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Culture medium and method for establishing and maintaining early embryonic-like cells

A technology of culture medium and hydroxyl method, which is applied in the direction of embryonic cells, cell culture active agents, non-embryonic pluripotent stem cells, etc., and can solve problems such as the inability to correctly develop gastrula

Pending Publication Date: 2022-05-13
MGI HLDG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current models require a mixture of cells of various types, rather than all cells arising from a single cell and self-organizing, and blastocysts behave differently from true blastocysts, such as failing to develop properly into gastrula

Method used

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  • Culture medium and method for establishing and maintaining early embryonic-like cells
  • Culture medium and method for establishing and maintaining early embryonic-like cells
  • Culture medium and method for establishing and maintaining early embryonic-like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0271] Materials and methods

[0272] 4CL basal medium

[0273] A 1:1 mixture of neural basal medium (Gibco) and advanced DMEM / F12 (Gibco), supplemented with N2 supplement (1X, Gibco), B27 supplement (1X, Gibco) (homemade N2 and B27), sodium pyruvate (1X, Hyclone), non-essential amino acids (NEAA) (Gibco), glutaminase TM (1X, Gibco) and penicillin-streptomycin (1X, Gibco).

[0274] 4CL Supplement

[0275] 4CL medium 1 is supplemented in 4CL basal medium:

[0276] SAH / PRC / EZH2 inhibitor (10nM DZNep), HDAC inhibitor (5nM TSA), L-ascorbic acid (50μg / mL), JAK / STAT3 activator (20ng / mL human LIF), MAPK / ERK inhibitor (1μM PD0325901 ), tankyrase inhibitor (5μM IWR), ACTIVIN A / NODAL activator (20ng / mL human ACTIVIN A), extracellular matrix (0.2% (v / v) Geltrex TM ), ROCK inhibitor (1 μM Y27632). Table 1 lists the trademarks and catalog numbers of all 4CL supplements.

[0277] Table 1

[0278]

[0279] REM medium

[0280] 1:1 mixture of advanced DMEM F12 (Gibco) and RPMI 1460...

Embodiment 2

[0300] Materials and methods

[0301] 4CL basal medium

[0302] Same as Example 1.

[0303] 4CL Supplement

[0304] Same as Example 1.

[0305] cell

[0306] Human ESC lines: H1 (male), HN10 (female), HUES1 (male) and WIBR3 (female); human iPSC lines: CBC14 (made by the inventor, female), C11 (made by the inventor, female), Phoenix ( Ulrich Martin laboratory gift, female), DiPS 1016SevA (purchased from Harvard Stem Cell Institute, male), STiPS O-XX1 (purchased from Harvard Stem Cell Institute, female), UH10 (male).

[0307] method

[0308] The same method as in Example 1 was used.

[0309] Experimental results

[0310] Figure 15 is a histogram of RT-qPCR data showing that pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 were significantly induced in ICLCs transformed from various primed human PSC lines. It proved that 4CL medium 1 has wide applicability for inducing human PSC.

Embodiment 3

[0312] Materials and methods

[0313] 4CL basal medium

[0314] Same as Example 1.

[0315] 4CL Supplement

[0316]Same as Example 1.

[0317] cell

[0318] H9 human ESC line.

[0319] method

[0320] The same method as in Example 1 was used, except that the cells were added in 1% (v / v) Geltrex TM DMEM-F12 coated dishes, but not feeder cells.

[0321] Experimental results

[0322] Figure 16 Histogram of RT-qPCR data, shown in Geltrex using 4cl medium 1 TM Pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 were significantly induced in transformed ICLCs on coated dishes, similar to ICLCs on feeder layers. This shows that 4CL Medium 1 is also effective without feeder cells.

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Abstract

The invention provides a culture medium and a method for establishing and maintaining early embryonic-like cells of mammals. The culture medium is used for culturing pluripotent stem cells (PSC) of mammals, has definite chemical components, contains a basic culture medium for culturing stem cells, and is supplemented with an S-adenosylhomocysteine hydrolase (SAH) / polycomb inhibition complex (PRC) / EZH2 inhibitor and an HDAC inhibitor. By means of the culture medium, primate (human and non-human) animal PSC can be converted into pre-implantation internal cell mass-like cells (ICLC) or 8-cell embryonic-like cells (8CLC).

Description

technical field [0001] The present invention relates to media and methods for establishing and maintaining mammalian early embryo-like cells. Background technique [0002] Mammalian embryogenesis is a complex process of cell division and differentiation that leads to the development of an embryo. Successful fertilization of the oocyte with sperm triggers embryogenesis. This tightly controlled process produces billions of cells with different functions and morphologies from a single fertilized egg. The high complexity of cell type and organization in all sexual reproductive organisms begins with embryogenesis. Initially, a single fertilized egg divides to form 2 cells. These 2 cells then divide to form 4 cells, 8 cells and 16 cells. After further proliferation and differentiation, the embryo becomes a blastocyst, which consists of two regions called the inner cell mass (ICM) and the trophectoderm (Trophectoderm, TE). At this point, the stage of embryonic development is c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N5/074C12N5/09C12N5/073
CPCC12N5/0606C12N5/0696C12N5/0693C12N5/0603C12N2500/38C12N2500/40C12N2500/30C12N2501/72C12N2501/73C12N2501/727C12N2533/90C12N2506/02C12N2506/45C12N2506/30
Inventor 米格尔·A·埃斯特班穆罕默德·阿卜杜勒·马西德李文娟骆志伟卡尔·沃德黎运盼赖毅维
Owner MGI HLDG CO LTD
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