New Molecular Evidence That Oct-4 Is Truly Expressed In a Rare Population Of Developmental Early Stem Cells In Human Umbilical Cord Blood (UCB) and That Epigenetic Modification Of Imprinting At Igf2-H19 Locus Regulates Their Quiescent State – Potential Implications For Regenerative Medicine

One of the most intriguing questions in stem cell biology is whether human umbilical cord blood (UCB) contains early-development stem cells that express markers of pluripotency and thus could be employed in regenerative medicine. Several groups have reported mRNAs for genes regulating stem cell pluripotency, such as Oct-4A, Nanog, and SSEA-1, in UCB cells. However, detection of the Oct-4A transcript may be hampered by the presence of several pseudogenes and Oct-4B isoform, which is not related to stem cell pluripotency. Another important question is: why are these primitive stem cells that are present in UCB highly quiescent and relatively resistant to ex vivo expansion? We previously identified a population of Oct-4⁺ CD133⁺Lin^–CD45^– cells in human UCB (Leukemia 2007;21:297–303) that may become specified into long-term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278) and mesenchymal stem cells (Stem Cell & Dev. 2013;22:622). These Oct-4⁺CD133⁺Lin^–CD45^– cells present in UCB correspond to a population of murine Oct-4⁺Sca-1⁺Lin⁺CD45^– cells that remain quiescent in bone marrow because of epigenetic modification of parentally imprinted genes, including the Igf-2-H19 tandem gene (Leukemia 2009;23:2042). The quiescence of these cells has been explained by erasure of imprinting in the regulatory differentially methylated region (DMR) at the Igf2-H19 locus. In appropriate animal models, these small cells also give rise to LT-HSCs (Exp. Hematol 2011;3:225), mesenchymal stem cells (Stem CellsDev 2010;19:1557), and lung epithelium (Stem Cells2013;doi: 10.1002/stem.1413). Moreover, as we demonstrated, the epigenetic reversal of the maternal type of imprinting to the somatic type in the DMR for the Igf2-H19 locus, which is necessary to maintain balanced expression between insulin-like growth factor 2 (Igf-2) and noncoding H19 RNA (precursor for several inhibitory miRNAs) from paternal and maternal chromosomes, respectively, is required for these cells to enter the cell cycle. The crucial role of Igf2-H19 imprinting in quiescence of the most-primitive stem cells in murine BM has been very recently confirmed by another group (Nature 2013, doi: 10.1038/nature12303).

To address whether human UCB Oct-4^± CD133^±Lin^–CD45^– cells truly express genes regulating pluripotency, we examined the DNA methylation state of the promoters for pluripotency/germ-line genes (Oct4, Nanog, and Sall4) and of the DMR for Igf2-H19.

UCB CD133⁺Lin^–CD45^– cells were isolated by multiparameter fluorescence-activated cell sorting (FACS) after intra-cellular staining for Oct4 protein in lineage-depleted human UCB mononuclear cells. Bisulfide modification of DNA followed by sequencing was employed to evaluate the methylation state of CpG islands in the promoters for Oct-4, Nanog, and Sall4 as well as in the DMR for the Igf2-H19 locus.

We observed that Oct4, Nanog, and Sall4 promoters in UCB Oct-4+CD133⁺Lin^–CD45^– cells were demethylated to a similar degree as the human teratocarcinoma NTERA2, which is evidence for true expression of these genes. Furthermore, in human UCB we observed Oct-4⁺CD133⁺Lin^–CD45^– cells that, like their murine counterparts, erase the imprinting in the DMR at the Igf2-H19 locus, which demonstrates that genomic imprinting could be a key mechanism for maintaining the quiescence of these cells. This imprinting data was subsequently confirmed by RQ-PCR analysis of gene expression, showing downregulation of autocrine Igf-2 and upregulation of noncoding H19 RNA.

Our methylation studies of the promoters for pluripotency/germ-line genes (Oct4, Nanog, and Sall4) provide for the first time strong molecular evidence that UCB contains cells that truly express pluripotent stem cell markers. Moreover, molecular analysis of the methylation state in the DMR for the Igf2-H19 locus also explains for the first time how the quiescent state of these cells is regulated by changes in parental imprinting at the Igf2-H19 locus. Thus, elucidation of this mechanism that controls and modifies genomic imprinting in VSELs will be crucial for developing strategies to expand these cells and employ them more efficiently in regenerative medicine and we are currently working on this.

Figure 1

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shows methylation status of Oct-4 promoter (A) and imprinting at Igf2-H19 locus (B) in Oct-4⁺Lin^- vs Oct-4^-Lin^- cells.

Figure 1

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shows methylation status of Oct-4 promoter (A) and imprinting at Igf2-H19 locus (B) in Oct-4⁺Lin^- vs Oct-4^-Lin^- cells.

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