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Culture system and method for inducing human pluripotent stem cells into neural mesoderm progenitor cells in vitro and maintaining self-renewal and application

A technology of human pluripotent stem cells and culture system, applied in the field of long-term culture of neuromesoderm progenitor cells

Pending Publication Date: 2021-05-04
THE NAVAL MEDICAL UNIV OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, it has been reported in the literature that pluripotent stem cells can be induced to generate neuromesoderm progenitor cells, but how to realize the self-renewal of neuromesoderm progenitor cells is still an unsolved problem
There is still a lack of effective induction methods in the field to induce hPSCs into stable self-renewing neuromesodermal progenitors (srNMPs), especially the in vitro stability of neuromesodermal progenitors cannot be achieved under chemically defined culture conditions self renewal

Method used

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  • Culture system and method for inducing human pluripotent stem cells into neural mesoderm progenitor cells in vitro and maintaining self-renewal and application
  • Culture system and method for inducing human pluripotent stem cells into neural mesoderm progenitor cells in vitro and maintaining self-renewal and application
  • Culture system and method for inducing human pluripotent stem cells into neural mesoderm progenitor cells in vitro and maintaining self-renewal and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Induction of srNMPs and identification of molecular phenotypes

[0036] 1. Recovery, culture and identification of hPSCs

[0037] The surface of the culture dish of the cells was pre-coated with DMEM / F12 medium containing 1% Matrigel (Corning, Cat#356231), and stored overnight at 4°C. The frozen hPSCs lines in our laboratory include H1 (passage 70-85) and HUES9 (passage 20-35). After taking it out from the -80°C refrigerator, shake it quickly in a 37°C water bath to dissolve until a little ice remains. Centrifuge the cells quickly in a low-temperature horizontal centrifuge, centrifuge at 1000rpm for 5 minutes, discard the frozen storage solution, resuspend with hPSCs medium, inoculate in matrigel-coated culture plates, and change the medium every day. After the cells were digested with TrypLE digestion enzyme (Gibco, Cat# 12605-010), they were subcultured at a ratio of 1:10, and 5 μM Blebbistatin (Selleck, S7099) was added to prevent cell apoptosis caused b...

Embodiment 2

[0050] Example 2: srNMPs have bidirectional differentiation potential of nerve and mesoderm

[0051] 1. Single cell differentiation experiment of srNMPs:

[0052] After srNMPs were digested into single cells, they were inoculated into Matrigel-coated six-well plates at a very low density (about 50 cells per well). After five days of growth under srNMPs culture conditions, most of the single cells formed colonies ( figure 2 A), and then changed to N2B27 basal medium conditions to allow spontaneous differentiation, and continued to culture for two to three weeks, and detected srNMPs clones derived from single cells by immunofluorescence staining, and expressed neural molecular marker MAP2 and mesoderm molecular marker αSMA ( figure 2 B), which shows that the clone formed by a single srNMPs cell can be differentiated to produce neurons and mesoderm cells, and it is proved from the single cell level that srNMPs have the bidirectional differentiation potential of nerves and mesod...

Embodiment 3

[0057] Example 3: Neural Directed Differentiation of srNMPs

[0058] 1. Spontaneous differentiation of srNMPs towards nerves

[0059]Neural differentiation of srNMPs was continuously induced for three weeks in a neural differentiation medium that did not contain any morphogens. The culture conditions were based on B27 medium, adding 0.2 μM γ-secretase inhibitor RO4929097 (EMD Chemicals), 10 ng / mL BDNF ( PeproTech, 450-02), 10ng / mL GDNF (PeproTech, 450-10), this neuron differentiation condition is recorded as BGRO ( image 3 C).

[0060] Neurons differentiated by srNMPs under BGRO conditions can express broad-spectrum neural molecular markers TUJ1, MAP2 and HOX family HOXB8, HOXB9 ( image 3 D. image 3 E), which shows that the induced neurons still maintain their localization at the posterior end of the spinal cord. The expression of DBX1 and PAX2, the molecular markers of V0 region, and the expression of glutamate transporter gene SLC1A1 were detected by RT-PCR ( image ...

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Abstract

The invention relates to the technical field of biomedical engineering, and provides a culture system for inducing human pluripotent stem cells into nerve mesoderm progenitor cells in vitro and maintaining self-renewal. The culture system comprises a basic culture medium and an induced differentiation factor added in the basic culture medium, comprising 0.01 to 500 [mu] m of MTGF [beta] inhibitor, 0.01 to 500 [mu] m of MBMP inhibitor, 0.01 to 50 [mu] m of GSK3 inhibitor, 0.01 to 500 ng / mL of FGF family growth factor, and 0.01 to 500 ng / mL of LEGF family growth factor, wherein the basic culture medium comprises a liquid basic culture medium, a 0-5*B27 additive, a 0-5*N2 additive, 1% penicillin / streptomycin and 0-500 [mu] g / mL 2-phosphoric acid-vitamin C. The induction method comprises the following steps of A, culturing hPSCs until the cell density is 50-70% by adopting an hPSCs culture medium; and B, carrying out cell induction by using the culture system, changing a half amount of liquid every day, carrying out cell passage according to the ratio of 1: 6 when the cell density reaches 90%, adding Blebbistatin into the culture system during subculture, and continuously inducing for more than 10 days.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, and relates to a system and method for inducing human pluripotent stem cells to differentiate into neuromesoderm progenitor cells in vitro and maintaining the long-term culture of neuromesoderm progenitor cells. Background technique [0002] Human pluripotent stem cells (hPSCs) can be divided into human embryonic stem cells and human induced pluripotent stem cells according to different sources. Human embryonic stem cells (hESCs) are isolated from the inner cell mass of early blastocysts, and the cell lines that can be stably cultured in vitro for a long time are optimized by culture conditions. hESCs can maintain stable self-renewal and multi-lineage differentiation potential in vitro, and have the potential to differentiate into all types of adult cells. The acquisition of hESCs and the establishment of cell lines provide potential cell sources for cell replacement therapy, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/0735C12N5/10C12N5/0793C12N5/077
CPCC12N5/0618C12N5/0619C12N5/0658C12N2501/15C12N2501/155C12N2501/115C12N2501/11C12N2501/727C12N2501/148C12N2501/113C12N2501/117C12N2501/119C12N2506/02C12N2506/45C12N2501/73C12N2500/38C12N2510/00C12N2506/08
Inventor 李文林孙平新袁媛虞欣璐吕竺蔓马昊鑫
Owner THE NAVAL MEDICAL UNIV OF PLA
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