Differentiation Of Es Cells Into Pancreatic Lineages

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Isolated islet and whole-organ pancreas transplants are accepted therapies for some patients with type I diabetes. Fewer than 100 islet cell transplants and approximately 1400 pancreas transplants are performed annually in the United States, and cadaver donor shortages severely limit more widespread application. The generation of glucose-responsive, insulin-secreting p cells from a renewable human source would thus be an ideal alternative to organs procured through donation. Unfortunately, current technology does not allow precise directed differentiation of ES cells, whether from mouse or human, into islet lineages. Several approaches have been taken toward the generation of cells of pancreatic lineage from mouse and human ES cells.

Work in our own lab (39) and others (49) has shown that, under nonselective conditions, ES cells are capable of differentiation into pancreatic endocrine cell lineages. Using murine ES cells, we followed a two-step nonselective culture protocol including EB formation for 5-7 days followed by plating and further differentiation in the presence of FBS. In these differentiated cultures, foci of pancreatic lineage cells emerge, including cells that stain positively for PDX1 (pancreatic duodenal homeobox 1, a homeodomain protein absolutely required for pancreas development in both humans and mice [50-52]), peptide YY (a marker of early endocrine cells), and endocrine hormones including insulin, glucagon, and somatostatin (39). Although lineage tracing has yet to be performed on cells grown under these experimental conditions, it appears that the culture system recreates the classical developmental stages of islet cytodifferen-tiation, because the timing of appearance of specific cell types is compatible with the derivation of islet cell types from pancreatic progenitor cells. For example, hormone-positive cells emerge from within PDXl-positive foci. In addition, the primary to secondary transition pattern seen in pancreas development in vivo appears to be recapitulated in these cultures, because there is an abrupt transition from cells that stain for both insulin and glucagon in early stage cultures to single hormone-positive cells found in later cultures. Ultimately, cells that are reminiscent of true p cells emerge in the cultures (39).

Under the same nonselective culture conditions used for mouse ES cells, we have obtained similar results with human ES cells. Given the longer doubling time of human ES cells, we empirically increased the lengths of both the EB formation step and the subsequent differentiation after plating EBs. As in the mouse cultures, discrete foci of PDXl-positive cells are observed, followed by the emergence of hormone-positive cells (unpublished data). The nonselective culture conditions allow, but importantly, do not appear to promote, directed differentiation toward a pancreas fate. The frequency with which pancreatic-lineage cells are generated under these conditions is less than 0.1%; however, modification of culture conditions using a growth factor addition scheme based on developmental mechanisms is likely to promote pancreatic fate determination from ES cell-derived endoderm, ultimately producing the desired pancreatic cell types including islet endocrine cells. Such a strategy has been effective in producing enriched populations of motor neurons (53), cardiomyocytes (54), and hematopoietic lineages (55).

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