Method for obtaining endothelial cells from pluripotent stem cells

Abstract

The invention relates to a method for obtaining endothelial cells from human pluripotent stem cells. In particular, the present invention relates to a method for obtaining three-dimensional endothelial cells from human pluripotent stem cells of embryonic origin or induced human pluripotent stem cells, compatible with GMP standards.

Description

Description Title: Method for obtaining endothelial cells from pluripotent stem cells

[0001] The present invention relates to a method for obtaining endothelial cells from embryonic pluripotent stem cells (Pluripotent Stem Cell or PSC) or induced pluripotent stem cells (IPS) in three dimensions. Context of the invention

[0002] Human pluripotent stem cells, whether embryonic or induced to pluripotency, have the ability to proliferate identically (each mother cell giving rise to two daughter cells identical to the first) indefinitely, without ever entering senescence like all other cells in the organism, and the ability, under other culture conditions, to differentiate to give rise to any cell of the organism (ectoderm, endoderm and mesoderm).

[0003] Stem cells are important in regenerative medicine (a major and promising source of interest for successfully manufacturing organs), for modeling, particularly of diseases, and for pharmacological screening.

[0004] Pluripotent stem cells appear as a possible alternative due to their unlimited proliferation and differentiation capacity, allowing for the production of all cell types of interest in large quantities from a single donor. Furthermore, pluripotent stem cells provide a homogeneous population of differentiated cells, unlike primary cell cultures (which are heterogeneous, limited in number, and risk senescence).

[0005] Methods for differentiating pluripotent stem cells into endothelial cells are already known. Protocols that yield approximately 70% of cells of interest at the end of differentiation are generally described in the literature (e.g., in Gu, Curr Protoc Hum Genet, 98(1):e64, (2018); Olmer et al. Stem Cell Reports. 2018 May 8; 10(5): 1657-1672). Therefore, to enrich the cell population, a cell sorting step after the differentiation protocol is generally necessary.

[0006] The known and used methods for sorting cells following a differentiation protocol are MACS and FACS. These methods are performed with steps carried out under a laminar flow hood and are sensitive to contamination.

[0007] On the one hand, the Magnetic-Associated Cell Sorting (MACS) method is described. It can be defined as separation using magnetic beads coupled to antibodies. Cell sorting requires several preparation steps, including washing and centrifugation, as well as an incubation step with specific antibodies. After a final washing and centrifugation step, the cells are deposited at the top of the column, and the labeled cells are retained. After several gravity washes, the cells are detached mechanically using a syringe plunger. These steps are lengthy and stressful for the cells, which represents a limitation of this method.In addition, the MACS method has several other disadvantages: sorting large quantities of cells requires a considerably long purification time and there is a risk of external contamination of the magnetic separation columns which makes the method incompatible with Good Manufacturing Practices (GMP).

[0008] The documents Masuda et al. (Regen Ther., 9:1-9, (2018)); Gu et al. (Curr Protoc Hum Genet, 98(1):e64, (2018)); Olmer et al. (Stem Cell Reports, 10, 1657-1672, (2018)); Mulfaul et al. (Stem Cell Res Ther 11, 409 (2020)) or Abutaleb et al. (STAR ​​Protocols, 2(2), (2021)) describe, for example, the use of the MACS method.

[0009] On the other hand, the FACS (Fluorescence-Associated Cell Sorting) method is well-known. This method can be defined as flow cytometry separation after labeling with a fluorescent antibody, using the same preparation techniques as the MACS method. The paper by Sriram et al. (Stem Cell Res TherQ, 261 (2015)) illustrates, for example, the use of the FACS method.

[0010] While the FACS method allows for the purification of cell populations based on combinations of markers rather than a single marker (enrichment of subpopulations of interest), it does have drawbacks. The processing time is very long (over 6 hours) for large quantities of cells, which is particularly detrimental to cell viability and makes the FACS method incompatible with industrial scale-up. The system also uses washable tubing, which is incompatible with GMP standards because it is not single-use. Solutions exist for single-use Flow Cytometry (FCM) sorting cartridges (MACSQuant® Tyto®; Miltenyi Biotec), but these solutions are not suitable for sorting a high proportion of cells of interest, i.e., a proportion greater than 50%.

[0011] The cell sorting stage is therefore a complex, critical and limiting step in the industrial scaling up of a cell differentiation process and in bringing it into compliance with pharmaceutical grade cell production standards under "clinical" conditions, i.e. conditions where the products used are manufactured according to GMP Standards and can therefore be used in clinical studies unlike "research" grade products which can only be used for research.

[0012] The sorting stage is therefore a lengthy and costly process, potentially impacting cell quality and leading to microbiological contamination. However, it is often necessary to enrich the cell population after a differentiation protocol, in order to then proceed to an amplification stage.

[0013] Human umbilical vein endothelial cells (HUVECs) are endothelial cells derived from human umbilical cord blood. They can be obtained in large quantities. However, they can exhibit heterogeneity issues, and there is a risk of batch variability between donors.

[0014] Obtaining endothelial cells from pluripotent stem cells therefore appears to be the preferred alternative, despite the sorting step to be carried out.

[0015] To date, there remains a need to obtain endothelial cells in large quantities as easily as possible, and under so-called "clinical" conditions. Indeed, the needs are increasingly numerous in various areas of clinical application, such as obtaining reconstituted tissues usable in therapy requiring the production of large quantities of cells (several hundred million, or even several billion).

[0016] The purity of the cell population is also an important factor: there is a need to obtain a cell population comprising more than 95% of the cells of interest, preferably more than 98% and more than 99%. This step represents a technological hurdle to the mass production of differentiated cells of interest from pluripotent stem cells.

[0017] Obtaining a cell population with approximately 90% purity has already been described (Hamad et al. Stem Cell Res Ther, 251 (2022)). The authors describe the differentiation of endothelial cells from human iPS cells without the use of serum. The described method eliminates the need for sorting and amplification steps.

[0018] The present invention is based on the Inventors' results demonstrating the production of an endothelial cell population comprising more than 95% of the cells of interest, preferably more than 99%, by performing an amplification and maturation step directly after the differentiation process. This process also eliminates the need for a cell sorting step. Surprisingly, the Inventors have shown that performing an amplification and maturation step, using microcarriers, directly after the differentiation process, yields a pure endothelial cell population. The Inventors have also shown that the purity of this endothelial cell population is improved when platelet lysate is used in the amplification and maturation step following differentiation.

[0019] The present invention therefore relates to a method of obtaining a pure population of endothelial cells from embryonic pluripotent stem cells (Pluripotent Stem Cell or PSC) or three-dimensional induced pluripotent stem cells (iPS) in which the sorting step is eliminated.

[0020] The present invention relates to a method for obtaining endothelial cells in three dimensions, under so-called "clinical" conditions, that is to say, conditions where the products used are manufactured according to Good Manufacturing Practices (GMP) and can therefore be used in clinical studies unlike "research" grade products which can only be used for research.

[0021] The "GMP" standards constitute a quality assurance concept established by the European (or American) Commission for the manufacture of medicines for human or veterinary use (EUDRALEX in France or FDA in the United States). They were created to limit the risks of cross-contamination of products, by emphasizing hygiene practices, as well as the risks of confusion: labeling / identification.

[0022] Good Manufacturing Practices (GMP) principles mandate the creation of standard operating procedures and instructions to ensure consistent quality production with compliant traceability. They also encompass processes, product quality, and personnel safety. GMP is currently organized into three parts: Good manufacturing practices for medicinal products for human use. Good manufacturing practices for active substances used as raw materials in medicines. Documents relating to good manufacturing practices providing recommendations on international requirements for batch certification.

[0023] To establish protocols for obtaining "clinical" endothelial cells, the raw materials must meet the same GMP standards. The products used are therefore fully defined in terms of their composition, concentration, origin, and sterility levels. Description of the invention

[0024] The present invention relates to a method for obtaining three-dimensional endothelial cells from human pluripotent stem cells of embryonic origin or induced human pluripotent stem cells which meets GMP standards.

[0025] The present invention relates to a method for obtaining in three dimensions a pure population of endothelial cells by differentiation of embryonic pluripotent stem cells (Pluripotent Stem Cell or PSC) or induced pluripotent stem cells (iPS) comprising an amplification and maturation step using microcarriers directly after differentiation.

[0026] More specifically, the present invention relates to a method for differentiating human pluripotent stem cells into endothelial cells, in which: a) at 0.01, human pluripotent stem cells, previously frozen in cell clusters, are seeded at a concentration of 100,000 to 800,000 cells / mL, preferably around 400,000 cells / mL, and cultured in the presence of a medium suitable for the culture of pluripotent cells and a ROCK inhibitor; b) at 0.02, the medium is replaced by a medium suitable for mesoderm induction, further comprising an inhibitor of GSK3 (Glycogen Synthase Kinase 3), BMP4 (Bone Morphogenetic Protein 4), and N2B27 CTS; c) at J5, the medium is replaced by a medium adapted to the maturation of mesodermal progenitors into endothelial cells, also including VEGF (Vascular Endothelial Growth Factor), forskolin and fibroblast growth factor 2 (“Fibroblast Growth Factor 2” or FGF2); d) at J7, the cells are dissociated and immediately put back into culture on microcarriers and amplified and matured in bioreactors for 8 to 11 days.

[0027] More specifically, the present invention relates to a method for differentiating human pluripotent stem cells into endothelial cells, in which: a) at J0, human pluripotent stem cells previously frozen in cell clusters are seeded at a concentration of 100,000 to 800,000 cells / ml, preferably around 400,000 cells / mL and cultured in the presence of a medium suitable for the culture of pluripotent cells and a ROCK inhibitor in a bioreactor; b) on day 2, the medium is replaced by a medium adapted for the induction of the mesoderm including in addition an inhibitor of GSK3 (Glycogen Synthase Kinase 3) of BMP4 (Bone Morphogenetic Protein 4); of N2B27 CTS; c) at J5, the medium is replaced by a medium adapted to the maturation of mesodermal progenitors into endothelial cells, also including VEGF (Vascular Endothelial Growth Factor), forskolin and fibroblast growth factor 2 (“Fibroblast Growth Factor 2” or FGF2); d) at J7, the cells are dissociated and immediately put back into culture on microcarriers and amplified and matured in bioreactors for 8 to 11 days.

[0028] Advantageously, all culture media and agents used are chemically defined and do not contain any additives of animal origin not controlled according to Good Manufacturing Practices (or GMP) standards.

[0029] By "pluripotent stem cells" is meant any undifferentiated cell capable of indefinite self-renewal and of differentiating into all cell types (ectoderm, endoderm, mesoderm). Preferably, the pluripotent stem cells according to the invention are human pluripotent stem cells ("human pluripotent stem cell" or "hPSC").

[0030] In one embodiment, pluripotent stem cells are human embryonic stem cells (hESCs). There are many hESC lines, including the RC-9 line (Roslin Cells 9), which was developed as a clinical-grade hESC line.

[0031] In a preferred embodiment, human pluripotent stem cells are obtained by methods that do not require the destruction of embryos.

[0032] In another embodiment, the stem cells are human induced pluripotent stem cells (hiPSCs). Numerous commercial hiPSC lines exist, as well as lines manufactured using various reprogramming techniques (episome, mRNA, Sendai viral vector).

[0033] According to one embodiment of the invention, pluripotent stem cells are donor-specific hiPSCs obtained by reprogramming peripheral blood mononuclear cells from said donor. The reprogramming protocols for peripheral blood mononuclear cells from the donor or for CD34+ cells isolated from umbilical cord blood are known to those skilled in the art.

[0034] By "medium suitable for the culture of pluripotent cells" we mean any medium which contains the nutrients and factors enabling the in vitro culture of pluripotent cells.

[0035] Preferably, the medium suitable for culture of pluripotent cells is chosen from iPS Brew XF medium (marketed by Miltenyi Biotec) or iPS Brew GMP (marketed by Miltenyi Biotec), and iPS Stempro medium (marketed by ThermoFisher).

[0036] In step a), the medium is supplemented with a ROCK inhibitor. Preferably, the ROCK inhibitor is provided within the RI supplement (Y-27632) marketed by Stemcells.

[0037] According to one embodiment, the final concentration of ROCK inhibitor is between 5 and 15 pM, preferably about 10 pM.

[0038] The term "mesoderm-induction medium" refers to any medium containing the nutrients and factors necessary for inducing the mesodermal pathway in pluripotent cells. Preferably, a mesoderm-induction medium includes N2 and B27 supplements. N2B27 CTS medium is a 1:1 mixture of DMEM-F12 CTS KO medium and Neurobasal CTS medium, supplemented with Glutamax CTS, N2 CTS, [3-mercaptoethanol], and B27 CTS.

[0039] In step b), the medium adapted for mesoderm induction also includes a GSK3 and BMP4 inhibitor.

[0040] The practitioner has at their disposal a number of agents known to inhibit the GSK3 kinase. Typically, the GSK3 inhibitor may be Chir99021, marketed by the company Tocris.

[0041] According to one embodiment, the final concentration of Chir99021 is between 5 and 10 pM, preferably about 6 pM.

[0042] According to one embodiment, the final concentration of BMP4 is between 15 and 50 ng / ml, preferably around 25 ng / ml.

[0043] The term "medium suitable for the maturation of mesodermal progenitors into endothelial cells" refers to any medium that allows specification towards the endothelial lineage. Preferably, this medium suitable for the maturation of mesodermal progenitors is also suitable for the culture of endothelial cells.

[0044] Even more preferred, this medium adapted to the maturation of mesodermal progenitors and also adapted to the culture of endothelial cells is the EndoGO medium (marketed by the company Sartorius).

[0045] According to one embodiment, the medium adapted for the maturation of mesodermal progenitors can be supplemented by a medium adapted for the culture of endothelial cells, or by any molecules or substances allowing the in vitro culture of endothelial cells, such as VEGF, forskolin and FGF2.

[0046] By "medium suitable for the culture of endothelial cells" we mean any medium which contains the nutrients and factors enabling the in vitro culture of endothelial cells.

[0047] Preferably, the medium suitable for culturing endothelial cells is the EndoGO medium marketed by the company Sartorius.

[0048] Preferably, VEGF is supplied at a final concentration between 100 and 300 ng / mL, even more preferably around 200 ng / mL.

[0049] Preferably, forskolin is supplied at a final concentration between 2 and 6 pM, even more preferably about 4 pM.

[0050] Preferably, FGF2 is supplied at a final concentration between 100 and 400 ng / mL, even more preferably around 200 ng / mL.

[0051] According to one embodiment of the invention, the medium is renewed at J6.

[0052] At day 6, a certain portion of the cell population differentiated into endothelial cells.

[0053] On day 7, at the end of differentiation, the percentage of cells positive for the CD34 and / or CD31 marker is 30 to 75%. CD34+ and / or CD31+ cells are dissociated and immediately recultured on microcarriers and amplified and matured in bioreactors for 8 to 11 days. The phrase "dissociation and immediate reculture" means that the cells are isolated from one another and then immediately recultured for an amplification and maturation step. This means that no cell sorting is performed in the process according to the invention before amplification / maturation. The cells are dissociated using any technique known to those skilled in the art.

[0054] Preferably, the dissociation step is carried out by enzymatic treatment with a reagent such as trypsin (for example, TrypLE™ marketed by Thermo Fisher Scientific). TrypLE™ (marketed by Thermo Fisher Scientific) is a recombinant enzyme without any components of animal origin. Its use advantageously avoids any risk of transmission of pathogens of animal origin, and thus meets GMP requirements.

[0055] The term "microcarriers" refers to any type of matrix, generally spherical, used as a support for cell culture. Their diameter can range, for example, from 100 to 300 µm. Preferably, the microcarriers used in the process according to the invention have undergone a treatment or have a coating. Preferably, the microcarriers are selected from among those that have received physical treatment, such as polystyrene microcarriers with a synthetic treatment suitable for stem cell culture, such as Synthemax®, marketed by Corning, and those with biochemical treatment, such as collagen-coated polystyrene microcarriers, also marketed by Corning.

[0056] The amplification and maturation stage begins on day 7 and lasts at least 8 days. This stage is typically performed using a medium suitable for endothelial cell culture.

[0057] Preferably, when the medium used in step c) mentioned above is a medium also suitable for the culture of endothelial cells, the medium suitable for the culture of endothelial cells used in the amplification and maturation step is the same as that used in step c).

[0058] According to a preferred embodiment, during the amplification and maturation step, the medium suitable for endothelial cell culture is supplemented with VEGF and / or a platelet lysate. Preferably, the medium suitable for endothelial cell culture is supplemented with VEGF and a platelet lysate.

[0059] According to a preferred embodiment, VEGF is supplied here at a final concentration of between 50 and 100 ng / mL.

[0060] According to a preferred embodiment, the platelet lysate is supplied at a concentration of at least 2%, in particular at least 5%, preferably at least 10%. Typically, the platelet lysate may be ELAREM™ ULTIMATE-FDi (marketed by PL BioScience).

[0061] The use of a platelet lysate optimizes the obtaining of cells positive for the CD34 and / or CD31 marker at the end of the amplification and maturation step.

[0062] According to a preferred embodiment, the invention relates to a method for differentiating endothelial cells from human pluripotent stem cells, in which: a) at J0, human pluripotent stem cells previously frozen in cell clusters are seeded at a concentration of 100,000 to 800,000 cells / mL, preferably about 400,000 cells / mL, and cultured in the presence of a medium suitable for the culture of pluripotent cells (IPS-Brew) and a ROCK inhibitor (Y-27632 (10 iM)) in a bioreactor; b) on day 2, the medium is replaced by a medium adapted for the induction of the mesoderm including in addition a GSK3 (Glycogen Synthase Kinase 3) inhibitor (CHIR99021 (6pM), BMP4 (Bone Morphogenetic Protein 4) (25ng / mL), N2B27 CTS; c) at day 5, the medium is replaced by a medium adapted to the maturation of mesodermal progenitors into endothelial cells including in addition VEGF (Vascular Endothelial Growth Factor) (200 ng / mL), forskolin (between 2 and 4 pM, preferably 4 pM), fibroblast growth factor 2 (“Fibroblast Growth Factor 2” or FGF2) (200 ng / mL); d) at J7, the cells are dissociated and immediately put back into culture on microcarriers and amplified and matured in bioreactors for 8 to 11 days in the presence of a medium suitable for the culture of endothelial cells including VEGF (100 ng / mL), and at least 10% platelet lysate.

[0063] The present invention also relates to a population of endothelial cells directly obtained by the process described above. Advantageously, the endothelial cell population at the end of the amplification and maturation phase is homogeneous, i.e., more than 95%, preferably more than 98%, or more than 99% of the cells are positive for the CD34 and / or CD31 marker, preferably CD31.

[0064] The present invention also relates to the use of the endothelial cell population for the manufacture of a tissue, preferably a dermal tissue or skin substitute.

[0065] Examples

[0066] Example 1: Obtaining endothelial cells derived from hPSCs and phenotypic characterization of said cells

[0067] Previously frozen hiPSCs in cell clusters are seeded at 400,000 cells / mL in StemMACS IPS Brew XF medium supplemented with 10pM of ROCK inhibitor in Ambr250 bioreactor. Two days later, on day 2, the medium is replaced with a medium adapted for mesoderm induction: N2B27 CTS medium (1:1 mixture of DMEM-F12 KO and Neurobasal CTS medium supplemented with Glutamax CTS, N2 CTS and B27) with Chir99021 at 6pM and BMP4 at 25ng / ml.

[0068] On day 5, the medium is replaced with a medium adapted for the maturation of mesodermal progenitors into endothelial cells, also containing 200 ng / ml of VEGF, 4 pM of forskolin and 200 ng / ml of FGF2. The medium used is EndoGO medium (marketed by Sartorius).

[0069] On day 7 of differentiation, the differentiated cells are dissociated by enzymatic treatment with TrypLE™ and immediately recultured on Synthemax® microcarriers. They are then amplified and matured in bioreactors for 11 days. Amplification and maturation are carried out at 37°C under continuous stirring at 100 rpm, at a controlled pH of 7.2 ± 0.2 and an OD of 50%.

[0070] The cells obtained by this method were characterized phenotyptically. The cells obtained by the method of the invention clearly express the CD31 and CD34 markers.

[0071] Example 2: Testing under different amplification / maturation conditions

[0072] A) Presence or absence of microcarriers. Three different conditions for the amplification and maturation stage are compared: - Amplification / maturation using Synthemax® microcarriers, as described in Example 1, - Amplification / maturation as described in Example 1, except that the microcarriers used are collagen-coated microcarriers, - Amplification / maturation using spheroids: as described in Example 1, except that on the seventh day of differentiation, the dissociated cells are recultured in a suitable medium, and their reorganization into spheroids is observed. The results are presented in Table 1 below.

[0073] Table 1 Microcarriers Microcarriers coated with Synthemax ® Spheroids collagen Quantity of cells 8.00 x 10 5 8.00 x 10 5 1.20 x 10 7 sown Quantity of cells / well 6.76 x 10 5 6.44 x 10 5 4.68 x 10 5 Viability (%) 84.1 78.0 96.3 Amplification factor 0.84 0.80 0.04 Harvest yield (%) 36.47 56.60 11.69

[0074] The results highlight better amplification with the use of microcarriers than with the use of spheroids. Indeed, amplification and yield are very low when the cells are reorganized into spheroids.

[0075] B) Different concentrations of platelet lysate.

[0076] The cells used to assess the impact of platelet lysate concentration were obtained using the following method: a) at JO, human pluripotent stem cells previously frozen in cell clusters are seeded at a concentration of 400,000 cells / mL, and cultured in the presence of a medium suitable for the culture of pluripotent cells and a ROCK inhibitor (10pM); b) on day 2, the medium is replaced by a medium adapted for mesoderm induction including an inhibitor of GSK3 (6pM), BMP4 (25ng / mL); and N2B27 CTS; c) on day 5, the medium is replaced with a medium adapted for the maturation of mesodermal progenitors into endothelial cells, including VEGF (200 ng / mL), forskolin (4pM), fibroblast growth factor 2 (200 ng / mL) until day 7; d) on day 7, the differentiated spheroid cells are dissociated and immediately placed back into 2D culture for 6 days with a medium adapted for the culture of endothelial cells, including platelet lysate (2% or 10%) and VEGF (50 ng / mL) for amplification and maturation.

[0077] The re-culturing is performed here in 2D. This is a simplified model allowing measurement of the impact of the concentration of the platelet lysate and transposable to 3D in a bioreactor.

[0078] The medium used for culturing endothelial cells is EndoGO medium (marketed by the company Sartorius).

[0079] The platelet lysate (PL) is ELAREM™ ULTIMATE-FDi (marketed by PL BioScience).

[0080] The percentages of cells positive for the CD31 and CD34 markers were analyzed by the FACS method.

[0081] Two cellular sources, designated "source 04" and "source 33", were analyzed.

[0082] At the start of amplification, at the end of step c) source 04 comprised about 30% CD31+ / CD34+ cells while source 33 comprised more than 50% CD31+ / CD34+ cells.

[0083] The results obtained at the end of the amplification and maturation stage are presented in Table 2 below.

[0084] Table 2 Source - CD31 + CD34+ Supplementation CD31+ / CD34+ Source 04 - Fetal Bovine Serum 10% 56.30 72.50 72.60 Source 04 - Platelet Lysate 2% 83.90 93.60 84.70 Source 04 - Platelet Lysate 10% 97.10 98.00 97.00 Source 33 - Fetal Bovine Serum 10% 98.50 99.30 98.50 Source 33 - Platelet Lysate 2% 99.30 99.30 99.20 Source 33 - Platelet Lysate 10% 99.30 99.50 99.10

[0085] These results thus show that the addition of platelet lysate (2 or 10%) improves the percentage of CD31+ / CD34+ cells compared to the initial levels at the end of step c).

[0086] Example 3: Obtaining a cell population comprising more than 99% of cells positive for the CD31 and / or CD34 marker

[0087] The cells were obtained by the process described in Example 1.

[0088] The medium used for culturing endothelial cells is EndoGO, marketed by Sartorius. The concentration of ELAREM™ ULTIMATE-FDi platelet lysate (marketed by PL BioScience) used is 10%. The VEGF concentration is 100 ng / mL.

[0089] The percentages of cells positive for the CD31 and CD34 markers were analyzed by the FACS method.

[0090] Table 3 Amplification / Maturation in an Ambr250 Bioreactor Quantity of seeded cells: 25 x 10 6 Percentage of CD31 cells and CD31: 50.75 CD34 at the beginning of CD34: 50.43 amplification / maturation Microcarrier Surface 2500 cm 2 Culture volume during 240 ml amplification / maturation Culture time for the phase 11 days of amplification / maturation Quantity of cells at the end 129 x 10 6 amplification / maturation Viability (%) 94.2 Amplification / maturation factor 5.16 Percentage of CD31 cells and CD31: 99.33 CD34 at the end CD34: 99.91 amplification / maturation

[0091] This example demonstrates that the endothelial cell population at the end of the amplification / maturation phase is homogeneous, meaning that more than 99% of the cells are positive for the CD34 and / or CD31 marker. This shows that it is possible to bypass cell sorting at the end of differentiation. Indeed, this example shows that during the amplification phase, the cells complete their maturation towards the endothelial lineage, since at the beginning of the amplification phase, the cell population comprised approximately 50% CD31+ and / or CD34+ cells, and at the end of amplification / maturation, the cell population comprises more than 99% CD31+ and / or CD34+ cells.

Claims

Demands

1. A method for differentiating pluripotent stem cells into endothelial cells, wherein: a) at JO, human pluripotent stem cells previously frozen in cell clusters are seeded at a concentration of 100,000 to 800,000 cells / mL, preferably about 400,000 cells / mL, and cultured in the presence of a medium suitable for the culture of pluripotent cells and a ROCK inhibitor; b) on day 2, the medium is replaced by a medium adapted for mesoderm induction including an inhibitor of GSK3, BMP4; and N2B27 CTS; c) at J5, the medium is replaced by a medium adapted to the maturation of mesodermal progenitors into endothelial cells, also including VEGF, forskolin, and fibroblast growth factor 2; d) at J7, the cells are dissociated and immediately put back into culture on microcarriers and amplified and matured in bioreactors for 8 to 11 days.

2. A method according to claim 1 characterized in that the ROCK inhibitor is used at a concentration of 5 to 15 pM, preferably about 10 pM, in step a).

3. A method according to any one of the preceding claims, characterized in that the GSK3 inhibitor is Chir99021, preferably used at a concentration of 5 to 10 pM, even more preferably at a concentration of about 6 pM, in step b)

4. A method according to any one of the preceding claims, characterized in that BMP4 is used at a concentration between 15 and 50 ng / ml, preferably about 25 ng / ml in step b).

5. A process according to any one of the preceding claims, characterized in that FGF2 is used at a concentration of 100 to 400 ng / ml, preferably about 200 ng / ml, in step c).

6. A process according to any one of the preceding claims, characterized in that VEGF is used at a concentration between 100 and 300 ng / ml, preferably about 200 ng / ml in step c).

7. A process according to any one of the preceding claims, characterized in that forskolin is used at a concentration between 2 and 6 pM, preferably about 4 pM in step c).

8. A method according to any one of the preceding claims, characterized in that the cells are amplified and matured in the presence of a medium suitable for the culture of endothelial cells further comprising VEGF and a platelet lysate, at least 2%, preferably at least 10% platelet lysate.

9. A method according to any one of the preceding claims, characterized in that the pluripotent stem cells are human stem cells induced to pluripotency.

10. Population of endothelial cells obtained by the process as defined in claims 1 to 9.

11. Use of the endothelial cell population of claim 10 for the manufacture of a dermal tissue.

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