Gene expression profiles at the single cell level of TNKS/PARP-inhibitor Regulated Naïve (TIRN) cells with or without transgenic inducible DUX4 (iDUX4) activation.

Here, we show that primed, conventional human pluripotent stem cells (hPSC) cultured continuously under non-specific TNKS1/TNKS2/PARP1-inhibited chemical naïve reversion conditions underwent epigenetic reprogramming to clonal blastomere-like stem cells. TIRN stem cells concurrently expressed hundreds of gene targets of the ZGA-priming pioneer factor DUX4, as well as a panoply of four-cell (4C)-specific (e.g., TPRXL, HOX clusters), eight-cell (8C)-specific (e.g., DUXA, GSC, GATA6), primitive endoderm-specific (e.g., GATA4, SOX17), trophectoderm-specific (e.g., CDX2, TFAP2C), and naïve epiblast-specific (e.g., DNMT3L, NANOG, POU5F1(OCT4)) factors; all in a hybrid, combinatorial single-cell manner. Mapping of proteomic and single-cell expressions of TIRN cells against human preimplantation embryo references identified them as relatively homogenous 4C-8C stage populations. The bulk RNA-seq and proteomics analyses of TIRN cells (+/- iDUX4) revealed acquisition of pre-lineage embryonic transcriptional programs. To determine if TIRN +/- iDUX4 cells were mixed populations or clonal blastomere-like cells, single cell (sc)RNA-seq was performed to investigate transcriptional heterogeneity at a single cell level. Overall, single, unmodified TIRN cells shared high transcriptional identity with single TIRN+iDUX4 cells, and single primed hPSC and TIRN +/- iDUX4 cells expressed comparable and uniformly homogenous levels of core pluripotency factors (e.g., NANOG, POU5F1 (OCT4), SOX2, KLF4, CMYC). However, single TIRN and TIRN+iDUX4 populations homogenously co-expressed not only higher levels of naïve ICM-specific genes (e.g., DNMT3L, SP5, ERVH48-1, GDF3, and IFITM1) (Park et al., 2020; Zimmerlin et al., 2016), but also hundreds of diverse and antagonistic lineage-specifying genes (e.g., HOX-PRD, T-BOX, FOX, GATA families) that are co-expressed simultaneously only during 4C-to-morula human embryonic stages (Petropoulos et al., 2016; Zou et al., 2022). Transcript levels of 4C-specific (i.e., TPRXL, HOXA1, EBF2), 8C-specific (i.e., GATA6, FOXB1), trophectoderm lineage-specific (i.e., CDX2, TFAP2C), and endoderm lineage-specific (i.e., GATA4, SOX17) pioneer factors were co-expressed in the same single cell populations; with expressions that were further augmented following iDUX4 activation.