We compared gene expression profiles of mouse and human ES cells by immunocytochemistry, RT-PCR, and membrane-based focused cDNA array analysis. Several markers that in concert could distinguish undifferentiated ES cells from their differentiated progeny were identified. These included known markers such as SSEA antigens, OCT3/4, SOX-2, REX-1 and TERT, as well as additional markers such as UTF-1, TRF1, TRF2, connexin43, and connexin45, FGFR-4, ABCG-2, and Glut-1. A set of negative markers that confirm the absence of differentiation was also developed. These include genes characteristic of trophoectoderm, markers of germ layers, and of more specialized progenitor cells. While the expression of many of the markers was similar in mouse and human cells, significant differences were found in the expression of vimentin, beta-III tubulin, alpha-fetoprotein, eomesodermin, HEB, ARNT, and FoxD3 as well as in the expression of the LIF receptor complex LIFR/IL6ST (gp130). Profound differences in cell cycle regulation, control of apoptosis, and cytokine expression were uncovered using focused microarrays. The profile of gene expression observed in H1 cells was similar to that of two other human ES cell lines tested (line I-6 and clonal line-H9.2) and to feeder-free subclones of H1, H7, and H9, indicating that the observed differences between human and mouse ES cells were species-specific rather than arising from differences in culture conditions.

Embryonic stem (ES) cells are of significant interest as a renewable source of therapeutically useful cells. ES cell aggregation is important for both human and mouse embryoid body (EB) formation and the subsequent generation of ES cell derivatives. Aggregation between EBs (agglomeration), however, inhibits cell growth and differentiation in stirred or high-cell-density static cultures. We demonstrate that the agglomeration of two EBs is initiated by E-cadherin-mediated cell attachment and followed by active cell migration. We report the development of a technology capable of controlling cell-cell interactions in scalable culture by the mass encapsulation of ES cells in size-specified agarose capsules. When placed in stirred-suspension bioreactors, encapsulated ES cells can be used to produce scalable quantities of hematopoietic progenitor cells in a controlled environment.

Since cardiac transplantation is limited by the small availability of donor organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. To determine the compatibility of human embryonic stem cellderived cardiomyocytes (hESC-CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca ] i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca 2؉ influx rather than on sarcoplasmic reticulum Ca 2؉ release. Third, in agreement with the notion that a voltage-dependent Ca 2؉ current is present in hESC-CMs and contributes to the mechanical function, verapamil completely blocked contraction. Fourth, whereas hESC-CMs expressed SERCA2 and NCX at levels comparable to those of the adult porcine myocardium, calsequestrin and phospholamban were not expressed. Our study shows for the first time that functional properties related to intracellular Ca 2؉ handling of hESC-CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity. STEM CELLS 2006;24:236 -245