小鼠从体细胞到多能干细胞的化学重编程
A cocktail of seven small-molecule compounds (VC6TFZ+TTNPB) can be used to generate pluripotent stem cells from mouse somatic cells (such as MEFs, MNFs, MAFs or ADSCs) at a frequency up to 0.2%. This chemical reprogramming technology has potential use in generating functional desirable cell types for clinical applications. [1] Pluripotent stem cells are self-replicating cell that can be induced from somatic cells by nuclear transfer into oocytes, transgene delivery, or treatments with chemical compounds and then differentiate into three primary germ layers. [1] To achieve complete chemical reprogramming without the Oct4-inducible system, these small molecules were further tested in the chemical reprogramming of OG-MEFs without transgenes. When DZNep was added 16 days after treatment with VC6TF (VC6TFZ), GFP-positive cells were obtained more frequently. The expression levels of most pluripotency marker genes were increased, but were still lower than in ESCs. After switching to 2i-medium with dual inhibition (2i) of glycogen synthase kinase-3 and mitogen-activated protein kinase signaling with CHIR-99021 (CT99021) and PD0325901, certain GFP-positive colonies developed an ESC-like morphology, which are chemically induced pluripotent stem cells (CiPSCs). An additional screen found a synthetic retinoic acid receptor ligand, TTNPB enhanced chemical reprogramming efficiency up to a frequency comparable to transcription factor–induced reprogramming (up to 0.2%). [1] In immunodeficient (SCID) mice injected with CiPSCs, the cells were able to differentiate into tissues of all three germ layers. When injected into eight-cell embryos or blastocysts, CiPSCs were capable of integration into organs of all three germ layers, including gonads and transmission to subsequent generations. The chimeric mice generated from CiPSCs were 100% viable and apparently healthy for up to 6 months, which suggested that the CiPSCs were fully reprogrammed into pluripotency. [1]
Reference:
1. Hou P, Li Y, Zhang X, Liu C, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013 Aug 9;341(6146):651-4.
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Stages |
Time |
Procedures |
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Plate |
Day -1 |
The initial cells (MEFs, MNFs, MAFs or ADSCs) were seeded at a density of 50,000 cells per well of a 6-well plate or 300,000 cells per 100 mm dish. |
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Stage 1 |
Day 0 |
The original medium was replaced with chemical reprogramming medium containing the small-molecule cocktails. The small-molecule cocktails-containing medium was changed every 4 days. |
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Re-plate |
Day 12-16 |
On day 12, these cells were washed in PBS and digested with 0.25% Trypsin-EDTA at 37°C for 3-5 min. After neutralization, the cell clumps were dissociated into single cells by thorough pipetting. The cells were harvested (300,000-1,000,000 cells per well of a 6-well plate) and replated at a density of 300,000-500,000 cells per well of a 6-well plate in the chemical reprogramming medium containing the small-molecule cocktails. |
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Stage 2 |
Day 16 or 20 |
DZNep was added to the cell cultures on day 16 or day 20. |
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Stage 3 |
Day 28 |
On day 28, the small-molecule cocktails including DZNep were removed. Meanwhile, the chemical reprogramming medium was replaced with 2i-medium (ESC culture medium supplemented with 2i (3 μM CHIR99021 and 1 μM PD0325901)). |
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Pick up |
Day 36-40 |
2i-competent, ESC-like and GFP-positive colonies were counted as primary CiPSC colonies. For CiPSC induction from wild-type cells without OG reporter, 2i-competent and ESC-like colonies were counted as primary CiPSC colonies. These CiPSC colonies were picked up for expansion and characterization. Alternatively, CiPSCs could be induced without replating on day 12. |
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Reference: 1. Hou P, Li Y, Zhang X, Liu C, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013 Aug 9;341(6146):651-4. |
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