Method for sorting differentiated cells 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 thus obtained and its use. (no figure)

Description

Title of the invention: 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. 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 of 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, which allows 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 cultures (heterogeneity, limited numbers, risk of senescence).

[0005] Methods for differentiating pluripotent stem cells are already known. However, to date, no differentiation protocol for pluripotent stem cells exists that allows obtaining a pure population at the end of differentiation. Protocols that, at best, yield approximately 70% cells of interest at the end of differentiation are generally described in the literature (for example, in Gu, Curr Protoc Hum Genet, 98(l):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 perform a cell sorting step after the differentiation protocol. In practice, it is generally desirable to have a cell population comprising at least 90% cells of interest, and 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 carried out manually, with steps performed under a laminar flow hood and sensitive to contamination.

[0007] On the one hand, the MACS (Magnetic-Associated Cell Sorting) method is described. It can be defined as separation using magnetic beads coupled to antibodies. It is relatively rapid and allows for the purification of a cell population of interest. Manual 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.Furthermore, the MACS method has several other drawbacks: sorting large quantities of cells requires a considerably long purification time, the capacity of the columns is reduced, the number of cells that can be sorted is limited, and increasing the number of columns increases the risk of handling errors or cell loss. In addition, since the magnetic separation columns are in an open system, there is a risk of external contamination, making the method incompatible with Good Manufacturing Practices (GMP). This risk is even greater with a larger number of columns used.

[0008] The documents Masuda et al. (Regen Ther., 9:1-9, (2018)); Gu et al. (Curr Protoc Hum Genet, 98(l):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] 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 as separation by flow cytometry after labeling with a fluorescent antibody using the same preparation techniques as the MACS method. The document Sriram et al. (Stem Cell Res Ther 6, 261 (2015)) illustrates, for example, the use of the FACS method.

[0011] While the FACS method allows for the purification of cell populations based on combinations of markers and not just a single marker (enrichment in subpopulations of interest), it does have drawbacks. Indeed, 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 difficult to comply with GMP standards because… non-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_cplloi2a8d3cb3jvrln69e5h7i?content-disposition=inline). This is an automated magnetic enrichment process for human cells expressing the CD34 marker, using blood products obtained after leukapheresis. According to the reported results, the process recovers a cell population 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 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.

[0014] Good Manufacturing Practices (GMP) standards constitute a quality assurance concept established by the European (or American) Commission for 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, emphasizing hygiene practices, as well as the risks of confusion regarding labeling / identification. GMP principles require the writing of standard operating procedures and instructions to ensure consistent quality production with compliant traceability. They also incorporate processes, product quality, and personnel safety.

[0015] GMPs 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 medicines. 3. Documents relating to good manufacturing practices giving recommendations on international requirements for batch certification.

[0016] To date, there is therefore no way to sort large quantities of cells of interest according to GMP standards, let alone following a differentiation process from pluripotent stem cells. However, the needs are increasingly numerous, In various clinical applications, such as obtaining reconstituted tissues for therapeutic use, which requires the production of large quantities of cells (several hundred million, or even several billion), it is essential 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 a need to obtain a cell population comprising at least 90% of the cells of interest, preferably at least 95%. This step represents a technological hurdle to the large-scale 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 adaptation to 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 differentiated cells 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 to a marker expressed on the surface of said cells of interest.

[0019] The process according to the invention has, in particular, the following advantages: 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 cell population with high purity.

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

[0021] The present invention also relates to the cell population, in particular endothelial cells, obtained by the process 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 selection of said cells of interest using magnetic beads coupled to at least one antibody specific to a marker expressed on the surface of said cells of interest.

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

[0025] By "differentiated cells" is meant any cell specialized into a cell type. Differentiated cells are identifiable, for example, by morphological characteristics or the expression of gene(s) specific to a cell type. Typically, these may be endothelial cells, fibroblasts, ...

[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] The term "marker" refers to any molecule located on the surface of a cell that allows the cell type to be identified. This includes, in particular, Cluster of Differentiation markers. In one embodiment, the marker is thus chosen from among CD31, CD34, and CD144, and is preferably CD34.

[0029] Preferably, the antibody according to the invention is a GMP-grade antibody (i.e., one that meets 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 to the CD34 marker expressed on the surface of said cells of interest.

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

[0033] In one embodiment, the present invention 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 in the cell population 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 to 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 in the cell population 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 labeling said cells of interest, within the cell population to be sorted, by 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 for 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 labeling step of said cells of interest, within the population of cells to be sorted, by 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 selection step of said cells labeled 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 labeling step of said cells of interest, within the population of cells to be sorted, by 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 cell population washing step sort, - (iii) at least one selection step of said cells labeled 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. This selection step makes it possible, in particular, to obtain a cell population 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 includes 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 to a marker expressed on the surface of the cells of interest prior to the labeling step. The cells of interest are then incubated with the magnetic beads, thus forming a suspension. The suspension is then washed and directed into a column that is near a magnetic field (magnet) to perform positive cell selection; that is, cells not bound 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 away 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 labeling said cells of interest, within the cell population to be sorted, by 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 selection step of said cells marked in step (i) and retained on the magnetic sorting column during step (iii).

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

[0042] Optionally, at least one optional washing step (ii) is followed by at least one filtration step, in particular to remove any cell aggregates that 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 for the release of positive (of interest) cells 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 at least one labeling step, the labeled cells are directed to 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® automaton.

[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 not to handle 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 process according to the invention allows the loading of a population of 1 billion cells, and the sorting to be carried out in a single step.

[0049] According to one embodiment, a single automated closed system is required and can perform the sorting of cells of interest in less than 2h30, allowing better survival and recovery of the sorted cells.

[0050] According to one embodiment, the differentiation of the 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 cell population comprising at least 90%, in particular 95%, of cells of interest, which was obtained according to the invention, may be followed by an amplification step. The amplification step is typically carried out with a medium suitable for the culture of endothelial cells.

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

[0053] In one embodiment, the differentiated cells are endothelial cells obtained by a production process characterized in that: - (a) at J0, said pluripotent stem cells are seeded at a density of 40,000 to 60,000 cells / cm2, preferably about 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 day 2, the medium is replaced by a medium adapted for mesoderm induction including an inhibitor of GSK3 (Glycogen Synthase Kinase 3) and BMP4 (Bone Morphogenetic Protein 4); - (c) at J5 the medium is replaced by a medium adapted for the culture of endothelial cells including in addition VEGF (Vascular Endothelial Growth Factor) and forskolin; - (d) at J6, the medium is again replaced by a medium adapted for the culture of endothelial cells including in addition VEGF (Vascular Endothelial Growth Factor) and forskolin. - (e) at day 7 the cells are dissociated, - (f) optionally a step of obtaining a population of cells to be sorted comprising at least 20% differentiated cells, in particular at least 40%, and preferably at least 60%.

[0054] The terms "matrix" or "coating" refer to any substrate that allows the culture of stem cells in a monolayer. Preferably, the matrix used in the process according to the invention is a defined protein matrix. Preferably, the matrix is ​​selected from the group consisting of Matrigel™, L7 coating™, laminin, and vitronectin. Particularly preferred, the matrix is ​​the L7™ matrix marketed by Lonza under the reference FP-5020.

[0055] The term "medium suitable for the culture of pluripotent cells" means any medium that contains the nutrients and factors necessary for the in vitro culture of pluripotent cells. Preferably, the medium suitable for the culture of pluripotent cells is selected from the iPS Brew XF GMP medium marketed by Miltenyi Biotec and the iPS Stempro medium marketed by Thermo Fisher Scientific.

[0056] In step a), the medium is supplemented with "fibroblast growth factor 2" or "FGF2" or "Fibroblast Growth Factor 2". Typically, FGF2 is used at a final concentration of 5 to 20 ng / ml, preferably about 10 ng / ml.

[0057] Optionally, in step a), the medium is supplemented with a ROCK inhibitor. Preferably, the ROCK inhibitor is provided within the Revitacell supplement marketed by Gibco.

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

[0059] By "medium suitable for mesoderm induction" is meant any medium that contains the nutrients and factors enabling the induction of the mesodermal pathway of pluripotent cells. Preferably, the medium suitable for mesoderm induction comprises the N2 and B27 supplements. The N2B27 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.

[0060] During step b), the medium adapted for mesoderm induction also includes an inhibitor of GSK3 and BMP4.

[0061] A person skilled in the art has at their disposal a number of agents known to inhibit the GSK3 kinase. Typically, the GSK3 inhibitor can be Chir99021, marketed by Tocris.

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

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

[0064] By “medium suitable for the culture of endothelial cells” is meant 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] During step c) and step d), the medium is supplemented with VEGF and forskolin.

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

[0068] Preferably, forskolin is supplied at a final concentration 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 according to Good Manufacturing Practices (or GMP).

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

[0071] Advantageously, the cell population, particularly endothelial cells, is homogeneous, meaning that 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 the 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. Examples

[0073] Example 1: Comparison of LS columns and the TS310 tubing system

[0074] Materials and methods

[0075] Obtaining differentiated cells (endothelial cells) is carried out according to the method described in application WO2021176178. Following the differentiation step Conversion of pluripotent stem cells into endothelial cells yields a population of approximately 70% cells of interest. A cell sorting step is then performed to purify the population into CD34+ cells of interest.

[0076] Application WO2021176178 describes the use of LS (Miltenyi Biotec) columns. These are sterile, single-use columns with a low capacity (having a maximum total load of 50 million cells) for use in a Microbiological Safety Cabinet (MSC), which corresponds to an "open" system. The application also describes the use of anti-CD144 antibodies of "for Research Use Only" (RUO) grade, meaning "intended for research purposes only."

[0077] The cell sorting step according to the invention is here carried out in an automated system, by the CliniMACS Prodigy® cell culture system (Miltenyi Biotec) and the CliniMACS Prodigy® TS 310 tubing system.

[0078] Comparison

[0079] Sorting using LS columns according to application WO2021176178 and sorting in a closed system 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.

[0080] [Tables 1] LS Column (manual) TS 310 Column (automated) Maximum experimental load (M total cells) 5.00E+07 1.00E+09 Sorting efficiency Approximately 80% Pressure Mechanical Fluidic Sorting time for 19 total cells 3 hours / operator (3 operators) 2 hours (1 operator) System type "open" under PSM Closed in tubing Sorting antibodies CD 144 or CD34 (RUO-grade) CD34 (GMP-grade)

[0081] Comparison of LS and TS310 columns

[0082] Scaling up towards a bioproduction process

[0083] The use of the TS 310 kit, including a magnetic sorting column, makes it possible to pass from 50 million cells loaded onto the column to 1 billion cells (x20), and perform the sorting in a single step. Only one operator is needed and can This sorting process can be completed in less than 2.5 hours, resulting in improved survival and recovery of the sorted cells. In comparison, 18 manual columns would be required, necessitating 2 to 3 operators working in parallel for a total time of over 3 hours per operator. Thus, this invention leads to a simpler (only one operator, involved solely during the preparation phase), faster, and more efficient process. The invention therefore overcomes the technological barrier to the mass production of endothelial cells that was the cell sorting step performed using protocols described in the literature.

[0084] Major reduction in pharmaceutical risk

[0085] One of the major aspects in pharmaceutical production is the reduction of the risk of contamination (bacteriological, virological...).

[0086] The use of a tubing system eliminates the need to handle cells in an "open" system under a biosafety cabinet (BSC). All steps are performed within the closed system. Labeled cells are directed to the sorting column via sterile tubing and the action of a peristaltic pump. In manual processes, the risk of bacterial contamination is highest during the sorting step. The TS310 kit allows for sterile sorting without the need to handle cells / columns under a BSC. Furthermore, no mechanical pressure is exerted on the cells to release positive cells retained on the column (peristaltic pump flow displacement), which is less stressful for the sorted cells. The process is safer compared to manual column sorting. The invention thus enables a major reduction in pharmaceutical risk.

[0087] The use of GMP-grade antibodies. The pharmaceutical risk associated with the use of antibodies for the cell sorting step was mitigated by replacing the CD144 RUO antibody with a CD34 GMP antibody, which has much more comprehensive supplier documentation, thus securing 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 (not retained) fractions exhibit similar phenotypes according to the two sorting methods. The results, obtained using LS columns, are presented in Table 2.

[0088] [Tables2] Antibody used (MACS) Anti-CD144 Anti-CD34 Total cells obtained after the differentiation step 327E6 Total cells sorted 120E6 Quantity of cells retained (positive fraction, cells expressing the marker) 68.1E6 (56.8%) 72.4E6 (60%) Quantity of cells not retained (negative fraction, cells not expressing the marker) 42.7E6 (35.5%) 53E6 (44.7%)

[0089] Comparison between two antibodies

[0090] Example 2: Purity of the cell population obtained

[0091] 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 checked by flow cytometry analysis. The results obtained are presented in Table 3.

[0092] [Tables3] Total cells obtained at the end of the differentiation step: 1.6E9 Total cells sorted: 1E9 Quantity of cells retained (positive fraction, cells expressing the marker): 401E6 Quantity of cells not retained (negative fraction, cells not expressing the marker): 480E6

[0093] Characteristics of sorted cells and results

[0094] 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%.

[0095] 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 are thus obtained.

[0096] The invention thus makes it possible to purify a population of endothelial cells in a better controlled, more efficient, faster and larger scale manner.

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 CD 144, 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. Method for sorting cells of interest according to any one of the preceding claims, comprising: - (i) at least one step of labeling said cells of interest, within the population of cells to be sorted, by at least one antibody specific for a marker expressed on the surface of said cells of interest, - (ii) optionally at least one step of washing the population of cells to be sorted, - (iii) at least one step of selecting said cells labeled 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) at 0, 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 with 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 with a medium suitable for the culture of endothelial cells further comprising VEGF (Vascular Endothelial Growth Factor) and forskolin; - (d) on D6, the medium is again replaced with a medium suitable for the culture of endothelial cells further comprising VEGF (Vascular Endothelial Growth Factor) and forskolin. - (e) on 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 R

10. 1 a O. Use of the endothelial cell population of claim 9 for the manufacture of dermal tissue or a skin substitute.