Prospective Isolation of Multipotent Pancreatic Progenitors Using Flow-Cytometric Cell Sorting

Atsushi Suzuki Hiromitsu Nakauchi Hideki Taniguchi During pancreatic development, neogenesis, and regeneration, stem cells might act as a central player to generate endocrine, acinar, and duct cells. Although these cells are well known as pancreatic stem cells (PSCs), indisputable proof of their existence has not been reported. Identification of phenotypic markers for PSCs leads to their prospective isolation and precise characterization to clear whether stem cells exist in the pancreas. By combining flow cytometry and clonal analysis, we show here that a possible pancreatic stem or progenitor cell candidate that resides in the developing and adult mouse pancreas expresses the receptor for the hepatocyte growth factor (HGF) c-Met, but does not express hematopoietic and vascular endothelial antigens such as CD45, TER119, c-Kit, and Flk-1. These cells formed clonal colonies in vitro and differentiated into multiple pancreatic lineage cells from single cells. Some of them could largely expand with self-renewing cell divisions in culture, and, following cell transplantation, they differentiated into pancreatic endocrine and acinar cells in vivo. Furthermore, they produced cells expressing multiple markers of nonpancreatic organs including liver, stomach, and intestine in vitro. Our data strongly suggest that c-Met/HGF signaling plays an important role in stem/progenitor cell function in both developing and adult pancreas. By using this antigen, PSCs could be isolated prospectively, enabling a detailed investigation of stem cell markers and application toward regenerative therapies for diabetes. Diabetes 53:2143-2152, 2004 - Tacinar tissue, and ductal epithelia. The islets are he pancreas is an organ consisting of three major different structures: the islets of Langerhans, composed of neatly arranged endocrine cell populations (glucagon-producing -cells, insulin-producing -cells, pancreatic polypeptide [PP]-producing -cells, and somatostatin-producing -cells). The acinar cells that secrete various enzymes, such as amylase and lipase, into the intestine, comprise a system of terminal or intercalary acini joined by ducts. During pancreatic organogenesis, these endocrine and acinar tissues seem to be developed from a common cell component associated with the pancreatic ductal epithelium (1 4). Thus, pancreatic stem cells (PSCs) responsible for both endocrine and acinar tissue formation are thought to reside in the pancreatic ducts. Identification and isolation of PSCs have generated much interest due not only to their putative developmental importance but also to their therapeutic potential. Candidate PSC in mouse and human has been reported (57). They were derived from pancreatic ductal cell components and maintained in long-term culture, where they could differentiate into multilineage cell types. These cells possess characteristics very similar to those of neural (8,9), epidermal (10,11), mesenchymal (12), myogenous (13), and retinal (14) stem cells that can also propagate in culture. The value of stem cells expanded in vitro is expected to be great not only in conventional studies of their differentiation or self-renewing potential but also in therapy, such as with virus-mediated gene transfer, or as a theoretically unlimited source of cells. Characterization of those PSC candidates, however, has always been carried out retrospectively, after expansion of crudely isolated cells in culture for a relatively long period. Therefore, it has still not been determined which cells possess stem cell activity in vitro as well as in vivo. To distinguish PSCs from other cell types precisely, their prospective identification and single cell based analysis are required. The hematopoietic stem cells, probably the best-characterized stem cell population, were prospectively identified and isolated based on expression of cell surface antigens by flow cytometry (1517). Although their self-renewing ability could not be maintained easily in vitro, prospective identification facilitated rapid progress toward an understanding of these cells properties and yielded information on genes specifically expressed in this cell population (18,19). Several recent studies using fluorescence-activated cell sorting (FACS) have been conducted to isolate stem cell populations in neural tissue (20,21) and liver (2224). These prospective studies have not only given us information about specific characteristics of stem cells, but have also allowed us to separate them selectively from differentiated cells. In the study reported here, we combined monoclonal antibodies and FACS to fractionate cells derived from neonatal and adult mouse pancreas based on surface marker expression. By using an in vitro clonal colonyforming assay system that was established previously (25), we analyzed cells sorted from distinct fractions and attempted to identify pancreatic stem/progenitor cells prospectively to determine their capacity for differentiation and proliferation. In order to enrich the yield of colonyforming cells and thereby permit clonal analysis of this cell class, we attempted in the present study to sort for cells expressing c-Met, the hepatocyte growth factor (HGF) receptor. Interaction between c-Met and HGF, which is mediated by a signal exchange between epithelial and mesenchymal cells (26), plays an essential part in pancreatic development (27,28). This interaction also is active in the regeneration and carcinogenesis of this organ (29 31). In addition, the number of islet-like structures budding out from cultured ductal epithelial cells is increased by HGF (6). These findings, taken together, suggest that c-Met/ HGF interaction is critically responsible for growth and differentiation of pancreatic stem and progenitor cells during development, homeostatic cell turnover, and regeneration. Our current data demonstrate that clonal colonies derived from c-Metpositive cells contained cells expressing several markers for endocrine, acinar, and ductal lineage cells. These results strongly suggest that cells initiating colony formation are defined as PSCs or common progenitors for those cell types. Several c-Metpositive sorted cells continued growing with self-renewing cell divisions, and, only from this cell subpopulation, many cell aggregates budded from monolayer cells emerged in long-term culture. These cell aggregateproducing cells (CAPCs) could differentiate clonally into multiple pancreatic lineage cells in vitro and in vivo. Furthermore, they could generate daughter cells expressing several marker genes for other organs of endodermal origin such as the liver, stomach, and intestine in vitro. These findings indicate that candidate PSCs or progenitors express c-Met, that their numbers can be enriched, and that they can be isolated using flow cytometry. RESEARCH DESIGN AND METHODS Dissociation of pancreatic cells. Single-cell suspensions of pancreasconstituting cells were prepared from C57BL/6 mouse neonate (...truncated)