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Effective method for differentiating from hPSCs to MSCs

A BMP-SMAD1, high-efficiency technology, applied in the field of cell culture, can solve complex and difficult to control problems, and achieve the effect of high reproducibility, simplified steps, and easy operation

Active Publication Date: 2018-09-25
OSINGLAY BIO PHARM CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But it will inevitably lead to the derivation of unwanted cell lineages
However, the differentiation of stem cells into the required cell lineage is a very complex and difficult to control process, especially in human embryonic biology (limited by ethics and morals), so it is necessary to highly simulate in vitro and follow the biological principles of human embryonic development to study stem cells Directed differentiation is a very difficult and very challenging scientific research work
Therefore, no complete protocol for the directed differentiation of hPSCs into highly mimicking embryonic MSCs has been reported so far.

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  • Effective method for differentiating from hPSCs to MSCs
  • Effective method for differentiating from hPSCs to MSCs

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Experimental program
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Effect test

Embodiment 1

[0037] 1) The undifferentiated hPSCs cells ( figure 1 with figure 2 ) Transfer to Matrigel (Matrigel, MG; a kind of extracellular matrix) coated with a 24-well adherent culture plate for 30 minutes, use 0.5ml pluripotent stem cell differentiation medium, 37℃, 5% CO 2 After 24 hours of culture (Day 0), change to 0.5ml pluripotent stem cell differentiation medium without TGF-β1. In addition, the medium also contains 40ng / ml BMP4 and 30ng / mlActivin A, 6μM CHIR99021, 100nMPIK90. Among them, BMP4 is bone morphogenetic protein 4, CHIR99021 is 6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidine-2 -Amino]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. Detect the expression of MIXL1, BRACHYURY, HAND1 and other mid-strip markers. It was found that the three middle primitive streak markers were all highly...

Embodiment 2

[0053] Carry out differentiation culture according to the method of Example 1, and make the following improvements: Step 2) Replace with 20ng / ml BMP4 (BMP-SMAD1 / 5 / 8 signal pathway activator), 3uM / ml SB505124 (TGF-β1 / Activin / Nodal- SMAD2 / 3 signaling pathway activator), 1uM / ml Wnt-C59 (Wnt inhibitor) differentiation medium for 24 hours, at this time the lateral mesoderm markers Nkx2.5, HAND1, FOXF1, etc. are highly expressed (Table 4) It can be concluded that the cells are at the stage of lateral mesoderm at this time, and then change to 0.5ml pluripotent stem cell differentiation medium containing 10uM / ml SB431542 without TGF-β1 and continue to culture. The medium is changed every other day without passage. SB505124 is 2-[4-(1,3-benzodiazol-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] benzacetamide (CAS: 12243243 89-1). In step 4), MSCs precursor cells with typical spindle-shaped mo...

Embodiment 3

[0060] Carry out differentiation culture according to the method of Example 1, and make the following improvements: Step 1) After adding basic fibroblast growth factor (bFGF) to the differentiation medium, at step 4) Will get more and more typical MSCs precursor cells with spindle-shaped morphology, such as Figure 5 Shown. The rest of the steps remain unchanged. Finally, after FACS identification, the cells can express CD105, CD90, CD73 and other MSC positive Markers at a high level; they basically do not express CD45, CD34, CD14, CD11b, CD79α, CD19 and HLA-DR and other MSC negative Markers, as shown in the table 6 shown.

[0061] Table 6

[0062]

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Abstract

The invention discloses an effective method for differentiation induction from hPSCs to MSCs. The effective method comprises the following steps: 1) after transferring a hPSCs cell strain which is notdifferentiated on a culture plate coated with an extracellular matrix and culturing, culturing by using a differentiation culture medium which contains BMP-SMAD1 / 5 / 8 signal channel activator, a TGF-beta 1 / Activin / Nodal-SMAD2 / 3 signal channel activator, a Wnt activator and a PI3K inhibitor; 2) removing an old culture medium, and continuing to culture by using a differentiation culture medium whichcontains a TGF-beta1 / Activin / Nodal-SMAD2 / 3 signal channel inhibitor; 3) dissociating and transferring cells to a new culture plate, and continuing to culture by using a differentiation culture mediumwhich contains a TGF-beta1 / Activin / Nodal-SMAD2 / 3 signal channel inhibitor; and 3) dissociating and transferring cells to a wall-attached culture plate and continuing to culture so as to obtain MSCs.The complete scheme and method for differentiating into MSCs from hPSC in an oriented manner through a middle-section primitive streak stage or a side mesoderm stage is established in vitro. Comparedwith a conventional method, the effective method is simple in technical step, simple and easy to operate and high in reproducibility, and MSCs with mature phenotype and high quality can be obtained within 12 days only.

Description

Technical field [0001] The present invention relates to the field of cell culture, in particular to an efficient method for differentiation of hPSCs into MSCs. Background technique [0002] As the cell source for clinical regenerative medicine transplantation, it mainly includes human pluripotent stem cells (hPSCs), namely human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), etc. These two categories are collectively referred to as adult stem cells. Adult stem cells include hematopoietic stem cells and mesenchymal stem cells. Mesenchymal stem cells derived from the mesoderm were first identified by Friedenstein et al. in 1966 when they found fibroblast-like cells in a mouse bone marrow extract. In 1991, Caplan originally called these cells mesenchymal stem cells ( mesenchymal stemcells, MSCs). In 2006, the International Society for Cellular Therapy (ISCT) proposed to officially name these cells'multipotent mesenchymal stromal cells' or'mesenchym...

Claims

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Application Information

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IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2501/115C12N2533/54C12N2533/52C12N2501/58C12N2501/599C12N2501/155C12N2502/1352C12N2533/90C12N2506/02C12N2506/45C12N2501/415C12N2501/15C12N5/0668
Inventor 王淋立李强陈月花莫健
Owner OSINGLAY BIO PHARM CO LTD
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