Fig. 5. Chemical reversion of conventional primed hPSC with a tankyrase/PARP inhibitor promotes stable rewiring to a human preimplantation epiblast-like state with intact epigenomic imprints.

The TIRN method is a two-step culture system^109,111 comprised of one brief adaptation step of conventional hPSC with LIF and five small molecules (LIF-5i) that includes XAV939 (tankyrase/PARP inhibition), CHiR99021 (GSK3β inhibition), PD0325901 (MEK inhibition), forskolin (adenylate cyclase activation), and purmorphamine (Hedgehog signaling activation); followed by continuous, stable culture in only LIF/XAV939/CHIR99021/PD0325901 (LIF-3i) for at least 30 passages. This tankyrase/PARP inhibitor-mediated modification of the classical LIF-2i method has been validated to stably revert over 30 hPSC lines, and is independent of genetic donor background^109,111. a (Left panels) Three representative hPSC lines^109,110 (RUES1 hESC, cord blood (CB)-hiPSC 6.2, and fibroblast (fib)-hiPSC C.2 are shown in their starting primed (i.e., E8 medium; PRIMED) conditions, and 6–10 passages post culture in continuous TIRN-reverted (NAÏVE) conditions^109,111. Monolayer bFGF-dependent hPSC colonies in primed conditions became tolerant to bulk single-cell passaging and acquired a typical dome-shape morphology following TIRN reversion. (Middle panels) TIRN-hPSC retained strong expression of TRA-1–81 and SSEA4 surface antigens by flow cytometry. (Right panels) Western blot analyses in these three primed (P) and TIRN (N) lines demonstrated that TIRN-hPSC acquired de novo expression of phosphorylated STAT3 and reduced ERK1/2 phosphorylation. b TIRN-hPSC uniformly shifted their transcriptomes toward a pre-implantation naïve epiblast identity. Mean expression ratios of naïve epiblast-specific genes from Illumina gene arrays from isogenic primed-to-naïve-reverted pairs of genetically independent hPSC (n = 9 lines)¹⁰⁹. c Western blot analysis of primed (P) and TIRN (N) CB-hiPSC (E5C3). TIRN-CB-hiPSC markedly upregulated naïve epiblast-specific DNMT3L whilst maintaining DNMT1 protein expressions following TIRN reversion^109,110. d Infinium CpG methylation heatmaps of genomic Imprinted promoter regions of a panel of primed and TIRN-reverted isogenic hPSC samples revealed that TIRN-hPSC retains normal somatic epigenomic imprint configurations, similar to their isogenic primed states. Heatmaps compare previously published Zambidis lab¹⁰⁹ methylation data alongside published Court et al.¹²¹ data of identical imprinted genomic regulatory regions as controls. Zambidis lab methylation beta values were subset to exact imprinted regions provided by Court et al.¹²¹ (methylation beta values: 0—completely hypomethylated probe; 1—completely methylated probe). Matching Infinium probes are sorted by chromosomal location and arranged into their adjacent primed (−) and naïve (+) hPSC isogenic pairs. The heatmaps of the same imprinted regions from Court et al.¹²¹ include abnormal androgenetic hydatidiform mole, normal human tissues (e.g., brain, muscle, placenta, and sperm), and primed hESC lines.