A method for in vitro induction of differentiation of pluripotent stem cells to generate vascular endothelial cells

Abstract

The application belongs to the technical field of regenerative medicine, and particularly relates to a method for generating vascular endothelial cells by differentiating induced pluripotent stem cells in vitro. The method comprises the following steps: culturing pluripotent stem cells in differentiation medium I for first culture to obtain cells A; culturing the cells A in differentiation medium II for second culture to form endoderm cells B; culturing the endoderm cells B in differentiation medium III for third culture to obtain differentiated cells C; culturing the differentiated cells C in differentiation medium IV for fourth culture to form differentiated cells D; culturing the differentiated cells D in differentiation medium V for fifth culture to obtain vascular endothelial cells; wherein the differentiation medium I-V does not contain activin A, VEGF and BMP4. The method can improve the differentiation efficiency of IPS cells to vascular endothelial cells, and has a broad prospect in the cell therapy industry.

Description

Technical Field

[0001] This invention belongs to the field of regenerative medicine technology, specifically relating to a method for inducing pluripotent stem cells to differentiate into vascular endothelial cells in vitro. Background Technology

[0002] Damage and functional abnormalities of vascular endothelial cells (VECs) are the initiating factors for cardiovascular and cerebrovascular diseases such as atherosclerosis. Using in vitro cultured VECs to study related mechanisms is a common and important experimental method. However, existing VEC cell lines themselves have defects, are susceptible to cross-infection, and some are even not the original cell lines. Furthermore, VECs cultured via umbilical cord require the use of the umbilical cord, making the source difficult, complex, and limited in quantity. Induced pluripotent stem cells (IPS) are cells that have been reprogrammed into morphologically and functionally similar to embryonic stem cells by introducing specific transcription factors into differentiated adult cells. Current research has found that using iPS cells to induce vascular endothelial cell culture is a very promising technology. The emergence of iPS cells provides a new approach for the preparation and culture of vascular endothelial cells.

[0003] Chinese patent application CN116574672A discloses a culture medium and method for inducing chemical differentiation of pluripotent stem cells into hematopoietic endothelial cells, including a first-stage culture medium and a second-stage culture medium. The first-stage culture medium includes: basal medium DMEM / F12, non-essential amino acids, glutamine, thioglycerol, insulin, transferrin, sodium selenite, ethanolamine, vitamin C, lipid mixture, human albumin, WNT pathway inhibitor, and ROCK pathway inhibitor. The second-stage culture medium includes: basal medium DMEM / F12, non-essential amino acids, glutamine, thioglycerol, insulin, transferrin, sodium selenite, ethanolamine, vitamin C, lipid mixture, human albumin, recombinant human bone morphogenetic protein-4, basic fibroblast growth factor, and vascular endothelial growth factor.

[0004] Chinese patent application CN104928230A discloses a method for culturing vascular endothelial cells, comprising: 1) differentiating pluripotent stem cells into mesoendothelial precursor cells in culture medium A; 2) differentiating the mesoendothelial precursor cells into vascular endothelial cell progenitor cells in culture medium B; and 3) differentiating the vascular endothelial cell progenitor cells into vascular endothelial cells in culture medium C. Culture medium A contains DMEM medium, F12 medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, activin A, bone morphogenetic protein 4, and a glycogen synthase kinase-3 inhibitor; culture medium B contains DMEM medium, F12 medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, vascular endothelial growth factor, and a transforming growth factor β signaling pathway inhibitor; and culture medium C contains DMEM medium, F12 medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, vascular endothelial growth factor, epidermal growth factor, and fibroblast growth factor. This method differentiates pluripotent stem cells into vascular endothelial cells.

[0005] Currently, the culture media used for induced pluripotent stem cells in this field have overly complex compositions, often requiring inhibitors such as glycogen synthase kinase-3 inhibitors, WNT pathway inhibitors, ROCK pathway inhibitors, and bone morphogenetic proteins. While these media can ensure excellent self-renewal capabilities of stem cells and their induced differentiation, the introduction of numerous inhibitors introduces uncertainties into the self-renewal and differentiation processes of stem cells and their induced differentiation, which is detrimental to subsequent cell therapy. Summary of the Invention

[0006] To achieve the above-mentioned technical objectives, the present invention proposes the following technical solution:

[0007] In one aspect, the present invention provides a method for in vitro induction of pluripotent stem cell differentiation into vascular endothelial cells, the method comprising the following steps:

[0008] (1) Pluripotent stem cells were cultured for the first time in differentiation medium I to obtain cell A;

[0009] (2) The cells A were cultured for a second time in differentiation medium II to differentiate and form endoderm cells B;

[0010] (3) The endoderm cells B were cultured for the third time in differentiation medium III to obtain differentiated cells C;

[0011] (4) The differentiated cells C were cultured in differentiation medium IV for the fourth time to differentiate into differentiated cells D;

[0012] (5) The differentiated cells D were cultured for the fifth time in differentiation medium V to differentiate into vascular endothelial cells;

[0013] The differentiation culture media I, II, III, IV and V do not contain activator A, VEGF and BMP4.

[0014] In some embodiments, the differentiation medium I, differentiation medium II, differentiation medium III, differentiation medium IV and differentiation medium V do not contain synthase kinase-3 inhibitors, WNT pathway inhibitors and ROCK pathway inhibitors.

[0015] In some embodiments, the pluripotent stem cells are induced pluripotent stem cells.

[0016] In some embodiments, the vascular endothelial cells are human vascular endothelial cells.

[0017] In some embodiments, the differentiation culture medium I, differentiation culture medium II, differentiation culture medium III, and differentiation culture medium V contain an extracellular matrix.

[0018] In some embodiments, the extracellular matrix is ​​selected from at least one of collagen, gelatin, laminin, fibronectin, hydrin, nestin, or polylysine.

[0019] In some preferred embodiments, the differentiation culture medium I contains fibronectin.

[0020] In some preferred embodiments, the differentiation culture medium II contains fibronectin.

[0021] In some preferred embodiments, the differentiation culture medium III contains collagen.

[0022] In some preferred embodiments, the differentiation culture medium III is type IV collagen.

[0023] In some preferred embodiments, the differentiation culture medium V contains fibronectin.

[0024] In some embodiments, the differentiation culture media I, II, III, IV and V comprise a basal culture medium.

[0025] In some preferred embodiments, the basal culture medium is selected from RPMI 1640, M199, M119, MCDB131, IMDM, EMEM, αMEM, DMEM or Ham F12.

[0026] In some preferred embodiments, the basal culture medium is M119, preferably M119 with a pH of 7.2.

[0027] In some embodiments, the differentiation culture medium I further comprises collagenase and fetal bovine serum.

[0028] In some embodiments, the differentiation medium II further comprises heparin, L-glutamine, antibiotics, and / or insulin.

[0029] In some embodiments, the differentiation culture medium III further comprises an endothelial cell growth supplement and / or fetal bovine serum.

[0030] In some embodiments, the differentiation culture medium IV contains basic fibroblast growth factor, endothelial cell growth supplement, histone acetyltransferase inhibitor and / or heparin.

[0031] In some preferred embodiments, the basic fibroblast growth factor is recombinant bovine basic fibroblast growth factor.

[0032] In some embodiments, the differentiation culture medium V further comprises collagenase, insulin, antibiotics, fetal bovine serum, and / or endothelial cell growth supplements.

[0033] In some preferred embodiments, the antibiotics in the differentiation medium II and differentiation medium V include, but are not limited to, penicillin and / or streptomycin.

[0034] In some preferred embodiments, the endothelial cell growth supplement in differentiation culture medium III, differentiation culture medium IV and differentiation culture medium V is ECGS.

[0035] In some preferred embodiments, the differentiation culture medium I comprises 4 ng / mL fibronectin, 0.1% collagenase and 20% fetal bovine serum, wherein the percentages are by volume.

[0036] In some preferred embodiments, the differentiation culture medium II comprises 0.1 mg / mL heparin, 3 ng / mL L-glutamine, 4 ng / mL fibronectin, 100 µg / mL penicillin, 100 µg / mL streptomycin and 5 mg / mL insulin.

[0037] In some preferred embodiments, the differentiation medium III comprises 0.03-0.05 µg / mL ECGS, 3 ng / mL type IV collagen, and 10 ng / mL fetal bovine serum.

[0038] In some preferred embodiments, the differentiation medium IV comprises 1 ng / mL recombinant bovine basic fibroblast growth factor, 0.03-0.05 µg / mL ECGS, 5 ng / mL histone acetyltransferase inhibitor, and 0.1 mg / mL heparin.

[0039] In some preferred embodiments, the differentiation medium V comprises 1% collagenase, 4 mg / mL insulin, 100 µg / mL penicillin, 100 µg / mL streptomycin, 4 ng / mL fibronectin, 0.03-0.05 µg / mL ECGS and 10 ng / mL fetal bovine serum, wherein the percentages are by volume.

[0040] In some implementations, the first culture time is 1 day.

[0041] In some implementations, the second culture time is 2-3 days.

[0042] In some implementations, the third culture period is 6-7 days.

[0043] In some implementations, the fourth culture period is 21-22 days.

[0044] In another aspect, the present invention provides vascular endothelial cells obtained by any of the foregoing methods.

[0045] In another aspect, the present invention provides the use of any of the foregoing vascular endothelial cells in the preparation of cell therapy drugs.

[0046] In another aspect, the present invention provides a culture medium combination for inducing pluripotent stem cells to differentiate into vascular endothelial cells in vitro, the culture medium combination comprising differentiation medium I, differentiation medium II, differentiation medium III, differentiation medium IV and differentiation medium V;

[0047] The differentiation culture media I, II, III, IV and V do not contain activator A, VEGF and BMP4.

[0048] In some embodiments, the differentiation medium I, differentiation medium II, differentiation medium III, differentiation medium IV and differentiation medium V do not contain synthase kinase-3 inhibitors, WNT pathway inhibitors and ROCK pathway inhibitors.

[0049] In some embodiments, the differentiation culture medium I, differentiation culture medium II, differentiation culture medium III, and differentiation culture medium V contain an extracellular matrix.

[0050] In some embodiments, the extracellular matrix is ​​selected from at least one of collagen, gelatin, laminin, fibronectin, hydrin, nestin, or polylysine.

[0051] In some preferred embodiments, the differentiation culture medium I contains fibronectin.

[0052] In some preferred embodiments, the differentiation culture medium II contains fibronectin.

[0053] In some preferred embodiments, the differentiation culture medium III contains collagen.

[0054] In some preferred embodiments, the differentiation culture medium III is type IV collagen.

[0055] In some preferred embodiments, the differentiation culture medium V contains fibronectin.

[0056] In some embodiments, the differentiation culture media I, II, III, IV and V comprise a basal culture medium.

[0057] In some preferred embodiments, the basal culture medium is selected from RPMI 1640, M199, M119, MCDB131, IMDM, EMEM, αMEM, DMEM or Ham F12.

[0058] In some preferred embodiments, the basal culture medium is M119, preferably M119 with a pH of 7.2.

[0059] In some embodiments, the differentiation culture medium I further comprises collagenase and fetal bovine serum.

[0060] In some embodiments, the differentiation medium II further comprises heparin, L-glutamine, antibiotics, and / or insulin.

[0061] In some embodiments, the differentiation culture medium III further comprises an endothelial cell growth supplement and / or fetal bovine serum.

[0062] In some embodiments, the differentiation culture medium IV contains basic fibroblast growth factor, endothelial cell growth supplement, histone acetyltransferase inhibitor and / or heparin.

[0063] In some preferred embodiments, the basic fibroblast growth factor is recombinant bovine basic fibroblast growth factor.

[0064] In some embodiments, the differentiation culture medium V further comprises collagenase, insulin, antibiotics, fetal bovine serum, and / or endothelial cell growth supplements.

[0065] In some preferred embodiments, the antibiotics in the differentiation medium II and differentiation medium V include, but are not limited to, penicillin and / or streptomycin.

[0066] In some preferred embodiments, the endothelial cell growth supplement in differentiation culture medium III, differentiation culture medium IV and differentiation culture medium V is ECGS.

[0067] In some preferred embodiments, the differentiation culture medium I comprises 4 ng / mL fibronectin, 0.1% collagenase and 20% fetal bovine serum, wherein the percentages are by volume.

[0068] In some preferred embodiments, the differentiation culture medium II comprises 0.1 mg / mL heparin, 3 ng / mL L-glutamine, 4 ng / mL fibronectin, 100 µg / mL penicillin, 100 µg / mL streptomycin and 5 mg / mL insulin.

[0069] In some preferred embodiments, the differentiation medium III comprises 0.03-0.05 µg / mL ECGS, 3 ng / mL type IV collagen, and 10 ng / mL fetal bovine serum.

[0070] In some preferred embodiments, the differentiation medium IV comprises 1 ng / mL recombinant bovine basic fibroblast growth factor, 0.03-0.05 µg / mL ECGS, 5 ng / mL histone acetyltransferase inhibitor, and 0.1 mg / mL heparin.

[0071] In some preferred embodiments, the differentiation medium V comprises 1% collagenase, 4 mg / mL insulin, 100 µg / mL penicillin, 100 µg / mL streptomycin, 4 ng / mL fibronectin, 0.03-0.05 µg / mL ECGS and 10 ng / mL fetal bovine serum, wherein the percentages are by volume.

[0072] In another aspect, the present invention provides the use of any of the aforementioned culture medium combinations in in vitro inducing the differentiation of iPS cells into human vascular endothelial cells.

[0073] This invention proposes a method for inducing iPS cells to differentiate into human vascular endothelial cells in vitro. By adding a corresponding differentiation additive to M119 culture medium, iPS cells are directionally induced to differentiate, which improves the differentiation efficiency of iPS cells into vascular endothelial cells. This method allows for the rapid and convenient preparation of vascular endothelial cells through iPS differentiation. Attached Figure Description

[0074] Figure 1 This is a microscopic image of a cell from Example 1. Detailed Implementation

[0075] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0076] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0077] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0078] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0079] As used herein, the terms “comprises” or “comprising” mean “including, but not limited to”. This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term “comprising” includes the more restrictive terms “consisting of” and “substantially consisting of”. In one embodiment, the term “comprising” as used throughout the application, particularly in the claims, may be replaced by the term “consisting of”.

[0080] As used herein, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation occurs or does not occur. As used herein, “an” and “a” refer to one or more grammatical objects.

[0081] The term “and / or” as used herein should be understood to mean any one of the options or any combination of two or more of the options.

[0082] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without a specified manufacturer are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0083] Example 1

[0084] (1) Take 1×10 6 IPS cells were cultured on differentiation medium I for 1 day at a temperature of 36-37℃ and an ambient air concentration of 5% CO2 to obtain cell type A with a cell confluence of 80%-90%.

[0085] (2) The cells obtained in step (1) are cultured on differentiation medium II for 2-3 days to differentiate and form endoderm cells B.

[0086] (3) The cells obtained in step (2) are cultured on differentiation medium III for 6-7 days to obtain differentiated cells C;

[0087] (4) The cells obtained in step (3) are cultured on differentiation medium IV for 6-7 days to differentiate into differentiated cells D;

[0088] (5) The cells obtained in step (4) are cultured on differentiation medium V for 21-22 days to differentiate into vascular endothelial cells.

[0089] The differentiation medium I was prepared by adding 4 ng / mL fibronectin, 0.1% collagenase (type I), and 20% fetal bovine serum to 500 mL M119 culture medium (pH 7.2).

[0090] The differentiation medium II was prepared by adding heparin (final concentration 0.1 mg / mL), L-glutamine (3 ng / mL), fibronectin (4 ng / mL), penicillin (100 µg / mL), streptomycin (100 µg / mL) and insulin (5 mg / mL) to 500 mL of M119 culture medium (pH 7.2).

[0091] The differentiation medium III was prepared by adding ECGS (0.03-0.05 µg / mL), type IV collagen (3 ng / mL), and fetal bovine serum (10 ng / mL) to 500 mL of M119 culture medium (pH 7.2).

[0092] The differentiation medium IV consisted of 500 mL of M119 culture medium (pH 7.2) with the addition of recombinant bovine basic fibroblast growth factor (1 ng / mL), ECGS (0.03-0.05 µg / mL), histone acetyltransferase inhibitor (5 ng / mL), and heparin (final concentration 0.1 mg / mL).

[0093] The differentiation medium V is prepared by adding type I collagenase (1%), insulin (4 mg / mL), penicillin (100 µg / mL), streptomycin (100 µg / mL), fibronectin (4 ng / mL), ECGS (0.03-0.05 µg / mL), and fetal bovine serum (10 ng / mL) to 500 mL of M119 culture medium (pH 7.2).

[0094] Comparative Example 1

[0095] The only difference between this comparative example and Example 1 is that the fibronectin in differentiation medium I, differentiation medium II and differentiation medium V is replaced with type IV collagen.

[0096] Comparative Example 2

[0097] The only difference between this comparative example and Example 1 is that the fibronectin in differentiation medium I, differentiation medium II, and differentiation medium V is replaced with laminin.

[0098] Comparative Example 3

[0099] The only difference between this comparative example and Example 1 is that the fibronectin in differentiation medium I, differentiation medium II and differentiation medium V is replaced with hydrin.

[0100] Comparative Example 4

[0101] The only difference between this comparative example and Example 1 is that type IV collagen in differentiation medium III is replaced with fibronectin.

[0102] Comparative Example 5

[0103] The only difference between this comparative example and Example 1 is that type IV collagen in differentiation medium III is replaced with laminin.

[0104] Comparative Example 6

[0105] The only difference between this comparative example and Example 1 is that no histone acetyltransferase inhibitor was added to the differentiation medium IV.

[0106] Comparative Example 7

[0107] The only difference between this comparative example and Example 1 is that ECGS is not added to the differentiation medium IV.

[0108] Comparative Example 8

[0109] The only difference between this comparative example and Example 1 is that histone acetyltransferase inhibitors and ECGS are not added to the differentiation medium IV.

[0110] Experimental Cell Flow Cytometry Analysis

[0111] Using the methods described in Example 1 and Comparative Examples 1-8, iPS cells were induced to differentiate into human vascular endothelial cells in vitro. Then, the cells were analyzed by flow cytometry 10 days after differentiation to obtain the proportion of cells expressing the endothelial cell marker protein CD31. The results were repeated 5 times and are shown in Table 1.

[0112] Table 1

[0113]

[0114] Note: Compared with Example 1, ** p < 0.01, *** p < 0.001.

[0115] The results showed that the proportion of the marker protein CD31 in Example 1 was the highest, reaching 92.5%. Compared with Comparative Examples 1-8, this indicates that the differentiation culture medium combination provided by the present invention can efficiently induce pluripotent differentiation into vascular endothelial cells, resulting in a conversion rate of over 90%.

[0116] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for inducing pluripotent stem cell differentiation into human vascular endothelial cells in vitro, characterized in that, The method includes the following steps: (1) Pluripotent stem cells were cultured in differentiation medium I for 1 day to obtain cell A; (2) The cells A were cultured in differentiation medium II for 2-3 days to differentiate and form endoderm cells B. (3) The endoderm cells B were cultured in differentiation medium III for 6-7 days to obtain differentiated cells C; (4) The differentiated cells C were cultured in differentiation medium IV for 6-7 days to differentiate into differentiated cells D; (5) The differentiated cells D were cultured in differentiation medium V for 21-22 days to differentiate into human vascular endothelial cells; The differentiation medium I was prepared by adding 4 ng / mL fibronectin, 0.1% type I collagenase and 20% fetal bovine serum to M119 culture medium, where the percentages are by volume. The differentiation medium II was prepared by adding 0.1 mg / mL heparin, 3 ng / mL L-glutamine, 4 ng / mL fibronectin, 100 µg / mL penicillin, 100 µg / mL streptomycin and 5 mg / mL insulin to M119 culture medium. The differentiation medium III was prepared by adding 0.03-0.05 µg / mL ECGS, 3 ng / mL type IV collagen, and 10 ng / mL fetal bovine serum to M119 culture medium. The differentiation medium IV consisted of M119 culture medium supplemented with 1 ng / mL recombinant bovine basic fibroblast growth factor, 0.03-0.05 µg / mL ECGS, 5 ng / mL histone acetyltransferase inhibitor, and 0.1 mg / mL heparin. The differentiation medium V is M119 culture medium supplemented with 1% type I collagenase, 4 mg / mL insulin, 100 µg / mL penicillin, 100 µg / mL streptomycin, 4 ng / mL fibronectin, 0.03-0.05 µg / mL ECGS and 10 ng / mL fetal bovine serum, where the percentages are by volume. The pH of the M119 culture medium is 7.

2.

2. A culture medium composition for inducing pluripotent stem cell differentiation into human vascular endothelial cells in vitro, characterized in that, The culture medium combination consists of differentiation medium I, differentiation medium II, differentiation medium III, differentiation medium IV, and differentiation medium V; The differentiation medium I was prepared by adding 4 ng / mL fibronectin, 0.1% type I collagenase and 20% fetal bovine serum to M119 culture medium, where the percentages are by volume. The differentiation medium II was prepared by adding 0.1 mg / mL heparin, 3 ng / mL L-glutamine, 4 ng / mL fibronectin, 100 µg / mL penicillin, 100 µg / mL streptomycin and 5 mg / mL insulin to M119 culture medium. The differentiation medium III was prepared by adding 0.03-0.05 µg / mL ECGS, 3 ng / mL type IV collagen, and 10 ng / mL fetal bovine serum to M119 culture medium. The differentiation medium IV consisted of M119 culture medium supplemented with 1 ng / mL recombinant bovine basic fibroblast growth factor, 0.03-0.05 µg / mL ECGS, 5 ng / mL histone acetyltransferase inhibitor, and 0.1 mg / mL heparin. The differentiation medium V is M119 culture medium supplemented with 1% type I collagenase, 4 mg / mL insulin, 100 µg / mL penicillin, 100 µg / mL streptomycin, 4 ng / mL fibronectin, 0.03-0.05 µg / mL ECGS and 10 ng / mL fetal bovine serum, where the percentages are by volume. The pH of the M119 culture medium is 7.

2.

3. Use of the culture medium combination according to claim 2 in in vitro induction of pluripotent stem cell differentiation into human vascular endothelial cells.

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