Stem cells represent basic units of development and regeneration characterized by nearly unlimited self-renewal and differentiation capacities, but the greatest developmental capacity is restricted to embryonic stem (ES) cells isolated from the inner cell mass of pre-implantation embryos(Wobus &Boheler, 2005). In this research project, we have extended traditional genomic analyses to identify cis-elements that might be implicated in the control of ES cell-restricted gene promoters. The strategy that we employed relied on the generation of a Problem Specific List (PSL) from Serial Analysis of Gene Expression (SAGE) profiles, and subsequent promoter analyses to identify frameworks of multiple cis-elements conserved in space and orientation among genes from the PSL. Subsequent experimental data suggested that two transcription factors, B-Myb and Maz, predicted from these models are implicated either in the maintenance of the undifferentiated stem cell state or in early steps of differentiation. We have since focused on the role of B-Myb. Importantly, the transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Our research has recently shown that the major cell cycle defect in murine embryonic stem (mES) cells lacking B-Myb occurs in G2/M phase of the cell cycle. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. These findings lead us to conclude that appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, while its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death. Based on recent ChIP-chip analyses which are not yet published, we now predict that B-MYB is implicated in the regulation of all three unique traits of embryonic stem cells: pluripotency, a short cell cycle, and an epigenetic state characterized by poised genes. Because of NIH funding from the NIH iPS initiative, we have now been able to expand this project to examine cell surface markers of human iPS cells. As a control, we originally proposed and began examining human ES cells as a control, but due to NIH directives, additional iPS cell lines have been included at the exclusion of hES cells. The rationale and aims of this program are as follows: Human induced pluripotent stem cell (iPSC) lines generated in vitro are heterogeneous and exhibit a wide variety of differentiation and therapeutic potentials. These traits are analogous to hematopoietic stem cell (HSC) sub-populations that differ in their repopulating and differentiation potentials. Functional hematopoietic reconstitution, however, can be predicted through the use of lineage markers. If, as with HSCs, human iPSC (hiPSC) therapeutic potential is deterministic and not stochastic, then it is logical to assume that novel cell surface protein panels will serve as surrogates for unique populations of reprogrammed cells that differ in potency and differentiation potential. To begin the process of identifying these lineage markers and marker panels, we will label, capture and identify cell surface glycoproteins using state of the art biochemical methods and mass spectrometry. Analyses of the cell surface proteome will include at least 3 to 5 human iPSC (skin fibroblast- and fat-derived) lines. Data will be compared to a proprietary Cell Surface Protein Atlas to identify proteins whose expression is restricted to pluripotent cells. Using flow cytometry through a reiterative process, we will test individual antibodies and then panels of antibodies for their ability to reliably identify and isolate subpopulations of pluripotent cells from ES and iPS cells induced to differentiate. Selected antibody panels will then be tested for their ability to isolate bona fide reprogrammed somatic cells generated in vitro with episomal constructs. Proof-of-principal studies have already been established in our lab using mouse ESCs (R1 and D3) and iPSCs (TTF1 and 2D4), where we successfully identified novel cell surface differences (quantitative and qualitative) by flow cytometry. Since the core molecular program underlying maintenance of pluripotency, specification and differentiation is well conserved between mouse and human, the results from our proposed work on human cells (ES and iPS) coupled with our completed mouse dataset will promote the isolation and characterization of patient specific iPSCs and subpopulations, regardless of age or gender, with a broader differentiation and enhanced therapeutic potential.
