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Method for differentiating induced pluripotent stem cells into mesenchymal stem cells

A technology for pluripotent stem cells and pluripotent stem cells, which can be applied to artificially induced pluripotent cells, biochemical equipment and methods, animal cells, etc., and can solve the problems of long induction time, low induction efficiency, and complicated induction process.

Active Publication Date: 2017-03-22
GUANGDONG XTEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells, which can solve the shortcomings of traditional methods such as complicated induction process, long induction time, and low induction efficiency, and provide a unified source, sufficient amount, and high-quality MSc lays the groundwork

Method used

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  • Method for differentiating induced pluripotent stem cells into mesenchymal stem cells
  • Method for differentiating induced pluripotent stem cells into mesenchymal stem cells
  • Method for differentiating induced pluripotent stem cells into mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1 Inducing the differentiation of human iPSCs into MSCs

[0048] 1. Culture of human iPS cells

[0049] In this example, the human iPS cells (DYR0100, Cell Bank of Chinese Academy of Sciences) used were cultured on Matrigel (BD, 354277), and maintained with mTeSRTM1 (STEMCELL TECHNOLOGIES, 05850), such as figure 2 as shown in a. The specific cultivation method includes the following steps:

[0050] ①Take out the culture medium and required reagents from the refrigerator, and preheat them in a 37°C water bath for about 15 minutes;

[0051] ② Take out the cells, discard the original medium, and wash the cells once with DPBS (no calcium, no magnesium) (Invitrogen, 14190250);

[0052] ③ Add 1 mg / ml collagenase IV (dissolved in HBSS buffer, sterilized by 0.22 μm filter) (Invitrogen17104019) for digestion, place in a 37°C cell culture incubator and incubate for about 5 minutes, observe under a microscope, the edges of the clones have not rolled up yet Terminate ...

Embodiment 2

[0064] Example 2 Detection of differentiation of human iPS cells into mesenchymal cells

[0065] (1) Expression of related genes during induction and differentiation

[0066] Trizol (Invitrogen) method was used to extract the total RNA of iPS-induced 0, 3, 6, 9, 12d cells and iPS-MSC-P2,

[0067] The M-MLV first-strand synthesis system (Invitrogen) was used to reverse transcribe into cDNA and SYBR Premix Ex Taq (Takara) for Realtime PCR to detect the expression of Nanog, Oct4, CD73, CD105 and the internal reference gene actin ( image 3 ). With the prolongation of induction time, the expression levels of pluripotency genes Nanog and Oct4 decreased, while the expression levels of CD73 and CD105 gradually increased. MSC-P2 only expressed a small amount of Nnaog and Oct4, while CD73 and CD105 were almost 100% expressed.

[0068] (2) MSC Surface Marker Identification

[0069] Digest the obtained MSC cells (P2) into a single cell suspension, add fluorescently labeled mouse anti-...

Embodiment 3

[0070] Example 3 Determination of the growth curve of MSC differentiated from human iPS cells

[0071] MSCs (P5) differentiated from human iPS cells were digested into a single cell suspension, counted and spread in 24-well cells at a density of 5×103 / cm2 for culture, and cells were counted in 3 wells every day after inoculation. Figure 5 It can be seen that the MSCs differentiated from human iPS cells grow in an "S" shape during the culture process, the logarithmic growth phase is 2-5 days, and enter the plateau phase on the 6th day. Can self-renew and proliferate well.

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Abstract

The invention discloses a method for differentiating induced pluripotent stem cells (iPSC) into mesenchymal stem cells (MSC). The method includes the steps that the induced pluripotent stem cells formed through induction are dissociated into single cells; the single cells are cultivated in a cell plate coated with I type collagen through an iPSC medium and one differential medium including platelet-derived growth factors, and then cultivated in another differential medium to obtain MSC-like cells; and finally, the MSC-like cells are maturated in an MSC medium to obtain the MSC. According to the induction approach, the iPSC is directly differentiated into the MSC, and the method has the beneficial effects of being short in period, high in efficiency, safe, stable and the like.

Description

technical field [0001] The invention relates to a method for differentiating induced pluripotent stem cells (induced pluripotent stem cells) into mesenchymal stem cells (mesenchymal stem cells). Background technique [0002] Mesenchymal stem cells (MSCs) originate from the mesoderm and are cells with self-renewal and multi-directional differentiation potentials. They were first found in bone marrow and later found in tissues such as umbilical cord, placenta, fat and muscle. Due to their expandability, multidirectional differentiation and low immunogenicity, MSCs can also regulate immune response and paracrine function, so they are considered to be ideal seed cells in tissue engineering and a way of clinical cell replacement therapy. However, MSC tissue sources are complex, resulting in differences in biological characteristics such as proliferation speed, secreted cytokine profile, and immune regulation ability. In addition, issues such as the feasibility of extraction and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/071
CPCC12N5/0662C12N2500/84C12N2501/135C12N2501/30C12N2501/999C12N2506/45
Inventor 彭特单磊陈勇乔志平
Owner GUANGDONG XTEM BIOTECH CO LTD
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