The recent hot debate on the existence in bone marrow (BM) of developmentally early stem cells with broader specification challenged the hierarchy within the stem cell compartment in murine BM. Evidence has accumulated that hematopoietic stem cells (HSCs) can become specified from a population of migrating primordial germ cells (PGCs) during embryogenesis. In support of this intriguing possibility, HSCs and PGCs are highly migratory cells, and specification of the first primitive HSCs in yolk sac blood islands as well as the origin of definitive HSCs in the aorta–gonado–mesonephros (AGM) region are chronologically and anatomically correlated with the developmental migration of PGCs in extra- and intra-embryonic tissues. Furthermore, several papers have described the sharing of chromosomal aberrations between germline tumors and leukemias or lymphomas, which suggests their clonal origin. Moreover, our recent work demonstrated the presence of quiescent, small, Oct-4+Nanog+Sca-1+Lin–CD45– stem cells in adult murine BM that express several markers shared with migratory PGCs (Leukemia 2010;24:1450) and can be specified into the hematopoietic lineage (Exp.Hematology 2011;39:225). These cells were named very small embryonic-like stem cells (VSELs).
The aim of our study was to test the hypothesis that VSELs are related to PGCs, which would support a potential developmental link between hematopoiesis and the germ line.
We employed transmission electron microscopy (TEM), immunohistochemical staining, RQ-PCR analysis of mRNA and miRNA expression, gene array studies, and promoter methylation analysis to evaluate the expression of genes characteristic of PGC specification. We evaluated the expression of sex hormone receptors in VSELs and HSCs, and by direct in vitro and in vivo studies, we studied the effect of androgens and pituitary gonadotropins on proliferation and expansion of VSELs and HSCs.
The TEM studies revealed VSELs to be small cells with a high nuclear/cytoplasmic ratio, euchromatin, and few mitochondria. VSELs isolated under steady-state conditions from BM highly express, at the mRNA and protein levels, genes involved in specification of the epiblast (e.g., Stella, Fragilis, Blimp1) in addition to genes involved in PGC specification, such as Dppa2, Dppa4, and Mvh, which characterize late-migratory PGCs. The expression of some of these genes has been confirmed at the protein level and at the promoter level to confirm chromatin structure characteristic of actively transcribed genes. To explain highly quiescent state of VSELs, we observed that VSELs, like migrating PGCs, modify imprinting of some early-development parentally imprinted gene loci, including Igf2-H19 and KCNQ1/p57Kip2, which results in their resistance to Igf-1/Igf-2 signaling and upregulation of the cyclin-dependent kinase inhibitor p57KIP2. In parallel, VSELs express several miRNAs that attenuate Igf-1/Igf-2 signaling in these cells (mir681, mir470, mir669b) as well as upregulate expression of p57KIP2 (mir25.1, mir19b, mir92). More importantly, we observed that VSELs and HSCs express mRNA for several pituitary and gonadal hormone receptors as well as highly express Sall4, an early-development marker shared by germ and hematopoietic cells. Finally, in direct in vitro and in vivo experiments, we confirmed that the quiescent population of BM-residing VSELs responds to stimulation by androgens (danazol) and pituitary gonadotropins (PSMG, FSH, and LH). In particular, we found that 10-day administration of all the sex hormones evaluated in this study directly stimulated expansion (∼2–3x) of VSELs and HSPCs in BM and enhanced BrdU incorporation (Figure 1 ).
Our data support the challenging, alternative concept that HSCs can be specified during development from epiblast/migrating PGCs and that VSELs that express several unique PGCs markers, are the most primitive population of stem cells in BM. Moreover, changes in the epigenetic signature of imprinted genes as well as the miRNA network involved in resistance of these cells to Igf-1/Igf-2 signaling keeps these cells quiescent in adult tissues and prevents teratoma formation. Finally, our in vitro and in vivo data clearly show that both VSELs and HSCs proliferate in response to sex hormones. Thus, we conclude that the PGC origin of HSCs warrants further study.
Ratajczak:Neostem Inc.: Membership on an entity’s Board of Directors or advisory committees, Research Funding.
Asterisk with author names denotes non-ASH members.