DNA Synthesis Is Required for Reprogramming Mediated by Stem Cell Fusion

Abstract

SummaryEmbryonic stem cells (ESCs) can instruct the conversion of differentiated cells toward pluripotency following cell-to-cell fusion by a mechanism that is rapid but poorly understood. Here, we used centrifugal elutriation to enrich for mouse ESCs at sequential stages of the cell cycle and showed that ESCs in S/G2 phases have an enhanced capacity to dominantly reprogram lymphocytes and fibroblasts in heterokaryon and hybrid assays. Reprogramming success was associated with an ability to induce precocious nucleotide incorporation within the somatic partner nuclei in heterokaryons. BrdU pulse-labeling experiments revealed that virtually all successfully reprogrammed somatic nuclei, identified on the basis of Oct4 re-expression, had undergone DNA synthesis within 24 hr of fusion with ESCs. This was essential for successful reprogramming because drugs that inhibited DNA polymerase activity effectively blocked pluripotent conversion. These data indicate that nucleotide incorporation is an early and critical event in the epigenetic reprogramming of somatic cells in experimental ESC-heterokaryons.

Two distinct modes for propagation of histone PTMs across the cell cycle

“…Whether the drop in histone PTM levels after DNA replication affects gene expression and could provide an opportunity to modify the transcription program remains an open question (Steffen and Ringrose 2014). We envision that it could provide a window of opportunity to rewire cell fate (Fisher and Mechali 2003;Tsubouchi et al 2013). By following histones from the moment of their incorporation, we found that most PTMs are not imposed on new histones in a rapid replication-coupled manner.…”

“…We further envision that this mode of PTM maintenance could explain how cell cycle speed and withdrawal impact on cellular plasticity. DNA replication could be instrumental to reset epigenetic states (Tsubouchi et al 2013), and, by revealing how histone PTMs are maintained across the cell cycle, our findings pave the way to understanding how cellular identity and reprogramming potential are connected with cell proliferation (Guo et al 2014). …”

Nuclear Reprogramming and Mitosis – how does mitosis enhance changes in gene expression?

“…18 It is interesting to note that replication of DNA also appears to aid certain cell fusion experiments. 19 DNA synthesis may contribute to reprogramming in a similar manner to mitotic transition in so much as transcriptional regulators may be displaced by the DNA replication machinery and histone decorations may be changed at the time of histone incorporation into replicated DNA. 20 In the same vein, other aspects of the cell cycle that are associated with changes in cellular identity exist 21 and are likely to impact reprogramming by facilitating gene expression change.…”

Cell Fusion Enhances Mesendodermal Differentiation of Human Induced Pluripotent Stem Cells

“…Foshay et al showed that only pluripotent stem cells can induce pluripotency in somatic cells [16]. Factors, which influence reprogramming by cell fusion, include polycomb group proteins [17], activation-induced cytidine deaminase (AID)-dependent demethylation [18], and DNA synthesis [16,19]. However, the differentiation capacity of hybrid cells compared with the parental pluripotent stem cells is less studied, especially in human hybrids.…”

“…Cell fusion was used as an approach to study reprogramming of somatic cells toward pluripotency [3,[9][10][11][12][13][14][15][16][17][18][19][20]. Shortly after fusion, hybrid cells acquire similar characteristics as the parental pluripotent stem cells.…”