entirety.

[0056] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0057] As used in the specification and in the claims, the term "comprising" may include the embodiments "consisting of" and "consisting essentially of." The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as "consisting of" and "consisting essentially of" the enumerated ingredients/steps, which allows the presence of only the named ingredients/steps, along with any impurities that might result therefrom, and excludes other ingredients/steps.

[0058] Numerical values in the specification and claims of this application should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0059] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of "from 2 to 10" is inclusive of the endpoints, 2 and 10, and all the intermediate values).

[0060] The term "about" can be used to include any numerical value that can vary without changing the basic function of that value. When used with a range, "about" also discloses the range defined by the absolute values of the two endpoints, e.g. "about 2 to about 4" also discloses the range "from 2 to 4." The term "about" may refer to plus or minus 10% of the indicated number.

[0061 ] Several references that may be relevant to the present disclosure include: Functional Diversity of Laminins (Domogatskaya, A., S. Rodin, and K. Tryggvason, Annu. Rev. Cell Dev. Biol., 2012, 28: pp. 523-53.); Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling (Lian, X. et al., Stem Cell Reports, 2014, 3(5): pp. 804-16.); Generation of Vascular Endothelial and Smooth Muscle Cells from Human Pluripotent Stem Cells (Patsch, C. et al., Nat. Cell. Biol., 2015); BMP4 regulates vascular progenitor development in human embryonic stem cells through a Smad-dependent pathway (Bai, H. et al., J. Cell Biochem., 2010, 109(2): pp. 363-74.); Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells (Sahara, M. et al., Cell Res., 2014, 24(7): pp. 820-41 .); Simple and highly efficient method for production of endothelial cells from human embryonic stem cells (Tatsumi, R. et al., Cell Transplant, 201 1 , 20(9): pp. 1423-30.); and Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo (Wang, Z.Z. et al., Nat. Biotechnol., 2007, 25(3): pp. 317-8.)

[0062] A stem cell is an undifferentiated cell from which specialized cells are subsequently derived. Pluripotent stem cells can be differentiated into any of the three germ layers: endoderm, mesoderm, or ectoderm. Post fertilization, pluripotent stem cells form every cell type in the human body, including less plastic stem cell populations such as adult stem cells, fetal stem cells, and amniotic stem cells. Embryonic stem cells are a type of pluripotent stem cell, and possess extensive self-renewal capacity and pluripotency. More recently another type of pluripotent stem cell, induced pluripotent stem cells, were produced from mammalian terminally differentiated cells by a process termed somatic cell reprogramming. The process by which a stem cell changes into a more specialized cell is referred to as differentiation.

[0063] Laminins are a family of heterotrimeric glycoproteins that reside primarily in the basal lamina. They function via binding interactions with neighboring cell receptors on the one side, and by binding to other laminin molecules or other matrix proteins such as collagens, nidogens or proteoglycans. The laminin molecules are also important signaling molecules that can strongly influence cellular behavior and function. Laminins are important in both maintaining cell/tissue phenotype, as well as in promoting cell growth and differentiation in tissue repair and development.

[0064] Laminins are large, multi-domain proteins, with a common structural organization. The laminin molecule integrates various matrix and cell interactive functions into one molecule. A laminin protein molecule comprises one a-chain subunit, one β-chain subunit, and one γ-chain subunit, all joined together in a trimer through a coiled-coil domain. The twelve known laminin subunit chains can form at least 16 trimeric laminin types in native tissues. Within the trimeric laminin structures are identifiable domains that possess binding activity towards other laminin and basal lamina molecules, and membrane-bound receptors. For example, domains VI, IVb, and IVa form globular structures, and domains V, lllb, and Ilia (which contain cysteine-rich EGF-like elements) form rod-like structures. Domains I and II of the three chains participate in the formation of a triple-stranded coiled-coil structure (the long arm).

[0065] There exist five different alpha chains, three beta chains and three gamma chains that in human tissues have been found in at least fifteen different combinations. These molecules are termed laminin-1 to laminin-15 based on their historical discovery, but a more recent alternative nomenclature describes the isoforms based on their chain composition, e.g. laminin-1 1 1 (laminin-1 ) that contains alpha-1 , beta-1 and gamma-1 chains. Four structurally defined family groups of laminins have been identified. The first group of five identified laminin molecules all share the β1 and γ1 chains, and vary by their a-chain composition (a1 to a5 chain). The second group of five identified laminin molecules, including laminin-521 , all share the β2 and γ1 chain, and again vary by their a-chain composition. The third group of identified laminin molecules has one identified member, laminin-332, with a chain composition of α3β3γ2. The fourth group of identified laminin molecules has one identified member, laminin-213, with the newly identified γ3 chain (α2β1 γ3).

[0066] As used herein, the term "laminin-41 1 " refers to the protein formed by joining α4, β1 , and γ1 chains together. The term should be construed as encompassing both recombinant laminin-41 1 and heterotrimeric laminin-41 1 from naturally occurring sources. [0067] As used herein, the term "laminin-421 " refers to the protein formed by joining α4, β2, and γ1 chains together. The term should be construed as encompassing both recombinant laminin-421 and heterotrimeric laminin-421 from naturally occurring sources.

[0068] As used herein, the term "laminin-51 1 " refers to the protein formed by joining α5, β1 , and γ1 chains together. The term should be construed as encompassing both recombinant laminin-51 1 and heterotrimeric laminin-51 1 from naturally occurring sources.

[0069] As used herein, the term "laminin-521 " refers to the protein formed by joining α5, β2, and γ1 chains together. The term should be construed as encompassing both recombinant laminin-521 and heterotrimeric laminin-521 from naturally occurring sources.

[0070] The term "intact" refers to the protein being composed of all of the domains of the a-chain, β-chain, and γ-chain, with the three chains being joined together to form the heterotrimeric structure. The protein is not broken down into separate chains, fragments, or functional domains. The term "chain" refers to the entirety of the alpha, beta, or gamma chain of the laminin protein. The term "fragment" refers to any protein fragment which contains one, two, or three functional domains that possesses binding activity to another molecule or receptor. However, a chain should not be considered a fragment because each chain possesses more than three such domains. Similarly, an intact laminin protein should not be considered a fragment. Examples of functional domains include Domains I, II, III, IV, V, VI, and the G domain.

[0071 ] In general, differentiated cells typically require two things to survive and reproduce: (1 ) a substrate or coating that provides a structural support for the cells; and (2) a cell culture medium to provide nutrition to the cell. The substrate or coating (1 ) is typically formed as a layer in a container, for example a petri dish or in the well of a multi-well plate. Application of different cell culture mediums at appropriate time intervals in combination with the substrates containing laminins result in endothelial cells with more natural functions/properties.

[0072] The stem cells that can be used with the methods and materials disclosed herein are pluripotent human stem cells. Such stem cells can include induced pluripotent stem cells, embryonic stem cells, adult stem cells, fetal stem cells, amniotic stem cells, and generally any pluripotent stem cell.

[0073] The present disclosure generally relates to methods of culturing human stem cells to obtain endothelial cells. The expression of various proteins can be used to track the development of endothelial cells from the hESCs. CD31 (also referred to as PECAM-1 ) is a type-l integral membrane glycoprotein that is expressed at high levels on mature endothelial cells. Thus, CD31 can be used as a mature endothelial marker. CD34 is a cell surface adhesion molecule with a role in early hematopoiesis by mediating the attachment of stem cells to the bone marrow extracellular matrix or directly to stromal cells. As such, CD34 can be used as an endothelial progenitor marker. CD144, also known as VE-Cadherin, is a classic cadherin that permits cell ability to adhere in homophilic manners, thereby allowing the control of the cohesion and organization of the intercellular junctions. Thus, CD144/VE-cadherin can be used as a mature endothelial marker. Sox2 is a transcription factor that is essential for maintaining pluripotency and can thus be used as a pluripotency marker. MixM is a transcription factor that plays an active role in mesoderm patterning and tissue specification. Generally, MixM "marks" cells destined to become part of the mesoderm. As such, MixM expression is necessary for both mesoderm development and hematopoiesis. In addition, MixM is a necessary intermediate for MBP4-induced mesoderm patterning and differentiation of ES cells. Thus, MixM can be used as a mesoderm marker. KDR (kinase insert domain receptor), also known as VEGFR2, is a vascular endothelial growth factor (VEGF) receptor that functions as the main mediator of VEGF-induced endothelial proliferation, survival and migration. As such, KDR/VEGFR2 can be used as an endothelial progenitor marker. Nanog can be used as a marker for pluripotency.

[0074] In the methods of the present disclosure that are used to obtain endothelial cells, two cell culture substrates and three different cell culture media are used. In particular, the stem cells are cultured on a first cell culture substrate that contains either laminin-521 or laminin-51 1 . The stem cells are then exposed to a mesoderm induction medium and a vascular specification medium. The stem cells are then purified to identify cells that are of endothelial lineage. These stem cells are replated on a second cell culture substrate that has one of four different coatings: (a) only laminin-521 ; (b) only laminin-51 1 ; (c) a mixture of laminin-521 and laminin-421 ; or (d) a mixture of laminin-51 1 and laminin-421. The stem cells are then exposed to an endothelial amplification medium. This results in the generation of high quality endothelial cells of homogenous quality that can be used in, for example, regenerative medicine. Test results indicate that these methods can yield the generation of about 95% endothelium- specific CD31 +CD144+ cells after 1 1 days, as determined by FACS analysis. These methods are completely xeno-free and use fully human materials to culture homogenous monolayer hESCs on pure laminin matrices. As such, they do not use animal-derived components, but can use human-derived components.

[0075] The two cell culture substrates described above are used in combination with three different cell culture media to obtain the desired endothelial cells. Those three cell culture media are referred to herein as a mesoderm induction medium, a vascular specification medium, and an endothelial amplification medium. These three media are based on a common basal medium.

[0076] The basal medium includes DMEM/F12 medium, 1 X chemically defined lipid concentrate, 0.1 X Insulin-Transferrin-Selenium-X, GlutaMAX, mono-thio glycerol, and L- ascorbic acid 2-phosphate. The chemically defined lipid concentrate is commercially available from Gibco (catalog no. 1 1905-031 ). The Insulin-Transferrin-Selenium-X is also commercially available from Gibco (catalog no. 51500-056). GlutaMAX is commercially available from Gibco (catalog no. 35050-061 ), and is added in the amount of about 10 micromolar (μΜ) to about 5 millimolar (mM), and in particular embodiments is about 2 mM. The amount of mono-thio glycerol can range from about 250 micromolar (μΜ) to about 750 μΜ, and in particular embodiments is about 450 μΜ. The amount of L-ascorbic acid 2-phosphate can range from 25 micrograms per milliliter ^g/mL) to about 75 μg/mL, and in particular embodiments is about 50 μg/mL.

[0077] DMEM/F12 medium is commercially available from Invitrogen (catalog nos. 10565 and 21331 ), and generally contains the following ingredients listed below:

[0078] The mesoderm induction medium is made by supplementing the basal medium with an activin, a bone morphogenic protein, and a GSK-3 inhibitor. A particularly suitable activin is activin A (from R&D Systems). A particularly suitable bone morphogenic protein is BMP4 (from R&D Systems). A particularly suitable GSK-3 inhibitor is CHIR99021 (Tocris Bioscience). The activin is present in the mesoderm induction medium in an amount of about 5 ng/mL to about 15 ng/mL, including about 10 ng/mL. The bone morphogenic protein is present in the mesoderm induction medium in an amount of about 10 ng/mL to about 30 ng/mL, including about 20 ng/mL. Finally, the GSK-3 inhibitor is present in the mesoderm induction medium at a concentration of about 3 micromolar (μΜ) to about 9 μΜ, including about 6 μΜ. The mesoderm induction medium does not include a Notch signaling inhibitor or a TGF-β inhibitor.

[0079] The vascular specification medium is made by supplementing the basal medium with a vascular endothelial growth factor, basic fibroblast growth factor (bFGF), a bone morphogenic protein, and a Notch signaling inhibitor. A particularly suitable vascular endothelial growth factor is VEGF ₁₆ 5 (from Life Technologies, catalog no. PHC9394). A particularly suitable bone morphogenic protein is BMP4. A particularly suitable Notch signaling inhibitor is DAPT (Tocris Bioscience). The vascular endothelial growth factor is present in the vascular specification medium in an amount of about 25 ng/mL to about 75 ng/mL, including about 50 ng/mL. The bFGF (R&D Systems) is present in the vascular specification medium in an amount of about 5 ng/mL to about 15 ng/mL, including about 10 ng/mL. The bone morphogenic protein is present in the specification medium in an amount of about 10 ng/mL to about 30 ng/mL, including about 20 ng/mL. Finally, the Notch signaling inhibitor is present in the vascular specification medium at a concentration of about 5 μΜ to about 15 μΜ, including about 10 μΜ. The vascular specification medium does not include a GSK-3 inhibitor or a TGF- β inhibitor.

[0080] The endothelial amplification medium is formed by supplementing the basal medium with a vascular endothelial growth factor, basic fibroblast growth factor (bFGF), and a TGF-β inhibitor. A particularly suitable vascular endothelial growth factor is VEGF ₁₆ 5 (from Life Technologies, catalog no. PHC9394). A particularly suitable TGF-β inhibitor is SB431542 (Tocris Bioscience). The vascular endothelial growth factor is present in the endothelial amplification medium in an amount of about 25 ng/mL to about 75 ng/mL, including about 50 ng/mL. The bFGF (R&D Systems) is present in the endothelial amplification medium in an amount of about 5 ng/mL to about 15 ng/mL, including about 10 ng/mL. Finally, the TGF-β inhibitor SB431542 is present in the endothelial amplification medium at a concentration of about 5 μΜ to about 15 μΜ, including about 10μΜ. The endothelial amplification medium does not include a GSK-3 inhibitor or a Notch signaling inhibitor.

[0081 ] Initially, human embryonic stem cells (hESC) are plated on the first cell substrate. The first cell substrate consists essentially of a single laminin, which is either laminin-521 (LN-521 ) or laminin-51 1 (LN-51 1 ), at a density of about 5 micrograms per milliliter (pg/mL) to about 15 pg/mL, including specifically about 10 pg/mL. The hESCs can be plated at a density of about 50,000 cells/well to about 100,000 cells/well on a 24- well plate, depending on the cell line used, or from about 25,000 cells/cm ² to about 50,000 cells/cm ² , and should be in a monolayer. In this regard, laminin isoforms LN- 51 1 and LN-521 are normal components of subendothelial basement membranes during development and postnatally.

[0082] The cells are then cultured using a xeno-free, serum-free culture medium to nourish the cells, until they have reached about 40% to about 50% confluency (i.e. the proportion of surface area covered by adherent cells) or greater. A suitable medium is NutriStem hESC XF medium (Biological Industries, Israel), which also maintains the pluripotency of the stem cells. The time to reach the desired confluency may be from about 48 hours to about 72 hours. Cells are subcultured by exposure to TrypLESelect (Gibco Invitrogen) for 8 minutes at 37°C, 5% C0 ₂ .

[0083] Differentiation then takes place under appropriate conditions (e.g. 37°C, 5% C0 ₂ ). Differentiation is initiated (i.e. on day 0) by removing the NutriStem maintenance culture medium and applying the mesoderm induction medium to the plated stem cells. The cells are cultured in the mesoderm induction medium for a period of about 48 hours to about 96 hours, including about 72 hours (i.e. 3 days). The medium may be periodically changed, for example every 24 hours. This encourages the development of the mesoderm during embryogenesis, from which vascular cell lineages emerge.

[0084] Next, the mesoderm induction medium is removed, and the vascular specification medium is applied to the mesoderm-committed cells on the first cell culture substrate. The cells are cultured in the vascular specification medium for a period of about 72 hours to about 120 hours, including about 96 hours (i.e. 4 days). The medium may be periodically changed, for example, every 24 hours. This directs the mesoderm- committed cells towards the endothelial lineage.

[0085] After this step, the cells on the first cell culture substrate are purified to identify adherent differentiated cells. Dissociation can be done using TrypLESelect (Gibco), and purification can be done by identifying cells that are expressing endothelial lineage markers. For example, the MACS CD31 Microbead Kit from Miltenyi Biotec can be used.

[0086] The identified differentiated cells are then plated on a second cell substrate. The second cell culture substrate that has one of four different coatings containing one or more laminins. The laminin(s) in those coatings are either: (a) only laminin-521 ; (b) only laminin-51 1 ; (c) a mixture of laminin-521 and laminin-421 ; or (d) a mixture of laminin-51 1 and laminin-421 . In the mixtures (c) and (d), the volume ratio of the LN- 521/51 1 to the laminin-421 (LN-421 ) is from about 1 :2 to about 2:1 , and in particular embodiments is about 1 :1 . The total density of the one or more laminins on the second cell substrate is about 5 micrograms per milliliter (pg/mL) to about 15 pg/mL, including specifically about 10 pg/mL. All four of these laminins are normal components of subendothelial basement membranes.

[0087] The endothelial amplification medium is applied to the vascular committed cells on the second cell culture substrate. The cells are cultured in the endothelial amplification medium for a period of at least 96 hours (i.e. 4 days) to allow the differentiated cells to expand. The medium may be periodically changed, for example, every 24 hours. This expands the endothelial cells to obtain a pure population of mature endothelial cells.

[0088] These methods result in a total differentiation time period of about 1 1 days minimum in which the stem cells differentiate into endothelial progenitor cells. As can be appreciated, the last stage, endothelial expansion or amplification, can extend beyond day 1 1 , and may extend up to 6 weeks after purification (i.e. up to day 53) for the cells to reach maturity.

[0089] In some additional embodiments, the cells on the first cell culture substrate are not purified prior to replating the cells onto the second cell culture substrate. Rather, in these embodiments, the purification step occurs after the cells have been replated on the second cell culture substrate and have been cultured using the endothelial amplification medium. After expansion, the cells are then purified using the same techniques described above.

[0090] The following table identifies all of the ingredients used in each medium:

[0091 ] The methods described herein allow for robust large-scale production of pluripotent stem cell derived endothelial cells that can find application in human regenerative medicine, vascular graft coatings, functional cell assays, in vitro blood vessel models, etc., since the system is xeno-free and completely chemically defined. For example, the ECs can be used to endothelialize (i.e., to coat) artificial vascular grafts such as large Dacron or polyester grafts or stents or in blood vessels generated in vitro. In addition, the stable human endothelial cells can be used for drug and toxicity studies. From a therapeutic standpoint, the hECs of the present disclosure may find use in cell therapy, for example, cardiac disease, where they can be implemented together with cardiomyocytes for building up new vascularized heart muscle. Furthermore, a stable source of ECs can be used in the generation of in vitro models such as for the blood brain barrier and the glomerular filtration barrier, as well as for blood capillary models. As can be appreciated, other in vitro models can be generated.

[0092] The following examples are for purposes of further illustrating the present disclosure. The examples are merely illustrative and are not intended to limit devices made in accordance with the disclosure to the materials, conditions, or process parameters set forth therein.

EXAMPLES EXAMPLE 1

[0093] Maintenance of hESCs

[0094] 24-well tissue culture plates (Costar) were coated overnight at 4°C with sterile LN-521 , LN-51 1 , LN-521/LN-421 or LN-51 1/LN-421 (1 :1 ratio) combinations at 10 pg/mL according to manufacturer's instructions (BioLamina AB).

[0095] hESC lines H1 (WiCell Research Institute) and HS1001 (Karolinska Institute) were cultured in monolayer on pre-coated plates and maintained in NutriStem hESC XF (Biological Industries, Israel) medium.

[0096] Upon confluency, the cells were subcultured by exposure to TrypLESelect (GIBCO Invitrogen) for 8 min at 37°C, 5% C02. The H1 cell line was routinely passaged at 10,000 cells/cm ² and HS1001 line at 30,000 cells/cm ²

[0097] Differentiation protocol

[0098] The passaged hESCs were then plated on pre-coated 24-well tissue culture plates pre-coated with either LN-521 or LN-51 1 , at a density of 50,000-100,000 cells/well (depending on the cell line, about 25,000 cells/cm ² ) and refreshed with NutriStem every day.

[0099] The Basal medium used throughout the differentiation process consisted of DMEM/F12 medium, 1 X chemically defined lipid concentrate, 0.1 X Insulin-Transferrin- Selenium-X, 2 mM GlutaMAX (all from Gibco), 450 μΜ mono-thio glycerol (Sigma) and 50 pg/mL L-ascorbic acid 2-phosphate (Sigma).

[0100] Day 0 of differentiation began when the cultures reached from about 40% to about 50% confluency (i.e., after about 2 to about 3 days depending on the cell line). Their pluripotency was confirmed by high mRNA expression of Nanog, 99% Oct3/4 ⁺ and 100% TRA-1 -60 ⁺ and weak expression of KDR in total cell population. On day 0 of differentiation, the NutriStem was replaced with mesoderm induction medium, consisting of basal medium supplemented with 10 ng/mL Activin A, 20 ng/mL BMP4 (both from R&D Systems), and 6 μΜ CHIR99021 (Tocris Bioscience). This medium yields high populations of mesoderm-committed cells.

[0101 ] After 3 days, the mesoderm induction medium was replaced by vascular specification medium, consisting of basal medium supplemented with 50 ng/mL VEGFi ₆₅ (Gibco), 10 ng/mL bFGF, 20 ng/mL BMP4 (both from R&D Systems), and 10 μΜ DAPT, a Notch signaling inhibitor (Tocris Bioscience). This medium drives the mesoderm-committed cells towards endothelial lineage.

[0102] Two different protocols were subsequently followed.

[0103] In the first protocol (Protocol I), on day 7 of differentiation, adherent differentiated cells were dissociated with TrypLESelect (Gibco) and MACS-purified using CD31 Microbead Kit according to manufacturer's instructions (Miltenyi Biotec).

[0104] CD31 + cells were replated on laminin-coated plates containing one of four different coatings: (a) only laminin-521 ; (b) only laminin-51 1 ; (c) a 1 :1 mixture of laminin- 521 and laminin-421 ; or (d) a 1 :1 mixture of laminin-51 1 and laminin-421 . The cells were then cultured in endothelial amplification medium, consisting of basal medium supplemented with 50 ng/mL VEGF ₁₆₅ , 10 ng/mL bFGF, and 10 μΜ SB431542, a TGFp signaling inhibitor (Tocris Bioscience), and expanded until day 1 1 when they were characterized. All media were replaced every other day.

[0105] In the second protocol (Protocol II), no purification occurred on day 7. Rather, all cells were cultured in endothelial amplification medium for 4 days. Then, on day 1 1 , CD31 + cells were purified and then replated on one of the four different coatings identified above and then expanded until day 15, or for as long as up to six (6) weeks, when they were characterized. All media were replaced every other day. [0106] Quantitative PCR analysis

[0107] Cells were collected at different time points during differentiation. Total RNA was purified using RNeasy Micro Kit (Qiagen) according to manufacturer's instructions. The yield was determined by the NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies). cDNA was synthesized from 500 ng of total RNA in 20 μΙ reaction mixture using TaqMan Reverse Transcription Reagents Kit (Applied BioSystems) according to manufacturer's instructions. Real-time quantitative RT-PCR was performed in duplicates with synthesized cDNA in assay mix containing iQ SYBR Green Super mix (BioRad) and primers for genes of interest. GAPDH was used as the normalizing control.

[0108] FACS analysis

[0109] Cells were collected at different time points during the differentiation process and single-cell suspensions were fixed with Fixation Reagent (Medium A; Life Technologies) for 15 minutes at room temperature, washed with FACS buffer (0.5% BSA, 2 imM EDTA in 1 x PBS), blocked with blocking buffer (5% goat serum in FACS buffer), immunostained with primary antibodies in Permeabilization Reagent (Medium B; Life Technologies) for 15 minutes at room temperature, washed with FACS buffer and incubated with secondary antibodies diluted in 1 % goat serum in FACS buffer. For fluorophore-conjugated antibodies, fixed cells were incubated with antibodies diluted in Medium B for 30 minutes at room temperature, then washed with FACS buffer. Stained cells were resuspended in FACS buffer and subjected to FACS analysis (MACSQuant VYB, Miltenyi Biotec) to detect the expression of different specific cell-surface markers and transcription factors. The antibodies used in this study were: anti-Oct3/4 (Santa Cruz, 1 :20), TRA-1 -60 (Millipore, 1 :50), APC-conjugated CD34, APC-conjugated CD144/VE-cadherin (both from eBioScience, 1 :50), BV421 -conjugated CD31 (BioLegend, 1 :100), and FITC-conjugated KDR/VEGFR2 (Miltenyi Biotec, 1 :100) antibodies. Alexa-Fluor 488-conjugated secondary antibody (Life Technologies, 1 :1 ,000) and fluorophore-conjugated isotype controls were used accordingly. CD34 was assessed via single staining while KDR, CD31 and CD144 were assessed via triple staining. KDR ⁺ population was determined via histogram analysis, followed by scatter plot analysis of both CD31 and CD144/VE-cadherin within the KDR ⁺ population. CD31 and CD144/VE-cadherin were analyzed separately with their respective isotype controls to determine the gate settings for the scatter plot. Data were analyzed using MACSQuantify (Miltenyi Biotec) and FlowJo software.

[0110] Immunocvtochemistry

[0111 ] On day 1 1 (Protocol I) or day 15 (Protocol II), adherent cells were fixed with 4% paraformaldehyde in 1 x PBS for 20 minutes at 4°C, permeabilized and blocked in 0.1 % Triton X, 5% goat serum, and 1 % bovine serum albumin (BSA) in 1 x PBS for 15 minutes at room temperature. Cells were immunostained with mouse anti-human CD31 antibody (BD Pharmingen, 1 :50), then labeled with Alexa-Fluor 488-conjugated goat anti-mouse secondary antibody (1 :1 ,000), 30 minutes each at room temperature. The antibodies were diluted in 5% goat serum, 1 % BSA in 1 x PBS, and cells were washed with PBS between incubations. Samples were preserved in ProLong Gold antifade reagent with DAPI (Life Technologies) and visualized under Leica DMi8 fluorescent microscope.

[0112] DilAcLDL uptake assay

[0113] On day 1 1 (Protocol I) or day 15 (Protocol II), cells were incubated with or without 1 pg/ml Acetylated Low Density Lipoprotein labeled with fluorescent probe Dil (DilAcLDL, Alfa Aesar) in endothelial amplification medium for 5 hours at 37°C. Thereafter, cells were washed three times with DPBS, dissociated in single-cell suspensions, fixed with Medium A, and permeabilized with Medium B. After the final wash with FACS buffer, cells were subjected to FACS analysis. To confirm the location of DilAcLDL taken up by hESC-derived ECs, after 5 hours of incubation, cells were fixed with 4% paraformaldehyde for 20 minutes at 4°C, washed three times with PBS, and preserved by ProLong Gold antifade reagent with DAPI. Fluorescent signals were detected using rhodamine filter of the Leica DMi8 fluorescent microscope.

[0114] Tube formation assay

[0115] Growth factor reduced Matrigel (Corning) was thawed on ice overnight, and 300 μΙ was added in each well of a pre-chilled 24-well plate. The plate was incubated at 37°C for 45 minutes for gel formation. On day 1 1 of differentiation (Protocol I), cells were dissociated by TrypLESelect and 7x10 ⁴ cells were added onto Matrigel in 500 μΙ endothelial amplification medium. After 4 days, tube formation was visualized under light microscrope. To determine the cell viability on Matrigel, cells were washed with warm HBSS twice and then incubated with 600 μΙ of Calcein AM dye diluted in HBSS (8 pg/ml; Life Technologies) for 30 minutes at 37°C, according to manufacturer's instructions. Fluorescent images were obtained from Leica DMi8 fluorescent microscope.

[0116] Statistical analysis

[0117] Data are presented as means ± SEM from independent differentiation batches. Differences in relative mRNA expression and surface marker expression at different time points of differentiation were assessed by one-way ANOVA analysis, corrected for multiple comparisons using Tukey post hoc test. All graphs and statistical analyses were generated by Prism Software 6.0 (GraphPad). Differences were regarded as significant at P < 0.05.

[0118] Results

[0119] FIGs. 2A-2F are six different graphs showing quantitative mRNA expression of specific markers for H1 cells from Day 0 to Day 1 1 of differentiation upon a LN-521 substrate for all 1 1 days, and without purification. As seen in FIG. 2A, high expression of Nanog indicated the presence of pluripotent stem cells. As seen in FIG. 2B, peak expression of MixM indicated mesoderm development on Day 3 of differentiation. Starting on Day 5, the expression of endothelial lineage markers CD34, CD31 , KD R, and CD144 increased. On Day 5, a significant increase in mRNA expression of KDR was observed, compared to Day 3 (P < 0.001 , FIG. 2E). On day 7, KDR mRNA expression increased further together with significant increases in CD34 and CD144 expression, compared to day 3 (P < 0.0001 , P < 0.05 and P < 0.05, respectively, FIGs. 2E, 2C, and 2F), suggesting that a majority of the cell culture contained endothelial progenitors. Flow cytometric analyses of hESCs-derived cells collected on Day 7 showed 74% CD34 ⁺ , 82% KDR ⁺ , 76% CD31 ⁺ , 76% CD144 ⁺ and overall 62% KDR ⁺ CD31 ⁺ CD144 ⁺ in total cell population (FIG. 8E), indicating that there was a significant population of cells expressing CD31 at this time point with relatively low mRNA expression. Mature endothelial markers CD31 and CD144 had their highest expression on Day 1 1 . [0120] FIGs. 1 A-1 D are bright-field images of the cells on Days 0, 3, 7, and 1 1 (with and without purification). A morphological change after 3 days of mesoderm induction is seen when comparing FIG. 1A and FIG. 1 B. FIG. 1 C and FIG. 1 D illustrate the difference before and after the endothelial amplification medium is applied. For comparison, FIG. 1 E is an image of cells that were cultured according to Protocol I (i.e. with purification on Day 7), and shows the result on Day 1 1 . FIG. 1 D and FIG. 1 E can be directly compared to each other.

[0121 ] Generally speaking, the quantitative PCR analysis (qPCR) carried out at various time points showed decreased expression of pluripotency markers (Sox2, Nanog, Oct3/4), as well as gradually increasing expression of endothelial lineage markers (KDR/VEGFR2 , CD34, CD31 , CD144/VE-cadherin).

[0122] Four different sets of data were obtained. All four sets used LN-521 as the first cell culture substrate. Two sets used LN-521 as the second cell culture substrate, while two sets used an LN-521 /421 combination (1 :1 v/v) as the second cell culture substrate. Two sets were performed according to Protocol I, while two sets were performed according to Protocol II. Flow cytometry analysis of pluripotency markers was conducted on each set.

[0123] Before differentiation started, hESCs were collected and immunostained for pluripotency markers Oct3/4 and TRA-1 -60, in addition to endothelial markers CD34, CD144, CD31 , and KDR, for antibody specificity testing. FACS analysis showed 99% Oct3/4 ⁺ and 100% TRA-1 -60 ⁺ while no CD34 ⁺ , CD31 ⁺ , CD144 ⁺ or KDR ⁺ populations were detected on day 0. The results of marker testing on Day 0 are shown in FIGs. 3A-3F. In these Figures, as well as FIGs. 3A-7D, the red line is the isotype control (labeled C), and the blue line is the positive population (labeled P).

[0124] After purification on day 7, CD31 ⁺ cells were allowed to proliferate for 4 days on either an LN-521 or LN-521/421 substrate (Protocol I). Almost 100% pure population of endothelial progenitor cells were obtained using either second cell culture substrate, expressing CD34 and co-expressing triple markers CD31 , CD144 and KDR.

[0125] FIGs. 4A-4D show the results for CD34, CD31 , CD144, and KDR for the set cultured initially on LN-521 , then replated on LN-521 using Protocol I. FIG. 4E is a graph showing CD31 ⁺ and CD144 ⁺ populations within the KDR ⁺ population. [0126] FIGs. 5A-5D show the results for CD34, CD31 , CD144, and KDR for the set cultured initially on LN-521 , then replated on LN-521/421 using Protocol I. FIG. 5E is a graph showing CD31 ⁺ and CD144 ⁺ populations within the KDR ⁺ population.

[0127] When the CD31 ⁺ population was purified on day 1 1 instead of day 7 (Protocol II), a decrease in KDR expression was observed 4 days later, while CD31 and CD144 still had high expression levels, indicating a more mature population was generated.

[0128] FIGs. 6A-6D show the results for CD34, CD31 , CD144, and KDR for the set cultured initially on LN-521 , then replated on LN-521 using Protocol II. FIG. 6E is a graph showing CD31 ⁺ and CD144 ⁺ populations within the KDR ⁺ population.

[0129] FIGs. 7A-7D show the results for CD34, CD31 , CD144, and KDR for the set cultured initially on LN-521 , then replated on LN-521/421 using Protocol II. FIG. 7E is a graph showing CD31 ⁺ and CD144 ⁺ populations within the KDR ⁺ population.

[0130] The results of these four sets of data are also summarized in the following table. In the table, "% CD34+ CD144+ in KDR+" refers to the percentage of cells in the KDR+ population that are also CD34+ and CD144+. In contrast, "% KDR ⁺ CD31 ⁺ CD144 ⁺ refers to the percentage of cells in the total population that are positive for all three markers.

[0131 ] Next, FIGs. 8A-8E compare the FACS results for six different time points of the various different sets of data. First, H1 cells were differentiated upon a LN-521 substrate for 1 1 days without purification. Letter A refers to the results on Day 7, and Letter B refers to the results on Day 1 1 of this data set where no purification occurred. Letter C refers to the results on Day 1 1 for cells cultured initially on LN-521 , then replated on LN-521 using Protocol I (i.e. FIGs. 4A-4E). Letter D refers to the results on Day 1 1 for cells cultured initially on LN-521 , then replated on LN-521/421 using Protocol I (i.e. FIGs. 5A-5E). Letter E refers to the results on Day 1 5 for cells cultured initially on LN-521 , then replated on LN-521 using Protocol II (i.e. FIGs. 6A-6E). Letter F refers to the results on Day 15 for cells cultured initially on LN-521 , then replated on LN-521 /421 using Protocol II (i.e. FIGs. 7A-7E).

[0132] FIG. 8A shows the results for CD34 ⁺ . FIG. 8B shows the results for CD31 ⁺ . FIG. 8C shows the results for CD144 ⁺ . FIG. 8D shows the results for KDR ⁺ . FIG. 8E shows the results for KDR ⁺ CD31 ⁺ CD144 ⁺ .

[0133] As seen in comparing columns A-B to columns C-F, purification on either day 7 (Protocol I) or day 1 1 (Protocol II) using CD31 ⁺ magnetic beads significantly improved the yields of CD34 ⁺ , KDR ⁺ , CD31 ⁺ and CD144 ⁺ populations. As seen in FIG. 8D, KDR expression was significantly reduced from day 7 to day 1 1 (without purification). A small reduction was observed when CD31 ⁺ cells were purified using Protocol I versus Protocol II on LN-521 substrate; compare column C to E in FIG. 8B. When purified cells were plated on an LN-521/421 combination substrate, there was no significant difference between Protocol I and Protocol II; CD31 ⁺ and CD144 ⁺ populations were maintained at almost 100% on either the LN-521 or LN-521/421 second cell culture substrate.

[0134] Using Protocol I, 97% KDR ⁺ , 95% CD34 ⁺ , 96% CD31 ⁺ , and 95% CD144 ⁺ populations were consistently obtained, and 94% of the final culture were triple positive for KDR, CD31 and CD144.

[0135] Using Protocol II, 94% CD34 ⁺ , 95% CD31 ⁺ , and 93% CD144 ⁺ populations were consistently obtained. The KDR ⁺ population was 86% on LN-521 substrate, and 88% on the LN-521/421 substrate, so the final triple positive populations for KDR, CD31 and CD144 were 84% on LN-521 substrate and 87% on LN-521/421 substrate .

[0136] Next, immunofluorescent analysis of the CD31 ⁺ population was performed on Day 1 1 for Protocol I, and on Day 15 for Protocol II, on both LN-521 and LN-521/421 substrates (used as the second cell culture substrate). FIGs. 9A-9D show the results; green is CD31 and blue is DAPI. CD31 was expressed at the cell membrane in all conditions.

[0137] One hallmark of endothelial cells is their ability to take up acetylated low density lipoprotein (AcLDL) via the "scavenger cell pathway" of LDL metabolism. At the end points of each protocol (Protocol I or II, replated on LN-521 or LN-521/421 ), the endothelial cultures were incubated with 1 g/ml AcLDL conjugated with Dil dye for 5 hours in endothelial amplification medium at 37°C, then harvested for FACS analysis and visualized via fluorescent microscopy.

[0138] FIGs. 10A-10D are the visualized microscopic images. FIG. 10A is for Day

1 1 , Protocol I, LN-521 substrate. FIG. 10B is for Day 1 1 , Protocol I, LN-521 /421 substrate. FIG. 10C is for Day 15, Protocol II, LN-521 substrate. FIG. 10D is for Day 15, Protocol II, LN-521 /421 substrate.

[0139] FIGs. 11A-11 E are the FACS results. FIG. 11 A is for Day 1 1 , Protocol I, LN- 521 substrate. FIG. 11 B is for Day 1 1 , Protocol I, LN-521/421 substrate. FIG. 11 C is for Day 15, Protocol II, LN-521 substrate. FIG. 11 D is for Day 15, Protocol II, LN-521 /421 substrate. FIG. 11 E compares the results of all four protocols with each other. In protocol I, 96% of endothelial cells in both LN-521 and LN-521/421 cultures were positive for DilAcLDL, while 94% were positive from protocol II, suggesting that they contained almost homogenous endothelial cells.

[0140] A Matrigel® tube formation assay was also performed to determine the angiogenic potential of the hESCs-derived cells following Protocol I and replated on LN- 521/421 substrate. On day 1 1 , cells were dissociated into single cell suspension and plated on Matrigel® (35,000 cells/cm ² ) in endothelial amplification medium. Tube-like structures were detected 4 days later. To determine the viability of the cells, Calcein- AM dye was added to the culture and incubated for 30 minutes to stain live cells. Strong green fluorescent signals were detected. FIG. 12A is a picture of the cells, and FIG. 12B is a picture of the stained cells.

EXAMPLE 2

[0141 ] Experiments were also performed following Protocol I (purification after 7 days) and Protocol II (purification after 1 1 days) as described above, using H1 cells. However, the initial cell culture substrate was LN-51 1 only, and the second cell culture substrate was either LN-51 1 or LN-51 1/421 .

[0142] FIGs. 13A-13E compare the FACS results for six different time points of the various different sets of data. First, for comparison, human embryonic stem cells were differentiated upon a LN-51 1 substrate for 1 1 days without purification. Letter A refers to the results on Day 7, and Letter B refers to the results on Day 1 1 of this data set where no purification occurred. Letter C refers to the results on Day 1 1 for cells cultured initially on LN-51 1 , then replated on LN-51 1 using Protocol I. Letter D refers to the results on Day 1 1 for cells cultured initially on LN-51 1 , then replated on LN-51 1 /421 using Protocol I. Letter E refers to the results on Day 1 5 for cells cultured initially on LN- 51 1 , then replated on LN-51 1 using Protocol II. Letter F refers to the results on Day 15 for cells cultured initially on LN-51 1 , then replated on LN-51 1 /421 using Protocol II.

[0143] FIG. 13A shows the results for CD34 ⁺ . FIG. 13B shows the results for CD31 ⁺ . FIG. 13C shows the results for CD144 ⁺ . FIG. 13D shows the results for KDR ⁺ . FIG. 13E shows the results for KDR ⁺ CD31 ⁺ CD144 ⁺ . KDR expression was significantly reduced from Day 7 to Day 1 1 (without purification). There were no significant differences in CD34 ⁺ , KDR ⁺ , CD31 ⁺ and CD144 ⁺ populations between the two protocols on either LN-51 1 or LN-51 1/421 . CD31 ⁺ and CD144 ⁺ populations maintained almost 100% on either substratum.

[0144] A DilAcLDL assay was also performed. At the end points of each protocol (Protocol I or II, replated on LN-51 1 or LN-51 1/421 ), the endothelial cultures were incubated with 1 pg/ml DilAcLDL dye for 5 hours in endothelial expansion medium at 37°C, then harvested for FACS analysis. Letter A refers to the results on Day 1 1 for cells cultured initially on LN-51 1 , then replated on LN-51 1 using Protocol I. Letter B refers to the results on Day 1 1 for cells cultured initially on LN-51 1 , then replated on LN- 51 1/421 using Protocol I. Letter C refers to the results on Day 15 for cells cultured initially on LN-51 1 , then replated on LN-51 1 using Protocol II. Letter D refers to the results on Day 15 for cells cultured initially on LN-51 1 , then replated on LN-51 1/421 using Protocol II. The results are shown in FIG. 14. In all conditions, 93% to 95% of DilAcLDL ⁺ population was obtained from the culture.

EXAMPLE 3

[0145] HS1001 cells were differentiated on LN-521 substrate for 1 1 days without purification. FIGs. 15A-15D are bright-field images of the cells on Days 0, 3, 7, and 1 1 (i.e. without purification). A morphological change after 3 days of mesoderm induction is seen when comparing FIG. 15A and FIG. 15B. FIG. 15C and FIG. 15D illustrate the difference before and after the endothelial amplification medium is applied. For comparison, FIG. 15E is an image of cells that were cultured according to Protocol I (i.e. with purification on Day 7), and shows the result on Day 1 1 . FIG. 15D and FIG. 15E can be directly compared to each other.

EXAMPLE 4

[0146] Human embryonic stem cells were subjected to Protocol I. On day 1 1 of differentiation, cells were collected, stained for EC-specific markers (CD34, KDR, CD31 and CD144), then subjected to FACS analysis. Four different sets of data, labeled A-D, were obtained which varied based on the laminin in the first cell culture substrate and the second cell culture substrate. The results are summarized in the table below, and demonstrate that a large proportion of the stem cells had differentiated to endothelial progenitor cells on day 1 1 , as shown by CD34 ⁺ staining and triple-stained KDR ⁺ CD31 ⁺ CD144 ⁺ .

EXAMPLE 5

[0147] Human embryonic stem cells were subjected to either Protocol I or Protocol II on an LN-521 substrate. The resulting EPCs were characterized with reference to hESCs and fully mature HUVECS that were also cultured on an LN-521 substrate.

[0148] In order to identify global transcriptomic changes across these cell states, RNA sequencing as well as transcriptome abundance data for the enrichment of biological processes in different cell types was analyzed and compared to hESCs and HUVECs. Principal components analysis (PCA), based on the overall transcriptome signatures from expressed genes (FPKM > 5 in at least one sample), revealed 63.7% of the total expression variability in the first principal component due to separation of hESCs from other cell types. The second principal component explained 31.3% of the variability originating from the separation of EPC samples from the mature HUVECS, as shown in FIG. 16A.

[0149] Gene set enrichment analysis (GSEA) was performed on the transcriptomic data to identify biological pathways that are enriched for up and down-regulated genes in EPC and HUVEC samples compared to hESCs. Using the KEGG and Gene Ontology Biological Process (GOBP) pathway repositories, several biological mechanisms were discovered that were significantly altered at false discovery rate (FDR) < 5%. From this list, a selection of key KEGG and GOBP pathways related to extracellular matrix (ECM) biology and endothelial lineage was further investigated to identify the pattern of changes in their constituent genes across hESCs, EPCs, and HUVEC samples. Specifically, from their gene-level expression patterns shown in heat maps in FIG. 16B, changes were highlighted in the GOBP pathways (wound healing and vasculature development) and in the KEGG pathways (JAK-STAT signaling and ECM-receptor interactions).

[0150] For each of these pathways, subsets of genes were observed that displayed cell stage-specific maximal expression, suggesting that different segments of the pathways were operative at different points of cellular differentiation. For example, considering the ECM-receptor interactions pathway, a subset of genes was identified that showed the lowest expression in the hESC state and highest expression in the HUVEC state (e.g., ITGA2, VWF, HSPG2, etc.). Another set of genes in the same pathway showed the highest levels of expression in hESCs and the most reduced expression in HUVECs (e.g., ITGA7, SPP1 , COL1 A2, etc.). A third category of genes showed maximal expression in EPC samples, compared to both hESCs and HUVECs (e.g., COL4A1 , COL4A2, ITGA4, etc.).

[0151 ] These findings were further elaborated in the gene-level plots of FIGs. 16C-E. Relevant genes within each pathway were extracted and their expression profiles plotted (in normalized FPKM) on the same scale. FIG. 16C illustrates the expressions of some major ECM genes during maturation of hESCs-derived endothelial lineage. The primary HUVECs exhibited high expression of laminin α4, β1 , β2, and γ1 chains, corresponding to LN-41 1 and LN-421 , revealing that mature ECs express high levels of isoforms that are specific for subendothelial BM. In comparison, α5, β2, and γ1 were the predominant laminin chains found in the hESC-derived EPCs. HUVECs also expressed high levels of perlecan (HSPG2) as well as matrix metallopeptidases MMP1 and MMP2, while hESC-derived EPCs significantly exhibited higher levels of collagen IV a1 and a2 subunits (COL4A1 and COL4A2), compared to those in HUVECs.

[0152] Major receptors for ECM proteins (e.g., integrin subunits) were analyzed and provided insight into cell-matrix interactions as shown in FIG. 16D. The data demonstrated a shift from α6β1 integrin (ITGA6, ITGB1 ) in hESCs to predominantly α5β1 integrin in the EPCs and to a lesser extent ανβδ (ITGAV, ITGB5), an integrin required for VEGF- or TGFa-induced angiogenesis.

[0153] As exhibited in FIG. 16E, the expression profile of the EPCs revealed up- regulation of specific genes along the endothelial lineage. Compared to mature HUVECs, the hESCs-derived EPCs from both protocols highly expressed early endothelial markers, such as CD34, VEGFR1 (FLT-1 ), KDR/VEGFR2, and neuropilin-1 (NRP1 ), which together with CD31 identified a population of hESC-derived cells capable of giving rise to stable cord-blood endothelial colony-forming cells. The expression of some mature endothelial markers, such as CD144/VE-cadherin, TIE1 , TIE2, MCAM and CD31 , was comparable between EPCs and HUVECs. These results suggested that hESC-derived EPCs from both protocols were still at a progenitor stage.

EXAMPLE 6

[0154] To determine whether hESC-derived EPCs had the potential to reach a fully mature phenotype, cells were allowed to mature further in endothelial amplification medium and their mRNA expression profiles analyzed at 2, 3, 4, 5 and 6 weeks after purification. These expression profiles are exhibited in FIGs. 17A-I. During the maturation, cell numbers did not decline and were similar in both protocols. A decrease in mRNA level of KDR/VEGFR2 (immature marker) and increases in the expression of VWF and LAMA4 (encoding laminin a4 chain) (mature endothelial markers) over time were observed, comparable to those of HUVECs. Importantly, CD31 and CD144/VE- cadherin maintained their high expression levels over long-term culturing. The major laminin β chain expressed by the EPCs was β2 (LAMB2), whereas HUVECs showed a much higher β1 mRNA level (LAMB1 ). Interestingly, one of the key matrix metallopeptidases MMP1 exhibited an increase in gene expression over time to a similar extent as seen in HUVECs, indicating a faster turnover rate once the cells become more and more mature.

[0155] Overall, it was demonstrated that hESC-derived EPCs from Protocol II were more mature than those from Protocol I, as evidenced by lower KDR/VEGFR2 and higher VWF, LAMA4 and MMP1 expression levels. It was also shown that the presence of TGFp inhibitor allowed hESC-derived EPCs to mature to ECs.

[0156] The present disclosure has been described with reference to exemplary embodiments. Obviously, modifications and alterations will occur to others upon reading and understanding the preceding detailed description. It is intended that the present disclosure be construed as including all such modifications and alterations insofar that they come within the scope of the appended claims or the equivalents thereof.