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A medium combination and method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells

A technology of pluripotent stem cells and mesenchymal stem cells, which is applied in the field of medium combinations for inducing pluripotent stem cells to differentiate into mesenchymal stem cells, and can solve the problems of high cost, low efficiency, and instability of induced differentiation

Active Publication Date: 2021-05-14
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the above-mentioned deficiencies in the prior art, the present invention provides a culture medium combination and method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells, so as to solve the problem of high cost, low efficiency and low efficiency of inducing differentiation of mesenchymal stem cells in the prior art. Instability and other technical issues

Method used

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  • A medium combination and method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells
  • A medium combination and method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells
  • A medium combination and method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038]experiment one

[0039]1. Experimental materials: induce multidocarbon stem cells (IPSCs). (Cells from laboratory system-induced IPSCs, primitive cells from normal human cells)

[0040]2, experimental associated medium and reagents: mtessTM 1 (Stemcell, Cat # 85850), DMEM / F-12 (Gibco, Cat # 11320033), Accutase (InvitrogenTM , Cat # 00-4555-56), Y-27632 (Sigma, Cat # Y0503), Matrigel (BD, CAT # 356243), D-PBS (Gibco, Cat # C14190500Bt).

[0041]3, the experiment step:

[0042]In -2 days, preheat (15-25 ° C) sufficient IPSCS medium MTESRTM 1, DMEM / F-12 and pancreatic enzyme substitutes for passage. MTESRTM The Y-27632 was added to promote the IPSCS adhesion wall, and the final concentration of Y-27632 was 10 μmol / L.

[0043]The 1 ml Matrigel package was pre-6-well plates half an hour before the start of the experiment.

[0044]The pores were washed with 1 ml D-PBS (excluding Ca ++ and Mg ++). Wash discard.

[0045]Add 1 ml Accutase to digest the IPSCs.

[0046]It was incubated at 37 ° C for 3 to ...

Embodiment 2

[0101]1. Objective: human mixed stem cell fluorescence quantitative PCR identification.

[0102]2. Experimental materials: mesenchymal stem cells divided by IPSCs and Example 1 (DAY represents the number of days from induction).

[0103]3, the experiment step:

[0104](1) Sample RNA extraction.

[0105](2) RNA concentration and quality assay.

[0106](3) Sample CDNA synthesis, synthesized cDNA in accordance with the reverse transcription standard system.

[0107](4) Dilute the sample template and follow the QPCR standard system. Each sample was repeated three times.

[0108](5) The relative expression of the gene of the calculated purpose.

[0109]OCT, Naong, SOX2, and EPCAM are embryonic stem cell-specific markers, while CD73, CD90, CD105, and CD 146 are markers of mesenchymal stem cell specificity. The fluorescent quantitative PCR results can be seen from the high expression specific marker of differentiated mature mesenchymal stem cells, while low expression of embryonic stem cell surface label. Descrip...

Embodiment 3

[0112]1. Objective: Human mixed stem cell immunofluorescence staining identification.

[0113]2. Experimental materials: Methylactic stem cells (DAY27) were completed in Example 1.

[0114]3, the experiment step:

[0115]The cell climbing in the 24-well plate was added, and the package was gelatin, 37 ° C 2 h.

[0116]Wrap the mesenchymal stem cells onto climbs, 37 ° C, 5% CO2Infit 1 to 2 days.

[0117]Abandon the culture medium, washed 1 free with PBS, 5 min every time.

[0118]Add 4% formaldehyde, 500 μl per well, and fixed at room temperature for 15 min.

[0119]Wash 3 times with PBS, 3 minutes per night, 30 rpm shake.

[0120]0.2% Triton-X100 room temperature is 15 min, 500 μL per well.

[0121]Wash 3 times with PBS, 3 minutes per night, 30 rpm shake.

[0122]3% BSA room temperature is closed for 1 to 2 hours, 500 μL per well.

[0123]Plus a anti-CD73 (Abcam, Cat # AB133582) 4 ° C overnight: antibody was diluted with 3% BSA, and 40 to 50 μl can be used according to each climb. The rubber strip is fixed to the d...

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Abstract

The invention discloses a culture medium combination and method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells. The medium combination includes a first-stage mesenchymal stem cell induction differentiation medium and a second-stage mesenchymal stem cell support medium, and the mesenchymal stem cell induction differentiation medium includes STEMdiff TM ‑ACF mesenchymal induction medium; the mesenchymal stem cell support medium includes basal medium, serum and additives, the additives include: L-glutamine, L-Ascorbic acid, sodium pyruvate, MEM NEAA. The method is to use the medium combination for culturing, including: using the mesenchymal stem cell differentiation medium to induce and culture the pluripotent stem cells for 3 days; on the 4th day, start to change to the mesenchymal stem cell support medium for culturing until maturity mesenchymal stem cells.

Description

Technical field[0001]The present invention relates to the field of biomedical technology, and more particularly to a medium combination and method for inducing polycogeneous stem cells into meta-mesenchymal stem cells.Background technique[0002]Induction of multi-energy stem cells (IPSCs) is a cells that are retrofitted by the transcription factor (OCT4, SOX2, KLF4 and C-myc) and have similar differentiation resistance to embryonic stem cells, which becomes a pathogenesis of human diseases. Cell replacement of important cell sources of treatment, and there is no ethical problem. As a source of source cells in IPSCs, it can be amplified and induced in vitro and differentiated tissue cells.[0003]Methylactic stem cells are a pluripotent stem cell, which has all common problems of stem cells, namely self-renewal and multi-differentiation. It is currently available in clinical applications to treat multiple diseases, therefore caught extensive attention. Metacchard stem cells have unique ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N5/074
CPCC12N5/0668C12N2500/30C12N2500/32C12N2500/38C12N2500/84C12N2506/45
Inventor 严庆丰陈虹贾则晓齐念民王皓
Owner ZHEJIANG UNIV
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