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Many of the currently established human embryonic stem (hES) cell lines have been characterized extensively in terms of their gene expression profiles and genetic stability in culture. Recent studies have indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Using both microarrays and quantitative PCR, we report here the differences in miRNA expression between undifferentiated hES cells and their corresponding differentiated cells that underwent differentiation in vitro over a period of 2 weeks. Our results confirm the identity of a signature miRNA profile in pluripotent cells, comprising a small subset of differentially expressed miRNAs in hES cells. Examining both mRNA and miRNA profiles under multiple conditions using cross-correlation, we find clusters of miRNAs grouped with specific, biologically interpretable mRNAs. We identify patterns of expression in the progression from hES cells to differentiated cells that suggest a role for selected miRNAs in maintenance of the undifferentiated, pluripotent state. Profiling of the hES cell "miRNA-ome" provides an insight into molecules that control cellular differentiation and maintenance of the pluripotent state, findings that have broad implications in development, homeostasis, and human disease states.

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Purpose-Human embryonic stem cell (hESC)-derived cardiomyocytes are a promising cell source for cardiac repair. Whether these cells can be transported long distance, survive, and mature in hearts subjected to ischemia/reperfusion with minimal infarction is unknown. Taking advantage of a constitutively GFP-expressing hESC line we investigated whether hESC-derived cardiomyocytes could be shipped and subsequently form grafts when transplanted into the left ventricular wall of athymic nude rats subjected to ischemia/reperfusion with minimal infarction. Co-localization of GFP-epifluorescence and cardiomyocyte specific marker staining was utilized to analyze hESCderived cardiomyocyte fate in a rat ischemia/reperfused myocardium.Methods-Differentiated, constitutively green fluorescent protein (GFP) expressing hESCs (HES3-GFP; Envy) containing about 13% cardiomyocytes were differentiated in Singapore, and shipped in culture medium at 4°C to Los Angeles (shipping time ~3 days). The cells were dissociated and a cell suspension (2×10 6 cells for each rat, n=10) or medium (n=10) was injected directly into the myocardium within the ischemic risk area 5 minutes after left coronary artery occlusion in athymic nude rats. After 15 minutes of ischemia the coronary artery was reperfused. The hearts were harvested at various time points later and processed for histology, immunohistochemical staining, and fluorescence microscopy. In order to assess whether the hESC-derived cardiomyocytes might evade immune surveillance, 2×10 6 cells were injected into immune competent Sprague-Dawley rat hearts (n=2), and the hearts were harvested at 4 weeks after cell injection and examined as in the previous procedures.Results-Even following 3 days of shipping, the hESC-derived cardiomyocytes within embryoid bodies (EBs) showed active and rhythmic contraction after incubation in the presence of 5% CO 2 at 37°C. In the nude rats, following cell implantation, H&E, immunohistochemical staining and GFP epifluorescence demonstrated grafts in 9 out of 10 hearts. Cells that demonstrated GFP epifluorescence also stained positive (co-localized) for the muscle marker alpha-actinin and exhibited cross striations (sarcomeres). Furthermore cells that stained positive for the antibody to GFP (immunohistochemistry) also stained positive for the muscle marker sarcomeric actin and Conclusions-hESCs-derived cardiomyocytes can survive several days of shipping. Grafted cells survived up to 4 weeks after transplantation in hearts of nude rats subjected to ischemia/reperfusion with minimal infarction. They continued to express cardiac muscle markers, exhibit sarcomeric structure, and were well interspersed with the endogenous myocardium. However, hESC-derived cells did not escape immune surveillance in the xenograft setting in that they elicited a rejection phenomenon in immune competent rats.

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Multiple cellular effects of human growth hormone (hGH) are mediated by an indirect mechanism requiring transcriptional activation of genes encoding protein effector molecules such as insulin-like growth factor-1. Such protein effector molecules then act directly to mediate the cellular functions of hGH. We report here that autocrine hGH production by mammary carcinoma cells specifically results in the transcriptional repression of the p53-regulated placental transforming growth factor-␤ (PTGF-␤) gene. Transcriptional repression of the PTGF-␤ gene does not require the p53-binding sites in the PTGF-␤ promoter, and autocrine hGH also desensitized the response of the PTGF-␤ promoter to p53 overexpression. Transcriptional repression of the PTGF-␤ gene is accompanied by consequent decreases in its protein product, Smad-mediated transcription, and its cellular effects that include cell cycle arrest and apoptosis. PTGF-␤ specifically inhibited the autocrine hGH-stimulated expression of cyclin D1 required for autocrine hGH-stimulated mammary carcinoma cell cycle progression. Thus, one mechanism by which autocrine hGH promotes an increase in mammary carcinoma cell number is by transcriptional repression of protein effector molecules that promote cell cycle arrest and apoptosis. Such transcriptional repression of negative regulatory factors, such as PTGF-␤, may also be requisite for direct stimulation of mammary carcinoma cell mitogenesis by hGH. The human growth hormone (hGH)1 gene is expressed in epithelial cells of the normal human mammary gland.2 Increased epithelial expression of the hGH gene is associated with the acquisition of pathological proliferation, and the highest level of hGH gene expression is observed in metastatic mammary carcinoma cells. 2 hGH receptor gene expression per mammary epithelial cell remains constant throughout the process of neoplastic progression (1), and therefore changes in the local concentration of ligand are likely to be pivotal to determine the effects of hGH on the behavior of the mammary epithelial cell. We have recently generated a model system to study the role of autocrine-produced hGH in mammary carcinoma by stable transfection of either the hGH gene or a translation-deficient hGH gene into mammary carcinoma (MCF-7) cells (2). The autocrine production of hGH by mammary carcinoma cells results in a hyperproliferative state with marked synergism observed between trophic agents such as IGF-1 (2). The increase in mammary carcinoma cell number as a consequence of autocrine production of hGH is a result of both increased mitogenesis and decreased apoptosis (3). Autocrine hGH production also results in enhancement of the rate of mammary carcinoma cell spreading on a collagen substrate (4), suggesting that it may affect cell motility and dissemination of the carcinoma. All of the studied effects of autocrine hGH on mammary carcinoma cell behavior are mediated via the hGH receptor (3). Thus, autocrine production of hGH by mammary carcinoma cells may direct mammary carcinoma cell behavio...