Method for sorting cells differentiated from pluripotent stem cells

Abstract

The invention relates to a method for sorting cells of interest in a closed system. The invention also relates to the cell population obtained in this way and to the use thereof.

Description

Title: Method for sorting differentiated cells from pluripotent stem cells

[0001] The present invention relates to a method for sorting differentiated cells from pluripotent stem cells. 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] Methods for differentiating pluripotent stem cells are already known. However, there is currently no protocol for differentiating pluripotent stem cells that allows obtaining a pure population at the end of differentiation. Protocols that allow obtaining at best approximately 70% of cells of interest at the end of differentiation are generally described in the literature (for example in Gu, Curr Protoc Hum Genet, 98(1):e64, (2018); Olmer et al. Stem Cell Reports. 2018 May 8; 10(5): 1657-1672). In order to enrich the cell population, it is therefore generally desirable to carry out a cell sorting step after the differentiation protocol. In practice, it is generally desirable to have a cell population comprising at least 90% of cells of interest, even more preferably at least 95%.

[0006] Two methods are already known for sorting cells following a differentiation protocol: MACS and FACS. These methods are performed manually, with steps carried out under laminar flow hoods and sensitive to contamination.

[0007] On the one hand, the MACS (Magnetic-Associated Cell Sorting) method is described. It can be defined by the separation by magnetic beads coupled to antibodies. It is relatively fast and allows the purification of a cell population of interest. Manual cell sorting requires several preparation steps, with washing and centrifugation, as well as an incubation step with specific antibodies. After a final washing and centrifugation step, the cells are placed at the top of the column and the labeled cells are retained. After several volumes of gravity washes, the cells are mechanically detached via the plunger of a syringe. These steps are long 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, the capacity of the columns is reduced, the number of cells to be sorted is limited, and increasing the number of columns increases the risk of handling errors or cell losses. In addition, since the magnetic separation columns are in an open system, there is a risk of external contamination, which makes the method incompatible with Good Manufacturing Practices (GMP). This risk is all the higher as the number of columns used is large.

[0008] For example, 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 the use of the MACS method.

[0009] Application WO2021176178 also describes a method for differentiating pluripotent stem cells into endothelial cells, comprising a cell sorting step (MACS) in an open system, on LS columns marketed by Miltenyi Biotec.

[0010] On the other hand, the FACS (Fluorescence-Associated Cell Sorting) method is known. This method can be defined by flow cytometric separation after labeling with a fluorescent antibody using the same preparation techniques as the MACS method. The document Sriram et al. (Stem Cell Res TherS, 261 (2015)) illustrates for example the use of the FACS method.

[0011] Although the FACS method allows for the purification of populations based on combinations of markers and not just a single marker (enrichment in subpopulations of interest), it does have drawbacks. Indeed, the process time is very long (over 6 hours) for large quantities of cells, which is particularly detrimental to cell viability, and which makes the FACS method incompatible with industrial scale-up. The system also uses a washable tubing system, which is difficult to comply with GMP standards because it is not single-use. Solutions are proposed 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% of cells of interest.

[0012] Automated cell sorting in a closed system has already been described (see Enrichment of human CD34+ cells - LP-34 Enrichment process - CliniMACS Prodigy® Tubing Set 310 - Miltenyi Biotec - https: / / static.miltenyibiotec.com / asset / 150655405641 / document_cp11ol2a8d3cb3Jvrln69e5h7i?cont ent-disposition=inline). This is an automated magnetic enrichment process for human cells expressing the CD34 marker, from blood products after leukapheresis. According to the results communicated, the process allows the recovery of a population of cells comprising between 52 and 74% of the cells of interest (expressing the CD34 marker).

[0013] The cell sorting step is therefore a complex, critical and limiting step in the industrial scaling up of a cell differentiation process and in its 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.

[0014] 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. GMP principles require the writing of operating procedures and instructions allowing for consistent quality production with compliant traceability. They also integrate processes, product quality and personnel safety.

[0015] The BPF are currently organized into 3 parts: 1. Good manufacturing practices for medicinal products for human use. 2. Good manufacturing practices for active substances used as raw materials in medicinal products. 3. Good manufacturing practice documents providing recommendations on international requirements for batch certification.

[0016] To date, there is no way to sort cells of interest in large quantities according to GMP standards, even less so at the end of a differentiation process from pluripotent stem cells. However, the needs are increasing in various fields of clinical application, such as obtaining reconstituted tissues usable in therapy requiring the production of large quantities of cells (several hundred millions, or even several billions). It is therefore necessary to be able to sort very large quantities of cells at the end of differentiation according to GMP standards. The purity of the cell population, obtained after sorting, is also an important factor: there is indeed a need to obtain a cell population comprising at least 90% of the cells of interest, preferably at least 95%.This step constitutes a technological barrier to the mass production of differentiated cells of interest from pluripotent stem cells. Brief description of the invention

[0017] The present invention relates to a new cell sorting method enabling the scaling up of cell sorting as well as its compliance with pharmaceutical grade production processes.

[0018] More particularly, the present invention relates to a method for sorting cells of interest in a closed system, characterized in that the cells of interest are cells differentiated from pluripotent stem cells, and said method comprising a step of selecting said cells of interest using magnetic beads coupled to at least one antibody specific for a marker expressed on the surface of said cells of interest.

[0019] The method according to the invention has the following advantages in particular: 1. The scaling up of cell sorting since this process makes it possible to sort a very large quantity of cells in a simpler, faster and more efficient way than by the processes described in the state of the art. 2. Compliance with pharmaceutical grade production processes according to GMP standards. 3. Obtaining a population of cells with high purity.

[0020] The method according to the invention thus makes it possible to remove the technological barrier to the mass production of cells of interest constituted by the cell sorting step carried out with the protocols described in the state of the art.

[0021] The present invention also relates to the population of cells, in particular endothelial cells, obtained by the method according to the invention.

[0022] The present invention also relates to the use of the endothelial cell population for the manufacture of dermal tissue or a skin substitute. Detailed description of the invention

[0023] The present invention relates to a method for sorting cells of interest in a closed system, characterized in that the cells of interest are cells differentiated from pluripotent stem cells, and said method comprising a step of selecting said cells of interest using magnetic beads coupled to at least one antibody specific for a marker expressed on the surface of said cells of interest.

[0024] By "pluripotent stem cells" is meant any undifferentiated cell, capable of infinite self-renewal, of differentiating into all cell types (ectoderm, endoderm, mesoderm). In one embodiment, said pluripotent stems are human pluripotent stem cells. In one embodiment, the pluripotent stem cells are human stem cells induced to pluripotency. According to one embodiment, the human pluripotent stem cells are obtained by methods that do not require the destruction of embryos.

[0025] The term "differentiated cells" refers to any cell that is specialized into a cell type. Differentiated cells can be identified, for example, by morphological characteristics or the expression of gene(s) specific to a cell type. Typically, these cells include endothelial cells, fibroblasts, etc.

[0026] In one embodiment, the cells differentiated from pluripotent stem cells are adherent or non-adherent cells.

[0027] In one embodiment, the cells differentiated from pluripotent stem cells are endothelial cells.

[0028] By "marker" is meant any molecule located on the surface of a cell which makes it possible to identify the cell type. This includes in particular Cluster of Differentiation. In one embodiment, the marker is thus chosen from CD31, CD34 and CD144, and is preferably CD34.

[0029] Preferably, the antibody according to the invention is a GMP grade antibody (i.e. complying with GMP standards). Even more preferably, the antibody according to the invention is a GMP grade antibody directed against CD34.

[0030] In a preferred embodiment, the differentiated cells are endothelial cells expressing the CD34 receptor.

[0031] In one embodiment, the present invention thus relates to a method for sorting cells of interest in a closed system, characterized in that the cells of interest are endothelial cells differentiated from pluripotent stem cells, and said method comprising a step of selecting said cells of interest using magnetic beads coupled to at least one antibody specific for the CD34 marker expressed on the surface of said cells of interest.

[0032] In one embodiment, the differentiated cells represent at least 20% of the cells within the cell population to be sorted, in particular at least 40%, and preferably at least 60%. At least 20% means all values ​​between 20% and 100%, for example values ​​20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%. In one embodiment, the differentiated cells represent between 20% and 80% of the cells within the cell population to be sorted. Advantageously, at the end of the sorting step, a cell population comprising at least 90% of the cells of interest is thus obtained. Preferably, at the end of the sorting step, a population of cells comprising at least 95% of the cells of interest is thus obtained.

[0033] In one embodiment, the present invention thus relates to a method for sorting cells of interest in a closed system, characterized in that the cells of interest are endothelial cells differentiated from pluripotent stem cells and represent at least 40% of the cells within the population of cells to be sorted, and said method comprising a step of selecting said cells of interest using magnetic beads coupled to at least one antibody specific for the CD34 marker expressed on the surface of said cells of interest. According to a preferred embodiment, said differentiated cells represent at least 60% of the cells within the population of cells to be sorted.

[0034] According to one embodiment of the invention, the method for sorting cells of interest comprises: - (i) at least one step of marking said cells of interest, within the population of cells to be sorted, with at least one antibody specific to a marker expressed on the surface of said cells of interest, - (ii) optionally at least one washing step of the cell population to be sorted, - (iii) at least one step of selecting said cells marked in step (i).

[0035] According to one embodiment of the invention, the method for sorting cells of interest comprises: - (i) at least one step of marking said cells of interest, within the population of cells to be sorted, with at least one antibody specific to a marker expressed on the surface of said cells of interest, said population comprising between 20% and 80% of cells of interest, - (ii) optionally at least one washing step of the cell population to be sorted, - (iii) at least one step of selecting said cells marked in step (i), and obtaining a population comprising at least 90% of cells of interest.

[0036] According to one embodiment of the invention, the method for sorting cells of interest comprises: - (i) at least one step of marking said cells of interest, within the population of cells to be sorted, with at least one antibody specific to a marker expressed on the surface of said cells of interest, said population comprising between 20% and 80% of cells of interest, - (ii) optionally at least one washing step of the cell population to be sorted, - (iii) at least one step of selecting said cells marked in step (i), and obtaining a population comprising at least 95% of cells of interest.

[0037] Preferably, "at least one selection step" means a selection step on a magnetic sorting column. Said selection step makes it possible in particular to obtain a population of cells comprising at least 90%, advantageously more than 95%, preferably more than 98% of the cells of interest.

[0038] According to one embodiment of the invention, the method for sorting cells of interest comprises, prior to step (i), a step of coupling the magnetic beads to at least one antibody specific to a marker expressed on the surface of the cells of interest.

[0039] According to one embodiment, the magnetic beads are coupled to at least one antibody specific for a marker expressed on the surface of the cells of interest prior to the labeling step. Then, the cells of interest are incubated with said magnetic beads and thus form a suspension. The suspension is then washed and then directed into the column which is close to a magnetic field (magnet) in order to carry out the positive selection of the cells, that is to say that the cells which are not attached to the antibody coupled to the magnetic bead are not retained on the column. The magnetic field is then removed and the cells of interest are carried by the liquid via a peristaltic pump.

[0040] According to one embodiment of the invention, the method for sorting cells of interest comprises: - (i) at least one step of marking said cells of interest, within the population of cells to be sorted, with at least one antibody specific to a marker expressed on the surface of said cells of interest, - (ii) optionally at least one washing step of the cell population to be sorted, - (iii) at least one step of loading the population of cells to be sorted onto a magnetic sorting column, - (iv) at least one step of selecting said cells marked in step (i) and retained on the magnetic sorting column during step (iii).

[0041] Optionally, the at least one optional washing step (ii) is followed by at least one centrifugation step, in particular to remove free beads.

[0042] Optionally, the at least one optional washing step (ii) is followed by at least one filtration step, in particular to remove any cell aggregates which could clog the column.

[0043] Optionally, the centrifugation step is followed by a filtration step.

[0044] According to one embodiment, no mechanical pressure is exerted on the cells to release the positive cells (of interest) retained on the column; the flow displacement is done by peristaltic pump, which is less stressful for the sorted cells.

[0045] According to one embodiment, following the at least one labeling step, the labeled cells are directed towards the sorting column by sterile tubing and the action of a peristaltic pump.

[0046] In one embodiment, the closed system is an automated system, for example the CliniMACS Prodigy® automation system.

[0047] In one embodiment, the closed system is a tubing system, for example the CliniMACS Prodigy® automated system used with the CliniMACS Prodigy® TS 310 kit. The use of a tubing system makes it possible in particular to avoid handling the cells in an “open” system in a Microbiological Safety Cabinet (MSC), which reduces the pharmaceutical risk and allows the sorting of cells according to GMP standards.

[0048] According to one embodiment, the method according to the invention allows the loading of a population of 1 billion cells, and to carry out the sorting in a single step.

[0049] According to one embodiment, only one automated closed system is required and can perform sorting of cells of interest in less than 2.5 hours, allowing for better survival and recovery of sorted cells.

[0050] According to one embodiment, the differentiation of cells of interest from pluripotent stem cells and the sorting of said cells of interest are carried out within the same closed system.

[0051] According to one embodiment, obtaining said population of cells comprising at least 90%, in particular 95%, of cells of interest, which has been obtained according to the invention, may be followed by an amplification step. The amplification step is typically carried out with a medium suitable for culturing endothelial cells.

[0052] Preferably, the medium suitable for the culture of endothelial cells is the CnT-Endo medium marketed by the company CelInTec. During the amplification step, this medium is supplemented with VEGF and fetal calf serum (2%).

[0053] In one embodiment, the differentiated cells are endothelial cells obtained by a production method characterized in that: - (a) at D0, said pluripotent stem cells are seeded at a density of 40,000 to 60,000 cells / cm 2 , preferably about 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); - (b) on D2, the medium is replaced by a medium suitable for mesoderm induction further comprising an inhibitor of GSK3 (Glycogen Synthase Kinase 3) and BMP4 (Bone Morphogenetic Protein 4); - (c) on D5 the medium is replaced by a medium suitable for the culture of endothelial cells also comprising VEGF (Vascular Endothelial Growth Factor) and forskolin; - (d) on day 6, the medium is again replaced by a medium suitable for the culture of endothelial cells also comprising VEGF (Vascular Endothelial Growth Factor) and forskolin. - (e) at D7 the cells are dissociated, - (f) optionally a step of obtaining a population of cells to be sorted comprising at least 20% of differentiated cells, in particular at least 40%, and preferably at least 60%.

[0054] 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.

[0055] The term “medium suitable for the culture of pluripotent cells” means any medium which contains the nutrients and factors enabling the in vitro culture of pluripotent cells. 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.

[0056] 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.

[0057] Optionally, during 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.

[0058] According to one embodiment, the pluripotent stem cells are thawed before seeding, in the presence of a ROCK inhibitor.

[0059] “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, β-mercaptoethanol, and B27 CTS.

[0060] In step b), the medium suitable for mesoderm induction also includes a GSK3 and BMP4 inhibitor.

[0061] 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.

[0062] Typically, the final concentration of Chir99021 is between 5 and 10 pM, preferably about 6 pM.

[0063] Typically, the final BMP4 concentration is between 15 and 50 ng / ml, preferably about 25 ng / ml.

[0064] “Medium suitable for the culture of endothelial cells” means any medium which contains the nutrients and factors enabling the in vitro culture of endothelial cells.

[0065] Preferably, the medium suitable for the culture of endothelial cells is the CnT-Endo medium marketed by the company CelInTec.

[0066] In step c) and step d), the medium is supplemented with VEGF and forskolin.

[0067] Preferably, the VEGF is provided at a final concentration of between 100 and 300 ng / ml, even more preferably about 200 ng / ml.

[0068] Preferably, forskolin is provided at a final concentration of between 1 and 3 pM, even more preferably about 2 pM.

[0069] 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.

[0070] The present invention also relates to the population of cells, in particular endothelial cells, obtained by the method described above.

[0071] Advantageously, the cell population, in particular endothelial cells, is homogeneous, i.e. more than 90%, advantageously more than 95%, preferably more than 98% of the cells are positive for the marker (preferably CD34). This illustrates the purity of the cell population obtained after cell sorting according to the invention. The cell population thus obtained therefore comprises at least 90% of cells of interest.

[0072] The present invention also relates to the use of the endothelial cell population for the manufacture of dermal tissue or a skin substitute.

[0073] Examples

[0074] Example 1: Comparison of LS columns and the TS310 tubing system

[0075] Materials and methods

[0076] Obtaining differentiated cells (endothelial cells) is carried out according to the method described in application WO2021176178. Following the step of differentiating the pluripotent stem cells into endothelial cells, a population of at best approximately 70% of cells of interest is obtained. A cell sorting step is thus carried out in order to purify the population into CD34+ cells of interest.

[0077] Application WO2021176178 describes the use of LS columns (Miltenyi Biotec). These are sterile, single-use, low-capacity columns (with a maximum load of 50 million total cells) for use in a Microbiological Safety Cabinet (MSC), which corresponds to a so-called "open" system. This application also describes the use of "for Research Use Only" (RUO) grade anti-CD144 antibodies.

[0078] The cell sorting step according to the invention is carried out here in an automated system, using the CliniMACS Prodigy® cell culture machine (Miltenyi Biotec) and the CliniMACS Prodigy® TS 310 tubing system.

[0079] Comparison

[0080] Sorting using LS columns according to application WO2021176178 and closed system sorting according to the invention were compared. These characteristics are summarized in Table 1. Sorting efficiency refers to the percentage of cells recovered after cell sorting (whether of interest or not) versus the total number of cells before sorting.

[0081] [Table 1]

[0082] Comparison of LS and TS310 columns

[0083] Scaling up to a bioproduction process

[0084] The use of the TS 310 kit, including a magnetic sorting column, allows to go from 50 million cells loaded on the column to 1 billion cells (x20), and to carry out the sorting in a single step. Only one manipulator is necessary and can carry out this sorting in less than 2h30, allowing a better survival and recovery of the sorted cells. In comparison, 18 manual columns would be necessary, requiring 2 to 3 operators in parallel for a total time of more than 3h per operator. Thus, this invention results in a simpler process (only 1 operator, only mobilized during the preparation phase), faster, and more efficient. The invention thus makes it possible to remove the technological barrier to the mass production of endothelial cells that constituted the cell sorting step carried out with the protocols described in the literature.

[0085] Major reduction in pharmaceutical risk

[0086] One of the major aspects in pharmaceutical production is the reduction of the risk of contamination (bacteriological, virological, etc.).

[0087] The use of a tubing system, eliminating the need to handle cells in an "open" system under PSM. All steps are carried out within the closed system. The labeled cells are directed to the sorting column by sterile tubing and the action of a peristaltic pump. In a manual process, the risk of bacteriological contamination is highest during the sorting step. The use of the TS310 kit allows sorting to be carried out in a sterile manner, without the need to handle the cells / columns under PSM. In addition, no mechanical pressure is exerted on the cells to release the positive cells retained on the column (flow displacement by peristaltic pump), which is less stressful for the sorted cells. The process is safer compared to sorting on a manual column. The invention thus allows a major reduction in pharmaceutical risk.

[0088] The use of GMP grade antibodies. The pharmaceutical risk regarding the use of antibodies for the cell sorting step was controlled by replacing the CD144 RUO antibodies with a CD34 GMP antibody, with much more complete supplier documentation, which secures the process. The population of interest expresses the CD144 and CD34 markers in a comparable manner and the sorting efficiencies with the two antibodies are also comparable. After sorting, the positive (retained) and negative (non-retained) fractions present similar phenotypes according to the 2 types of sorting. The results, obtained on LS columns, are presented in Table 2.

[0089] [Table 2]

[0090] Comparison between two antibodies

[0091] Example 2: Purity of the cell population obtained

[0092] The differentiated cells are obtained by the method described in Example 1. The purity before and after the cell sorting step according to the invention is controlled by flow cytometry analysis. The results obtained are presented in Table 3.

[0093] [Table 3]

[0094] Characteristics of sorted cells and results

[0095] The following results are obtained: - The percentage of cells expressing the CD34 marker in the total fraction before cell sorting is 42.5%. - The percentage of cells expressing the CD34 marker in the fraction retained after cell sorting is 96%. - The percentage of cells expressing the CD34 marker in the fraction not retained after cell sorting is 3%.

[0096] The cell population obtained after cell sorting according to the invention thus expresses the CD34 marker at more than 95%. A cell population comprising more than 95% of cells of interest is thus obtained.

[0097] The invention thus makes it possible to purify a population of endothelial cells in a better controlled manner, more efficiently, more quickly and on a larger scale.

Claims

Claims

1. Method for sorting cells of interest in a closed system, characterized in that the cells of interest are cells differentiated from pluripotent stem cells, and said method comprising a step of selecting said cells of interest using magnetic beads coupled to at least one antibody specific for a marker expressed on the surface of said cells of interest.

2. A method for sorting cells of interest according to claim 1, wherein said marker is selected from CD31, CD34 and CD144, preferably CD34.

3. A method for sorting cells of interest according to any preceding claim, wherein said cells differentiated from pluripotent stem cells are endothelial cells.

4. A method for sorting cells of interest according to any one of the preceding claims, wherein said differentiated cells represent at least 20% of the cells within the population of cells to be sorted, in particular at least 40%, and preferably at least 60%.

5. A method for sorting cells of interest according to any preceding claim, comprising: - (i) at least one step of marking said cells of interest, within the population of cells to be sorted, with at least one antibody specific to a marker expressed on the surface of said cells of interest, - (ii) optionally at least one washing step of the cell population to be sorted, - (iii) at least one step of selecting said cells marked in step (i).

6. A method for sorting cells of interest according to any preceding claim, wherein said closed system is a tubing system.

7. Method for sorting cells of interest according to any one of the preceding claims, in which the differentiated cells are endothelial cells obtained by a method of obtaining characterized in that: - (a) on D0, said pluripotent stem cells are 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 (“Fibroblast Growth Factor 2” or FGF2); - (b) on D2, the medium is replaced by a medium suitable for mesoderm induction further comprising an inhibitor of GSK3 (Glycogen Synthase Kinase 3) and BMP4 (Bone Morphogenetic Protein 4); - (c) on D5 the medium is replaced by a medium suitable for the culture of endothelial cells also comprising VEGF (Vascular Endothelial Growth Factor) and forskolin; - (d) on day 6, the medium is again replaced by a medium suitable for the culture of endothelial cells also comprising VEGF (Vascular Endothelial Growth Factor) and forskolin. - (e) at D7 the cells are dissociated.

8. Method for sorting cells of interest according to any one of the preceding claims, characterized in that the pluripotent stem cells are human stem cells induced to pluripotency.

9. Population of cells, in particular endothelial cells, obtained by the method as defined according to any one of claims 1 to 8.

10. Use of the endothelial cell population of claim 9 for the manufacture of dermal tissue or a skin substitute.

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