Background. The field of regenerative medicine is still looking for a pluripotent or multipotent stem cell with trans-germ-layer differentiation potential that could be employed in repairing damaged organs and expanded into transplantable hematopoietic stem cells (HSCs). Our group as first isolated a population of small CD133+lin-CD45- early-development stem cells from hematopoietic tissues, including bone marrow (BM), mobilized peripheral blood (mPB), and umbilical cord blood (UCB). Based on expression of early-development markers, these cells were named "very small embryonic-like stem cells" (VSELs). Currently, several groups worldwide who have carefully followed a multicolor-staining cell-sorting strategy described by us ( Current Protocols in Cytometry 2010, 9.29.1-9.29.15) have successfully isolated these cells and shown their contribution to all three germ layer lineages in appropriate tissue/organ injury models in vivo, including their specification into HSCs. However, efficient ex vivo expansion of these cells has been a problem due to erasure of certain parentally imprinted genes involved in insulin/insulin-like growth factor signaling (e.g., Igf-H19, Rasgrf1), which is a mechanism very well known for keeping for example the primordial germ cells in a quiescent state. In order to endow cells with proliferative potential, these erased loci must be again re-methylated by "de novo DNA methyltransferases - DNMTL3, DNMT3A and DNMT3B" (DNMTs). Hypothesis. Based on our own observations that i) DNMT3L are highly expressed in VSELs and ii) certain inhibitors of histone deacetylases (HDACs) such as valproic acid (VPA) or nicotinamide (NA) activate DNMTs and promote DNA re-methylation of erased loci in early development stem cells (Cell Reports 2017, 18, 1930-1945) we hypothesized that HDACs inhibitors could be employed for ex vivo expansion of these cells in highly defined media without third party supportive feeder layer cells . Materials and Methods. Human umbilical cord blood- or mobilized peripheral blood-derived VSELs highly purified by FACS were placed in DMEM supplemented with 10% serum in the presence of VPA or NA + factors that as we demonstrated in vivo may promote egress of VSELs fromquiescent state such as follicle stimulating hormone (FSH), luteinizing hormone (LH), bone morphogen protein 4 (BMP-4), kit ligand (KL), fibroblast growth factor-2 (FGF-2) and insulin-like growth factor 2 (Igf2) ( Stem Cells & Development 2015, 24, 927-937 ). Cells were cultured for 2-3 months and every 7 days, medium was exchanged. Cells in the expansion cultures were counted and studied for i) remethylation of erased parentally imprinted loci, ii) expression of Oct-4 (using specific primers to avoid amplification of Oct-4 pseudogenes) as well as for expression of the germline markers Stella, Fragilis, and Blimp-1. After 1 month of expansion, aliquots of these cells were tested for their hematopoietic potential. Results. We found that over time these small cells (slightly smaller than red blood cells) became enlarged and after 2 weeks began to undergo symmetric and some of them asymmetric divisions (Figure 1). The number of cells in the cultures increased up to 2000 times, and while the majority of cells retained small-cell morphology and expressed markers of pluripotency or multipotency at the protein and mRNA levels, some became enlarged in size and expressed markers typical of cells involved in germ line development (Stella, Blimp-1, and Fragilis). Moreover, cells proliferating in our ex vivo-expansion system re-methylated paternally erased loci (e.g., at the Igf2-H19 locus). Importantly, they did not expand indefinitely and after 3-4 months started to lose pluripotency markers and to cease proliferation and did grow teratoma in immunodeficient mice. Conclusions. We provide an evidence that small CD133+lin-CD45- cells are a population of highly quiescent cells that are kept in a dormant state by erasure of certain paternally imprinted growth-promoting genes and that these loci can be remethylated de novo by the presence of HDAC inhibitors such as VPA or NA in a DNMT3L-dependent manner. This data also support a hypothetical link between primordial germ cells and hematopoiesis. We propose that the currently established expansion system for VSELs isolated from postnatal tissues provides a new and exciting opportunity to employ cells isolated from adult tissues in regenerative medicine.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.