Fibroblasts-derived from Pluripotent Cells Harboring a Single Allele Knockout in Two Pluripotency Genes Exhibit DNA Methylation Abnormalities and pluripotency induction Defects

A complete knockout (KO) of a single key pluripotency gene has been shown to drastically affect embryonic stem cell (ESC) function and epigenetic reprogramming. However, knockin (KI)/KO of a reporter gene only in one of two alleles in a single pluripotency gene is considered harmless and is largely used in the stem cell field. Here, we sought to understand the impact of simultaneous elimination of a single allele in two ESC key genes on pluripotency potential and acquisition. We established multiple pluripotency systems harboring KI/KO in a single allele of two different pluripotency genes (i.e. Nanog+/-; Sall4+/-, Nanog+/-; Utf1+/-, Nanog+/-; Esrrb+/- and Sox2+/-; Sall4+/-). Interestingly, although these double heterozygous mutant lines maintain their stemness and contribute to chimeras equally to their parental control cells, fibroblasts derived from these systems show a significant reduction in their capability to induce pluripotency either by Oct4, Sox2, Klf4 and Myc (OSKM) or by nuclear transfer (NT). Tracing the expression of Sall4 and Nanog, as representative key pluripotency targeted genes, at early phases of reprogramming could not explain the seen delay/blockage. Further exploration identifies abnormal methylation landscape around pluripotent and developmental genes in the double heterozygous mutant fibroblasts. Accordingly, treatment with 5-azacytidine two days prior to transgene induction rescues the reprogramming defects. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction and suggests that insufficient levels of key pluripotency genes leads to DNA methylation abnormalities in the derived-somatic cells later on in development.