Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.

Use of human hepatocytes for therapeutic and drug discovery applications is hampered by limited tissue source and the inability of hepatocytes to proliferate and maintain function long term in vitro. Human embryonic stem (hES) cells are immortal and pluripotent and may provide a cell source for functional human hepatocytes. We report here that hES cells can be induced to differentiate into hepatocyte-like cells. Treatment with sodium butyrate induced hepatic differentiation as well as significant cell death, resulting in approximately 10-15% yield of a homogeneous population of cells. The differentiated cells have morphological features similar to that of primary hepatocytes and 70-80% of the cells express liver-associated proteins (albumin, alpha-1-antitrypsin, cytokeratin 8 and 18), accumulate glycogen, have inducible cytochrome P450 activity, and do not express alpha-fetoprotein. Because of the inherent proliferative capacity of hES cells, these cells may provide a reliable source of normal human hepatocytes for research and transplantation.

In vivo MRI of stem cells (SCs) is an emerging application to evaluate the role of cell therapy in restoring the injured myocardium. The high spatial and temporal resolution combined with iron-oxide-based intracellular labeling techniques will provide a sensitive, noninvasive, dual imaging modality for both cells and myocardium. In order to facilitate this novel imaging approach, much effort has been directed towards developing efficient transfection methods. While techniques utilizing poly-L-lysine (PLL), protamine sulfate (PS), and electroporation (ELP) have been proposed, the fundamental biological effects of these methods on mouse embryonic SCs (mESC) have not been investigated systematically. In this study a longitudinal in vitro evaluation of cellular viability, apoptosis, proliferation, and cardiac differentiation of magnetically labeled mESC was conducted. No significant difference was seen in these biological parameters among the three transfection methods. However, cardiac differentiation was most attenuated by ELP, and iron Growing evidence from preclinical and clinical studies suggests that cell therapy can improve cardiac function (1-4). Although this therapeutic option is a promising approach to repair the injured myocardium, the exact mechanism of functional restoration of the injured myocardium is unknown. While differentiation of embryonic stem cells (ESC) into functional cardiomyocytes has been attributed as one of the possible underlying mechanisms in restoring the injured myocardium, a more fundamental issue regarding cellular viability following delivery into the myocardium also needs to be addressed (5,6). At present, most cell therapy protocols require histological analysis to determine viable engraftment of the transplanted cells. The development of sensitive, noninvasive technologies to monitor this fundamental engraftment parameter will aid clinical implementation of cell therapy. Imaging modalities such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging (MRI), and optical bioluminescence imaging (BLI) have been reported to be capable of evaluating cellular engraftment parameters (4,7,8).In vivo MRI of SCs is an emerging application to monitor SC engraftment. The high spatial and temporal resolution combined with robust iron-oxide-based intracellular labeling enables dual assessment of myocardial function and cellular location. Much effort has been directed towards developing efficient transfection methods to facilitate intracellular magnetic labeling with superparamagnetic iron oxide (SPIO). Although techniques utilizing poly-L-lysine (PLL), protamine sulfate (PS), and electroporation (ELP) have been proposed, the effects of these three transfection methods on engraftment parameters of ESC have not been investigated systematically (9 -11). In this study a comparison of the engraftment parameters of magnetically labeled mouse embryonic SCs (mESC) using the three transfection methods was conducted. Fundamental biological ...

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