Method for generating endothelial cells from pluripotent stem cells

Description

[0001] The present invention relates to a method for obtaining endothelial cells from embryonic pluripotent stem cells (PSCs) or induced pluripotency stem cells (iPSCs). Background to the invention

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

[0003] Stem cells are important in regenerative medicine (a source of major interest and promise 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 property and their differentiation capacity, allowing to obtain from a single donor, all cell types of interest in large quantities. In addition, pluripotent stem cells allow to obtain a homogeneous population of differentiated cells, unlike primary cultures (heterogeneity, limit in number, risk of senescence).

[0005] The present invention relates to a method for obtaining endothelial cells, under so-called "clinical" conditions, i.e. 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.

[0006] "GMP" standards are a quality assurance concept established by the European (or American) Commission in the context of the manufacture of medicinal products 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.

[0007] GMP principles require the writing of operating procedures and instructions that enable consistent, high-quality production with compliant traceability. They also integrate processes, product quality, and personnel safety. GMPs are 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 medicinal products. Good manufacturing practice documents providing recommendations on international requirements for batch certification.

[0008] To establish protocols for obtaining "clinical" endothelial cells, raw materials must meet the same GMP standards. The products used are therefore fully defined in terms of their composition, concentrations, origins, and sterility levels.

[0009] Currently, there is no way to obtain endothelial cells in large quantities according to GMP standards. However, the need is growing in various areas of clinical application, such as obtaining reconstituted tissues usable in therapy.

[0010] HUVECs (Human Umbilical Vein Endothelial Cells) are endothelial cells derived from human umbilical cord blood. They can be obtained in large quantities. However, they can have heterogeneity issues and there is a risk that batches may exhibit variability between donors. The documents:

[0011] Makoto Sahara et al., Cell Research, vol 24, no7, 2014, p820 841; Liu Xiaopeng et al, Differentiation, vol 92, 2006, p 225-236; Hannah K. Wilson et al., Stem Cells, vol 32, 2014, p3037-3045; Gopu Sriram et al., Stem Cell Research & Therapy, vol 6, 2015; US2016 / 086135A1 CN109797132A and Xiaojun Lian et al., Stem Cell Reports, vol 3, 2014 p804-816 describe different protocols for the differentiation of human pluripotent cells into endothelial cells.

[0012] Pluripotent stem cells appear as a possible alternative due to their unlimited proliferation property and their differentiation capacity allowing to obtain from the same donor, all cell types of interest in large quantities.

[0013] To date, there is no method for differentiating human pluripotent stem cells into endothelial cells that meets GMP standards. Statement of the invention

[0014] The present invention relates to a method for obtaining endothelial cells from human pluripotent stem cells of embryonic origin or induced human pluripotent stem cells which meets GMP standards.

[0015] More particularly, the present invention relates to a method for differentiating endothelial cells from human pluripotent stem cells, characterized in that: a) on D0, the human pluripotent stem cells are dissociated, seeded at a density of 40,000 to 60,000 cells / cm 2< , preferably approximately 50,000 cells / cm 2< , on a matrix and cultured in the presence of a medium suitable for the culture of pluripotent cells, further comprising fibroblast growth factor 2 (“Fibroblast Growth Factor 2” or FGF2) and a ROCK inhibitor, b) on D1, the medium is replaced with a medium suitable for the induction of the mesoderm further comprising an inhibitor of GSK3 (Glycogen Synthase Kinase 3) and BMP4 (Bone Morphogenetic Protein 4); c) on D4, the medium is replaced with a medium suitable for the culture of endothelial cells further comprising VEGF (Vascular Endothelial Growth Factor) and forskolin; d) at D6, the cells are dissociated and selected for the expression of the CD144 marker.

[0016] Advantageously, all culture media and agents used are chemically defined and do not contain any additives of animal origin not controlled by Good Manufacturing Practice (or GMP) standards.

[0017] By "pluripotent stem cells" is meant any undifferentiated cell, capable of infinite self-renewal, 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").

[0018] In one embodiment, the pluripotent stem cells are human embryonic stem cells (hESCs). There are many hESC lines, including the RC-9 line (Roslin Cells 9), which has been developed as a clinical-grade hESC line, cited as a reference cell line.

[0019] Human pluripotent stem cells are obtained by methods that do not require the destruction of embryos.

[0020] In another embodiment, the stem cells are human induced pluripotent stem cells (hiPSCs). There are many commercial hiPSC lines or those manufactured using various existing reprogramming techniques (episome, mRNA, Sendai viral vector).

[0021] According to one embodiment of the invention, the pluripotent stem cells are donor-specific hiPSC cells obtained by reprogramming peripheral blood mononuclear cells from said donor. Protocols for reprogramming peripheral blood mononuclear cells from the donor or CD34+ cells isolated from umbilical cord blood are known to those skilled in the art.

[0022] By "matrix" or "coating" is meant any substrate allowing the culture of stem cells in a monolayer. Preferably, the matrix used in the method according to the invention is a defined protein matrix. Preferably, the matrix is chosen from the group consisting of Matrigel ™< , L7 coating ™< , laminin and vitronectin. Particularly preferably, the matrix is the L7 ™< matrix marketed by the company Lonza under the reference FP-5020.

[0023] By "medium suitable for the culture of pluripotent cells" is meant any medium which contains the nutrients and factors enabling the culture in vitro of pluripotent cells.

[0024] Preferably, the medium suitable for the culture of pluripotent cells is chosen from the iPS Brew XF GMP medium marketed by the company Miltenyi Biotec and the iPS Stempro medium marketed by the company ThermoFisher.

[0025] In step a), the medium is supplemented with “fibroblast growth factor 2” or “FGF2”. Typically, FGF2 is used at a final concentration of 5 to 20 ng / ml, preferably about 10 ng / ml.

[0026] In step a), the medium is supplemented with a ROCK inhibitor. Preferably, the ROCK inhibitor is provided in the Revitacell supplement marketed by the company Gibco.

[0027] “Mesoderm induction medium” means any medium that contains nutrients and factors that enable the induction of the mesodermal pathway in pluripotent cells. Preferably, the mesoderm induction medium comprises N2 and B27 supplements. N2B27 medium is a 1:1 mixture of DMEM-F12 CTS KO medium and Neurobasal CTS medium, supplemented with Glutamax CTS, N2 CTS and B27 CTS.

[0028] In step b), the medium suitable for mesoderm induction also includes a GSK3 and BMP4 inhibitor.

[0029] After step c), on day 5 the medium is again replaced by a medium suitable for the culture of endothelial cells, also comprising VEGF (Vascular Endothelial Growth Factor) and forskolin;

[0030] Those skilled in the art have at their disposal a number of agents known to inhibit GSK3 kinase. Typically, the GSK3 inhibitor may be Chir99021 marketed by the company Tocris.

[0031] Typically, the final concentration of Chir99021 is between 5 and 10 µM, preferably about 6 µM.

[0032] Typically, the final BMP4 concentration is between 15 and 50 ng / ml, preferably about 25 ng / ml.

[0033] By "medium suitable for the culture of endothelial cells" we mean any medium which contains the nutrients and factors enabling the culture in vitro of endothelial cells.

[0034] Preferably, the medium suitable for the culture of endothelial cells is the CnT-Endo medium marketed by the company CellnTec.

[0035] In step c), the medium is supplemented with VEGF and forskolin.

[0036] Preferably, the VEGF is provided at a final concentration of between 100 and 300 ng / ml, even more preferably about 200 ng / ml.

[0037] Preferably, forskolin is provided at a final concentration of between 1 and 3 µM, even more preferably approximately 2 µM.

[0038] According to one embodiment of the invention, the medium is renewed on D5.

[0039] At D6, a certain portion of the cell population has differentiated into endothelial cells.

[0040] In order to enrich the endothelial cell population, a selection step is carried out in step d). This selection can be done by cell sorting.

[0041] According to one embodiment, the selection is carried out by flow cytometry.

[0042] According to another embodiment, the selection is carried out using magnetic beads coated with an antibody specific for the endothelial cell marker such as CD34+ or CD144 or CD31+, preferably CD144.

[0043] The present invention also relates to a population of endothelial cells directly obtained by the method described above. Advantageously, the population of endothelial cells is homogeneous, that is to say that more than 90%, preferably more than 95%, more than 98%, or more than 99% of the cells are positive for the CD144 marker.

[0044] The present invention also relates to the use of the endothelial cell population for the manufacture of a tissue, preferably a dermal or skin substitute tissue. Brief description of the Figures

[0045] Other features, details and advantages of the invention will appear on reading the attached Figures. Fig. 1 [ Fig. 1] represents the characterization of reference endothelial cells at P5. (A) Morphology of reference endothelial cells HUVEC, HDMEC and iEC CDI. (B) Transcriptomic profile of reference cells, qPCR analysis of endothelial cell-specific markers PECAM1 (or CD31), CD34, KDR, VE-cadherin (or CD144) and vWF. (C) Protein profile, FACS analysis of CD31 / CD34 and CD31 / CD144 and immunofluorescence analysis of CD31, VE-cadherin (or CD144) and vWF. a) HUVEC b) HDMEC c) iEC CDI (D) Functionality: tubule formation on GFR-matrigel, endocytosis of Ac-LDL, response to inflammation by TNFα treatment and observation of ICAM expression. Fig. 2 [ Fig. 2] represents the method according to the invention and the characterization of the endothelial cells obtained. (A) Validated clinical protocol. (B) FACS CD144-APC at D6 (before selection). (C) FACS during the passages for the analysis of CD31 / CD144 and CD31 / CD34 co-markings with the results at P2 representative of the different passages. (D) Functionality of the cells produced during the passages and formation of tubules on GFR-matrigel. Example 1 : Characterization of reference endothelial cells

[0046] Primary endothelial cells HUVEC (Human Umbilical Vein Endothelial Cells) and HDMEC (Human Dermal Microvascular Endothelial Cells) as well as iPS-derived endothelial cells produced by Cellular Dynamics International (iEC CDI) were used as reference cells to perform all the setup and validation of quality controls of iPS-derived endothelial cells: transcriptomic profile (qPCR), protein profile (FACS, immunofluorescence) and functionalities (tubule formation on matrigel, response to inflammation, endocytosis capacity).

[0047] The markers analyzed to characterize endothelial cells are CD31 encoded by the PECAM1 gene and CD144 encoded by the VE-Cadherin gene, two membrane proteins involved in intercellular junctions between endothelial cells and which allow the integrity of the endothelium. The cytoplasmic marker vWF is involved in the recruitment of platelets during vessel injury and is present in mature endothelial cells. It therefore provides an idea of the maturity of the cells. The CD34 protein is a common marker for endothelial and hematopoietic cells because it is expressed from the hemangioblast stage, the precursor of both cell types. The last marker analyzed is VEGFR2 encoded by KDR, a VEGF receptor which itself is involved in the stimulation of angiogenesis and the survival of endothelial cells.

[0048] There Figure 1Ashows that the 3 reference cell types have the same morphology. They have a similar transcriptomic profile except for the vWF marker which shows a lower maturity of iEC CDI compared to HUVEC and HDMEC ( Figure 1 ). HUVECs and HDMECs express the membrane markers CD31 and CD144 at more than 98%. CDI iECs express them at 93.7%. As for the CD34 marker, a double population is observed for the 3 cell types, one positive and one negative at approximately 50%. Immunofluorescence makes it possible to verify that the vWF maturity marker is indeed expressed in the cytoplasm of the cells ( Figure 1C a, b and c ).

[0049] To verify the functionality of the cells, their ability to organize themselves into a "tubule-like" structure is tested on matrigel for 24 hours. The 3 control cell lines form network structures ( Figure 1D ).

[0050] The metabolic activity of endothelial cells is tested using the fluorochrome-coupled LDL-Ac endocytosis assay. This is added to the cell culture medium for 4 hours and then the cells are rinsed. The endocytosed LDL-Ac remains in the cells and can be observed by the presence of fluorescence inside the cells, demonstrating their endocytosis capacity ( Figure 1D ).

[0051] The final test of endothelial cell functionality aims to show their ability to respond to inflammation via TNFα stimulation for 24 hours. This treatment allows the overexpression of ICAM, a marker of inflammation (stimulator of adhesion and transmigration of leukocytes across the endothelial epithelium). Without treatment, no expression of ICAM is observed in the cells (not shown) whereas with treatment the cells express ICAM and in a similar way between the 3 cell types ( Figure 1D ).

[0052] These results show that the reference cells appear functional and express endothelial cell-specific markers. They are therefore good controls for cells derived from differentiations from pluripotent stem cells. Example 2 : Obtaining hPSC-derived endothelial cells and phenotypic characterization of said cells

[0053] The hiPSC cells are dissociated with accutase and then seeded at 50,000 c / cm 2 < on L7 coating in Stempro hESC SFM medium supplemented with 10ng / ml of FGF2 and Revitacell at 1 / 100. The next day, on D1, the medium is replaced with mesoderm induction medium: N2B27 CTS medium (1:1 mixture of KO DMEM-F12 and Neurobasal CTS medium supplemented with Glutamax CTS, N2 CTS and B27) with Chir99021 at 6uM and BMP4 at 25ng / ml.

[0054] On day 4, the medium is replaced with endothelial specification medium: CnT-ENDO medium supplemented with 200ng / ml of VEGF and 2µM of forskolin. The medium is renewed the next day. On day 6 of differentiation, differentiated cells are dissociated with accutase and selected using CD144+ magnetic beads with LS columns marketed by Miltenyi Biotec. Positive cells, i.e. differentiated endothelial cells, are collected and then frozen in a cryostor.

[0055] The cells are then thawed and seeded onto a 10µg / ml collagen I matrix at 20000c / cm 2< in CnT-ENDO medium supplemented with 50ng / ml VEGF. The medium is changed every 2 days and the cells are passaged every 3-4 days.

[0056] All of these steps are outlined in the Figure 2A .

[0057] The cells thus obtained by this method were characterized from the phenotypic and functional point of view. The cells obtained by the method of the invention express the markers CD31, CD144 and CD34 well ( Figure 2C ) and are functional up to p3 ( Figure 2D ). Example 3 : Comparison of different culture media and different coatinas

[0058] The protocol of Example 2 was carried out using different culture media and different coatings, as shown in Table 1 below.

[0059] The yields obtained (in % of CD144-positive (or CD144+) cells at the end of the differentiation step) were compared.

[0060] These tests show that both Stempro hES SFM and iPS Brew XF GMP media are effective as culture media for pluripotent cells.

[0061] The clinical grade L7 coating is as effective as the GFR-matrigel coating.

[0062] For the mesoderm induction step, CnT-ENDO medium is more effective than the other tested media.

Claims

1. A method for differentiating into endothelial cells from pluripotent stem cells, characterized in that: a) on D0, the pluripotent stem cells are dissociated, seeded at a density of 40 000 to 60 000 cells / cm2, preferably approximately 50 000 cells / cm2, on a matrix and cultured in the presence of a medium suitable for the culture of pluripotent cells, further comprising fibroblast growth factor 2 (FGF2) and a ROCK inhibitor; b) on D1, the medium is replaced with a medium suitable for mesoderm induction, further comprising a GSK3 (Glycogen Synthase Kinase 3) inhibitor and BMP4 (Bone Morphogenetic Protein 4); c) on D4, the medium is replaced with a medium suitable for the culture of endothelial cells, further comprising VEGF (Vascular Endothelial Growth Factor) and forskolin; d) on D6, the cells are dissociated and selected for expression of the CD144 marker, wherein said process meets good manufacturing practices (GMP) standards.

2. The method according to claim 1, characterized in that the FGF2 is used at a concentration from 5 to 20 ng / ml, preferably approximately 10 ng / ml, in step a).

3. The 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 µM, even more preferably at a concentration of approximately 6 µM, in step b).

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

5. The method according to any one of the preceding claims, characterized in that the VEGF is used at a concentration between 100 and 300 ng / ml, preferably approximately 200 ng / ml, in step c).

6. The method according to any one of the preceding claims, characterized in that the forskolin is used at a concentration between 1 and 3 µM, preferably approximately 2 µM in step c).

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