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1.  A Molecular Signature for Purified Definitive Endoderm Guides Differentiation and Isolation of Endoderm from Mouse and Human Embryonic Stem Cells 
Stem Cells and Development  2012;21(12):2273-2287.
Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as liver, pancreas, and intestine. However, purification of DE in mammals has not been achieved, limiting the molecular “definition” of endoderm, and hindering our understanding of DE development and attempts to produce endoderm from sources such as embryonic stem (ES) cells. Here, we describe purification of mouse DE using fluorescence-activated cell sorting (FACS) and mice harboring a transgene encoding enhanced green fluorescent protein (eGFP) inserted into the Sox17 locus, which is expressed in the embryonic endoderm. Comparison of patterns of signaling pathway activation in native mouse DE and endoderm-like cells generated from ES cells produced novel culture modifications that generated Sox17-eGFP+ progeny whose gene expression resembled DE more closely than achieved with standard methods. These studies also produced new FACS methods for purifying DE from nontransgenic mice and mouse ES cell cultures. Parallel studies of a new human SOX17-eGFP ES cell line allowed analysis of endoderm differentiation in vitro, leading to culture modifications that enhanced expression of an endoderm-like signature. This work should accelerate our understanding of mechanisms regulating DE development in mice and humans, and guide further use of ES cells for tissue replacement.
PMCID: PMC3411363  PMID: 22236333
2.  Extensive and coordinated transcription of noncoding RNAs within cell cycle promoters 
Nature genetics  2011;43(7):621-629.
Transcription of long noncoding RNAs (lncRNAs) within gene regulatory elements can modulate gene activity in response to external stimuli, but the scope and functions of such activity are not known. Here we use an ultra-high density array that tiles the promoters of 56 cell cycle genes to interrogate 108 samples representing diverse perturbations. We identify 216 transcribed regions that encode putative lncRNAs--many with RT-PCR-validated periodic expression during the cell cycle, show altered expression in human cancers, and are regulated in expression by specific oncogenic stimuli, stem cell differentiation, or DNA damage. DNA damage induces five lncRNAs from the CDKN1A promoter, and one such lncRNA, named PANDA, is induced in a p53- dependent manner. PANDA interacts with the transcription factor NF-YA to limit expression of pro-apoptotic genes; PANDA depletion markedly sensitized human fibroblasts to apoptosis by doxorubicin. These findings suggest potentially widespread roles for promoter lncRNAs in cell growth control.
PMCID: PMC3652667  PMID: 21642992
3.  Targeting SOX17 in human embryonic stem cells creates unique strategies for isolating and analyzing developing endoderm 
Cell stem cell  2011;8(3):335-346.
Investigating development of inaccessible human tissues like embryonic endoderm with embryonic stem cell (ESC) has been hindered by a lack of methods for marking and isolating endodermal cells, and tracing fates of their progeny toward differentiated lineages. Using homologous recombination in human ESC, we inserted an enhanced green fluorescent protein (eGFP) transgene into a locus encoding a postulated marker of human endoderm, SOX17, permitting purification of SOX17+ hESC progeny by fluorescence activated cell sorting (FACS). Microarray studies revealed a unique gene expression profile of human SOX17+ cells including endodermal marker enrichment, and unveiled specific cell surface protein combinations that permitted FACS-based isolation of primitive gut tube endodermal cells produced from unmodified human ESCs and from induced pluripotent stem cells (iPSC). FACS-isolated SOX17+ endodermal cells differentiated to progeny expressing markers of liver, pancreas, and intestinal epithelium, providing unprecedented evidence that human gastrointestinal lineages derive from SOX17+ cells. Thus, prospective isolation, lineage tracing, and developmental studies of hESCs described here have revealed fundamental aspects of human endodermal biology.
PMCID: PMC3063711  PMID: 21362573
4.  Deconstructing Pancreas Development to Reconstruct Human Islets from Pluripotent Stem Cells 
Cell stem cell  2010;6(4):300-308.
There is considerable excitement about harnessing the potential of human stem cells to replace pancreatic islets that are destroyed in type 1 diabetes mellitus. However, our current understanding of the mechanisms underlying pancreas and islet ontogeny has come largely from the powerful genetic, developmental, and embryological approaches available in nonhuman organisms. Successful islet reconstruction from human pluripotent cells will require greater attention to “deconstructing” human pancreas and islet developmental biology and consistent application of conditional genetics, lineage tracing, and cell purification to stem cell biology.
PMCID: PMC3148083  PMID: 20362535

Results 1-4 (4)