WO2019144605A1

WO2019144605A1 - High performance method for differentiation of hpscs into mscs

Info

Publication number: WO2019144605A1
Application number: PCT/CN2018/101076
Authority: WO
Inventors: 王淋立; 李强; 陈月花; 莫健
Original assignee: 皓昇莱生物制药有限公司; 王淋立
Priority date: 2018-01-26
Filing date: 2018-08-17
Publication date: 2019-08-01
Also published as: CN108570448B; CN108570448A

Abstract

Disclosed in the present invention is a high performance method for inducing differentiation of hPSCs into MSCs, comprising the steps of: 1) transferring undifferentiated hPSC cell lines onto a culture plate coated with an extracellular matrix for cultivation; and then cultivating same by using a differentiation culture medium containing BMP-SMAD1/5/8 signaling pathway activator, TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway activator, Wnt activator and PI3K inhibitor; 2) removing the old culture medium, and continuing to cultivate by using a differentiation culture medium containing TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway inhibitor; 3) digesting and transferring the cells onto a new cultivation plate, and continuing to cultivate by using a differentiation culture medium containing TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway inhibitor; and 4) digesting the cells and transferring same onto an adherent culture plate for continued cultivation, so as to obtain MSCs. The present invention establishes in vitro a complete method for the directed differentiation of hPSCs into MSCs through the middle primitive streak stage or the lateral mesoderm stage. Compared with the conventional methods, the technical steps of the present method are simple, easy to operate, and highly reproducible, and MSCs having mature phenotype and high quality can be obtained within only 12 days.

Description

A method for efficiently differentiating hPSCs into MSCs

Technical field

The invention relates to the field of cell culture, in particular to a method for efficiently differentiating hPSCs into MSCs.

Background technique

The source of cells for clinical regenerative medicine transplantation mainly includes human pluripotent stem cells (hPSCs), namely human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The general term and adult stem cells are two major categories, including adult stem cells including hematopoietic stem cells and mesenchymal stem cells. Mesenchymal stem cells derived from mesoderm were originally isolated and identified by fibroblast-like cells from mouse bone marrow extracts by Friedenstein et al. in 1966. In 1991, Caplan initially referred to these cells as mesenchymal stem cells ( Mesenchymal stem cells, MSCs). In 2006, the International Society for Cellular Therapy (ISCT) proposed to officially name these cells as 'multipotent mesenchymal stromal cells' or 'mesenchymal stem cells' (MSCs); and define MSCs as 1) to grow adherently; 2) can differentiate into mesoderm lineages such as osteoblasts, cartilage and adipocytes; 3) high-level expression of CD105, CD90, CD73 and other MSCs positive markers; basically do not express CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR Such as MSCs negative markers. In addition to self-renewal, MSCs differentiate into the muscle, bone, cartilage, fat, tendon and ligament of the mesoderm lineage, and even transdifferentiate into the multi-lineage differentiation and immune regulation of neurons and islet cells, and avoid tumors. According to the National NIH Clinical Trial Registry ClinicalTrials.gov, the 2016 Clinical Trial Database (with approximately 250,000 trial records in 201 countries) shows that ~500 clinical trials are related to MSCs, many of which have evaluated MSC vs. The effects of various diseases, including osteoarthritis, wound healing, degenerative diseases, autoimmune diseases, etc. This shows that MSCs are important in cell regenerative medicine. Although the sources of MSCs are widely available, clinical applications are generally obtained from bone marrow, umbilical cord and adipose tissue, but the isolation of MSCs from tissues will result in low yield, presence of mixed cells and purification of cells, and a more mature table. The longer time required for the type (about 1 month) is likely to cause cell aging, allogeneic immune rejection (the source of the umbilical cord) and the like. Therefore, in terms of clinical needs, the shortage of donor sources is still a problem that must be solved. In contrast, MSCs derived from autologous or immunomatch-matched and immortalized differentiation of hiPSCs are better at addressing these clinical cell therapies than MSCs derived from fat, cord blood, bone marrow, and ESCs. .

At present, the most classical method for the derivation of MSCs (hPSC-MSCs) from hPSCs (hiPSCs and hESCs) is to block the ALK5,4,7 kinase in the transforming growth factor TGF-β (Transforming Growth Factor) signaling pathway by the small molecule compound SB431542. The effect of suppressing the SMAD2/3 signal is achieved. Thus, the pluripotency of hiPSCs or hESCs is degraded, EMT (Epithelial-to-Mesenchymal Transition) is induced, and hPSC-MSCs are promoted, thereby obtaining phenotypic mature and high-quality hPSC-MSCs. But it will inevitably lead to the derivation of unwanted cell lineages. However, the differentiation of stem cells into the desired cell lineage is a complex and difficult process to control, especially human embryo biology (limited to ethics), so it is highly simulated in vitro, following the biological principles of human embryo development to study stem cells. Directional differentiation is a very difficult and challenging research work. Therefore, there has not been a complete method for the differentiation of hPSCs into highly mimetic embryonic MSCs.

Summary of the invention

The object of the present invention is a method for efficiently differentiating hPSCs into MSCs.

The technical solution adopted by the present invention is:

A method for efficiently inducing differentiation of hPSCs into MSCs, the steps of which are:

1) Transfer undifferentiated hPSCs cell line to extracellular matrix-coated culture plates; switch to BMP-SMAD1/5/8 signaling pathway activator, TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway Activator, Wnt activator, PI3K inhibitor differentiation medium culture; 2) Remove old medium, continue to culture with TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway inhibitor differentiation medium; 3) Cell Digest and transfer to a new culture plate, and continue to culture with TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway inhibitor differentiation medium; 4) Digest the cells and transfer them to the adherent culture plate for further cultivation. Obtain MSCs.

Preferably, in step 3), the new culture plate is coated with a gelatin-based extracellular matrix for cell screening.

Preferably, the cells obtained by cell screening are cells having a spindle-type morphological feature, positive surface markers CD90, CD73, CD105, negative for CD34, CD45, CD14 or CD11b, CD79α or CD19, and HLA-DR.

Preferably, the gelatin-based extracellular matrix is gelatin for cell culture or type I, III, IV, V-type collagen and elastin, and laminin.

Preferably, in step 1), culture is carried out using a differentiation medium containing BMP-SMAD1/5/8 signaling pathway activator, TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway activator, Wnt activator, PI3K inhibitor. The time is 12 to 72 hours.

Preferably, in step 1), at least one of BMP-SMAD1/5/8 signaling pathway activators BMP2, BMP4, BMP7.

Preferably, in step 1), the TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway activator is at least one of ActivinA, Activin B, TGF-β1, and Nodal.

Preferably, in step 1), the Wnt activator is at least one of CHIR99021, BIO, WNT-3a, and R-spondin-2.

Preferably, in step 1), the PI3K inhibitor is at least one of TG100713, PIK90, and PI-103.

Preferably, the TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway inhibitor is at least one of SB431542, SB505124, A8301, and RepSox.

Preferably, in step 3), the culture is continued for 2 to 30 days.

Preferably, prior to continuing the culture in step 2), culture with a differentiation medium containing BMP-SMAD1/5/8 signaling pathway activator, TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway activator, Wnt inhibitor .

Preferably, in step 2), the culture is continued for 2 to 10 days.

Preferably, the Wnt inhibitor is at least one of Wnt-C59 and XAV-939.

Preferably, the method of continuing the culture in step 4) is: culturing with a medium containing mesenchymal stem cells.

Preferably, in step 1) to step 4), at least one medium contains basic fibroblast growth factor bFGF.

Preferably, the obtained MSCs are obtained by inducing hPSCs to differentiate into MSCs through the middle stage or the mesoderm stage.

An MSCs cell prepared by the above differentiation method.

The beneficial effects of the present invention are as follows: 1) The present invention is the first method in the world to establish intact hPSCs in vitro to differentiate into MSCs through the middle stage or the lateral mesoderm stage. To provide a reliable technical resource for studying the differences between strains derived from different cells of human pluripotent stem cells and the differences between MSCs in vitro and in vivo.

2) The method for direct differentiation of hPSC-MSCs of the present invention has the advantages of simple steps, simple operation and high reproducibility, and phenotypic mature and high quality MSCs derived from mesodermal lineage can be obtained in only 12 days.

DRAWINGS

Figure 1 shows undifferentiated cells of hiPSCs.

Figure 2 shows undifferentiated cells of hESCs.

Figure 3 shows the differentiation of MSCs precursor cells obtained from the middle segment.

Figure 4 shows MSCs precursor cells obtained by differentiation through the mesoderm.

Figure 5 is a MSCs precursor cell obtained by adding bFGF to differentiate into a middle segment.

Figure 6 shows MSCs cells differentiated from hiPSCs into phenotypic and spindle-shaped forms.

Figure 7 shows MSCs cells differentiated from hESCs into phenotypic and spindle-shaped forms.

Figure 8 shows no cells found to adhere to growth.

Detailed ways

Culture method for differentiation of hPSCs into MSCs:

Example 1:

1) Transfer undifferentiated hPSCs cells (Fig. 1 and Fig. 2) to matrigel (Matrigel, MG; one of extracellular matrices), coated with a 30-well adherent plate for 30 min, and differentiated with 0.5 ml of pluripotent stem cells. The medium was cultured at 37 ° C, 5% CO _{2 for} 24 hours (Day 0), and changed to 0.5 ml of pluripotent stem cell differentiation medium containing no TGF-β1. In addition, the medium also contained 40 ng/ml BMP4 and 30 ng/ml Activin A, 6 μM CHIR99021, 100 nM PIK90. Among them, BMP4 is bone morphogenetic protein 4, and CHIR99021 is 6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidine-2 -ylamino]ethylamino]pyridine-3-carbonitrile (CAS: 252917-06-9), Activin A is activin A, PIK90 is N-(2,3-dihydro-7,8-dimethoxy Imidazo[1,2-c]quinazolin-5-yl)-3-pyridinecarboxamide (CAS: 677338-12-4). Cultivate for 24 hours. The expression levels of the raw strip markers in the middle segments such as MIXL1, BRACHYURY, and HAND1 were detected. It was found that all the three middle-section original strip markers were highly expressed, and it can be concluded that the cells were in the middle stage of the original strip (Table 1).

2) The old solution was removed, and 0.5 ml of the pluripotent stem cell differentiation medium containing no TGF-β1 containing 10 uM/ml SB431542 was continuously cultured for 5 days, and the solution was changed every other day without passage. SB431542 is 4-[4-(1,3-benzothiazol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate (CAS: 301836-41 -9). After treatment with SB431542, the expressions of pluripotency markers Oct3/4, Nanog, Sox2, etc. gradually decreased (Table 2), BRACHYURY decreased to no expression, and the mesodermal markers Nkx2.5, HAND1, FOXF1, etc. continued to be highly expressed. It can be concluded that the cells are in the stage of the mesoderm at this time (Table 1).

Table 1

Table 2

3) Remove the old solution, add 2 ml of 0.05% Trypsin-EDTA, digest the cells after 5 days of SB treatment for 5 min, add 4 ml of DMEM/F12 (containing 10% FBS) to terminate, collect 15 ml centrifuge tubes, and centrifuge at 200 g for 5 min.

4) Remove the supernatant, add 1 ml of pluripotent cell differentiation medium containing 10 uM/ml SB431542 without TGF-β1, gently suspend the cells, transfer to gelatin (GT) for 30 min, add 10 uM/ MLC SB431542 was cultured on a P100 adherent plate of 9 ml of pluripotent stem cell differentiation medium containing no TGF-β1 for 48 hours to obtain cells having adherent properties and spindle-shaped morphology (Fig. 3). The culture was continued for 3 days at 37 ° C, 5% CO ₂ , and the solution was changed every other day, which is the precursor cell stage of MSCs. Gelatin is gelatin for cell culture or type I, III, IV, V collagen and elastin, and laminin.

5) The cell confluence can reach ～90% by GT stage, and the cells are digested with 2ml of 0.05% Trypsin-EDTA for 3min, and then added with 4ml DMEM/F12 (containing 10% FBS). The 15ml centrifuge tube is recovered and centrifuged at 200g for 5min.

6) Remove the supernatant, add 1 ml of pluripotent stem cell differentiation medium containing 10 uM/ml SB431542 and no TGF-β1 inhibitor, gently suspend the cells, and transfer to (1:5) without any coating. 9 ml of P100 adherent medium containing 10 uM/ml SB431542 and no TGF-β1 inhibitor was cultured on a culture plate for 24 hours and then replaced with 10% FBS α-MEM, 37 ° C, 5% CO ₂ The culture was carried out for 3 days, and the results of flow identification were shown in Table 3. After FACS identification, the cells can express MSC positive Markers such as CD105, CD90 and CD73 at high levels; they do not express MSC-negative Markers such as CD45, CD34, CD14 or CD11b, CD79α, CD19 and HLA-DR. In addition, the cells have a typical spindle-shaped morphology, and the cells can be identified as MSCs cells (Fig. 6 and Fig. 7).

table 3

In addition, BMP4 was replaced with BMP2 and BMP7; Activin A was replaced with Activin B, TGF-β1, Nodal, and CHIR99021 was used with BIO (CAS: 667463-62-9), WNT-3a (WNT signaling protein 3a), R-spondin- 2 (R-spondin signal protein 2) substitution; SB431542 was replaced with SB505124 (CAS: 694433-59-5), A8301 (CAS: 909910-43-6), RepSox (CAS: 446859-33-2) PI-103; A similar effect can also be produced by replacing PIK90 with TG100713 (CAS: 925705-73-3), PI-103 (CAS: 371935-74-9).

Example 2:

The differentiation culture was carried out in accordance with the method of Example 1, and the following improvements were made: step 2) containing 20 ng/ml BMP4 (BMP-SMAD1/5/8 signaling pathway activator), 3 uM/ml SB505124 (TGF-β1/Activin/Nodal- The differentiation medium of SMAD2/3 signaling pathway activator and 1uM/ml Wnt-C59 (Wnt inhibitor) was further cultured for 24 hours, at which time the mesodermal markers Nkx2.5, HAND1, FOXF1 and the like were highly expressed (Table 4). It can be concluded that the cells are in the stage of mesoderm at this time, and then cultured in 0.5 ml of pluripotent stem cell differentiation medium containing no TGF-β1 containing 10 uM/ml SB431542, and the cells were changed every other day without passage. SB505124 is 2-[4-(1,3-benzobisazol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methylpyridine (CAS: 694433-59-5), Wnt-C59 is 4-(2-methyl-4-pyridyl)-N-[4-(3-pyridyl)phenyl]phenylacetamide (CAS: 1243243- 89-1). At the time of step 4), typical spindle-shaped MSCs precursor cells are obtained, as shown in FIG. The remaining steps were unchanged. Finally, after FACS identification, the cells could express MSC-positive Markers such as CD105, CD90 and CD73 at high levels; MSC-negative Markers such as CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR were not expressed at all. 5 is shown.

Table 4

table 5

Example 3:

The differentiation culture was carried out in accordance with the method of Example 1, and the following improvements were made: Step 1) Starting with the addition of basic fibroblast growth factor (bFGF) on the basis of the differentiation medium, at step 4) More and more typical spindle-shaped morphological MSCs precursor cells will be obtained, as shown in Figure 5. The remaining steps were unchanged. Finally, after FACS identification, the cells could express MSC-positive Markers such as CD105, CD90, CD73, etc.; MSC-negative Markers such as CD45, CD34, CD14, CD11b, CD79α, CD19 and HLA-DR were not expressed at all. 6 is shown.

Table 6

Comparative example

1) The undifferentiated hPSCs cells were transferred to a 24-well adherent culture plate coated with extracellular matrix (MG) for 30 min, and cultured with 0.5 ml of pluripotent stem cell differentiation medium at 37 ° C, 5% CO _{2 for} 24 hours ( Day 0), 0.5 ml of pluripotent stem cell differentiation medium containing no TGF-β1, containing 30 ng/ml Activin A, 6 μM CHIR99021, and 100 nM PIK90 for 24 hours. The expression levels of the protocolonary markers such as MIXL1, BRACHYURY, and TBX6 were detected, and it was found that high expression was obtained, and the cells were in the pre-segment phase (Table 7).

Table 7

2) The old solution was removed, and 0.5 ml of the pluripotent stem cell differentiation medium containing no TGF-β1 containing 10 uM/ml SB431542 was continuously cultured for 5 days, and the solution was changed every other day without passage.

4) Remove the supernatant, add 1 ml of pluripotent cell differentiation medium containing 10 uM/ml SB431542 without TGF-β1, gently suspend the cells, transfer to gelatin (GT) for 30 min, and add 10 uM/ml SB431542. 9 ml of the P100 adherent culture medium containing no TGF-β1 pluripotent stem cell differentiation medium was cultured for 48 hours, and no cells capable of adhering to growth were found (Fig. 8).

Claims

A method for efficiently inducing differentiation of hPSCs into MSCs, the steps of which are:

1) Transfer undifferentiated hPSCs cell line to extracellular matrix-coated culture plates; switch to BMP-SMAD1/5/8 signaling pathway activator, TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway Differentiation medium culture of activator, Wnt activator, PI3K inhibitor;

2) Remove the old medium and continue to culture with the differentiation medium containing TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway inhibitor;

3) Digest and transfer the cells to a new culture plate, and continue to culture with TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway inhibitor differentiation medium;

4) The cells were digested and transferred to an adherent culture plate for further culture to obtain MSCs.
The method of differentiation induction according to claim 1, wherein in the step 3), the new culture plate is coated with a gelatin-based extracellular matrix for cell selection.
The differentiation method according to claim 2, wherein the cells obtained by the cell screening have a spindle-shaped morphological characteristic, and the surface markers CD90, CD73, and CD105 are positive, CD34, CD45, CD14 or CD11b, CD79α or CD19, and HLA-DR. Negative cells.
The differentiation method according to claim 2, wherein the gelatin-based extracellular matrix is gelatin for cell culture or type I, III, IV, V-type collagen and elastin, and laminin.
The differentiation method according to claim 1, wherein in step 1), a BMP-SMAD1/5/8 signaling pathway activator, a TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway activator, Wnt is used. The differentiation medium culture time of the activator and the PI3K inhibitor is 12 to 72 hours.
The differentiation method according to claim 1, wherein in step 1), at least one of BMP-SMAD1/5/8 signaling pathway activators BMP2, BMP4, BMP7.
The differentiation method according to claim 1, wherein in step 1), the TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway activator is at least one of Activin A, Activin B, TGF-β1, and Nodal. Kind.
The differentiation method according to claim 1, wherein in the step 1), the Wnt activator is at least one of CHIR99021, BIO, WNT-3a, and R-spondin-2.
The differentiation method according to claim 1, wherein in the step 1), the PI3K inhibitor is at least one of TG100713, PIK90, and PI-103.
The differentiation method according to claim 1, wherein the TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway inhibitor is at least one of SB431542, SB505124, A8301, and RepSox.
The differentiation method according to claim 1, wherein in the step 3), the culture is continued for 2 to 30 days.
The differentiation method according to claim 1, characterized in that before the step 2), the BMP-SMAD1/5/8 signaling pathway activator, TGF-β1/Activin/Nodal-SMAD2/3 signaling pathway is used. The differentiation medium of the activator and the Wnt inhibitor is cultured.
The differentiation method according to claim 1, wherein in the step 2), the culture is continued for 2 to 10 days.
The differentiation method according to claim 12, wherein the Wnt inhibitor is at least one of Wnt-C59 and XAV-939.
The differentiation method according to claim 1, wherein the step of continuing the culture in the step 4) is: culturing with a medium containing mesenchymal stem cells.
The differentiation method according to claim 1, wherein in the step 1) to the step 4), the at least one medium contains basic fibroblast growth factor bFGF.
The differentiation method according to claim 1, wherein the obtained MSCs are obtained by inducing differentiation of hPSCs into MSCs by a middle stage or a mesoderm stage.
An MSCs cell prepared by the differentiation method according to any one of claims 1 to 17.