“VC6TF + EPZ004777+AM580”, “VC6TFZ+AM580+SGC0946+5-aza-dC” and “N2B27-2i+LIF” are three novel small molecules cocktails that enhanced the chemical reprogramming efficiency of an newly identified extraembryonic endoderm (XEN)-like state (i.e. an intermediate during the early stage of chemical reprogramming). [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]
There are 3 essential stages in the chemical reprogramming process. In stage 1, RA agonist-AM580 (A) and DOT1L inhibitor- EPZ004777 (E) enhances the formation of XEN-like colonies by 2- to 3-fold. In a cocktail of seven small molecules (VC6TFAE: VPA, CHIR99021, 616452, tranylcypromine, forskolin, AM580 and EPZ004777), the number of XEN-like colonies is increased by >5-fold. During stage 2, using another DOT1L inhibitor- SGC0946 (S) that replacing EPZ004777, the reprogramming efficiency is enhanced further by up to 5-fold , especially when an optimized 2i-medium (N2B27-2iL medium) is applied. In additions, CiPSC (chemically induced pluripotent cell) colonies generates in stage 3 only when supplemented with 5-aza-dC (D) in stage 2. Using the small-molecule cocktail VC6TFAZDS during stage 2 for 12 days induce ~100–600 CiPSC colonies from 50,000 re-plated cells at the final stage of chemical reprogramming. [1]
All six tested CiPSC lines can form teratoma after injection into SCID mice and produce chimeric mice after blastocyst injection. Four lines displays germline integration potential in chimeric mice, and germline transmission offspring are gained from chimeric mice. [1]
Reference:
1. Zhao Y, Zhao T, Guan J et al. A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming. Cell. 2015 Dec 17;163(7):1678-91.
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Related Biological Data
Phase and fluorescence images of primary CiPSC colonies in stage 3 (day 40) with treatment with VC6TFAZ plus 5-aza-dC and SGC0946 in stage 2. Scale bar, 100 mm.
Related Biological Data
Numbers and phase images of XEN-like colonies after treatment with control cocktail (VC6TF with CHIR, 20 μM) and that with additional small molecule EPZ004777 (E) and AM580 (A) for 16 days. Cells were re-plated at day 12 by 1:2. Scale bar, 100 mm.
Related Biological Data
Numbers of CiPSC colonies at day 44 induced with a control cocktail (VC6TFAZ) and with 5-azadC, 5-aza-dC plus EPZ004777 (EPZ), or SGC0946 (SGC) in stage 2 of 12 days.
Related Biological Data
Imaging tracing of the formation process of two CiPSC colonies (stage 3) in stage 1 and stage 2 during chemical reprogramming. Scale bars, 100 mm.
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