The function of metabolic state in stemness is poorly understood. Mouse embryonic stem cells (ESC) and epiblast stem cells (EpiSC) are at distinct pluripotent states representing the inner cell mass (ICM) and epiblast embryos. Human embryonic stem cells (hESC) are similar to EpiSC stage. We now show a dramatic metabolic difference between these two stages. EpiSC/hESC are highly glycolytic, while ESC are bivalent in their energy production, dynamically switching from glycolysis to mitochondrial respiration on demand. Despite having a more developed and expanding mitochondrial content, EpiSC/hESC have low mitochondrial respiratory capacity due to low cytochrome c oxidase (COX) expression. Similarly, in vivo epiblasts suppress COX levels. These data reveal EpiSC/ hESC functional similarity to the glycolytic phenotype in cancer (Warburg effect). We further show that hypoxiainducible factor 1a (HIF1a) is sufficient to drive ESC to a glycolytic Activin/Nodal-dependent EpiSC-like stage. This metabolic switch during early stem-cell development may be deterministic.

The umbilical cord contains an inexhaustible, noncontroversial source of stem cells for therapy. In the U.S., stem cells found in the umbilical cord are routinely placed into biohazardous waste after birth. Here, stem cells derived from human umbilical cord Wharton's Jelly, called umbilical cord matrix stem (UCMS) cells, are characterized. UCMS cells have several properties that make them of interest as a source of cells for therapeutic use. For example, they 1) can be isolated in large numbers, 2) are negative for CD34 and CD45, 3) grow robustly and can be frozen/thawed, 4) can be clonally expanded, and 5) can easily be engineered to express exogenous proteins. UCMS cells have genetic and surface markers of mesenchymal stem cells (positive for CD10, CD13, CD29, CD44, and CD90 and negative for CD14, CD33, CD56, CD31, CD34, CD45, and HLA-DR) and appear to be stable in terms of their surface marker expression in early passage (passages 4 -8). Unlike traditional mesenchymal stem cells derived from adult bone marrow stromal cells, small populations of UCMS cells express endoglin (SH2, CD105) and CD49e at passage 8. UCMS cells express growth factors and angiogenic factors, suggesting that they may be used to treat neurodegenerative disease. To test the therapeutic value of UCMS cells, undifferentiated human UCMS cells were transplanted into the brains of hemiparkinsonian rats that were not immune-suppressed. UCMS cells ameliorated apomorphine-induced rotations in the pilot test. UCMS cells transplanted into normal rats did not produce brain tumors, rotational behavior, or a frank host immune rejection response. In summary, the umbilical cord matrix appears to be a rich, noncontroversial, and inexhaustible source of primitive mesenchymal stem cells. STEM CELLS 2006;24:781-792

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107 (20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

Low oxygen levels have shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia inducible factor (HIF), can induce a hESC-like transcriptional program, including the iPSC inducers, OCT4, NANOG, SOX2, KLF4, cMYC and miRNA-302 in eleven cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver and breast tumors). Further, non-degradable forms of HIFα, combined with the traditional iPSC inducers are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4- and HIF1α-positive regions. Further, NANOG and OCT4 expression positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells neurospheres and hESC markers were induced in hypoxia but not in normoxia. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathological conditions.

Activation of checkpoint arrest and homologous DNA repair are necessary for maintenance of genomic integrity during DNA replication. Germ-line mutations of the ataxia telangiectasia mutated (ATM) gene result in the well-characterized ataxia telangiectasia syndrome, which manifests with an increased cancer predisposition, including a 20% to 30% lifetime risk of lymphoid, gastric, breast, central nervous system, skin, and other cancers. Somatic ATM mutations or deletions are commonly found in lymphoid malignancies, as well as a variety of solid tumors. Such mutations may result in chemotherapy resistance and adverse prognosis, but may also be exploited by existing or emerging targeted therapies that produce synthetic lethal states.