It has recently been established that pancreas is capable of limited regeneration in response to damage. Thus in the adult pancreas there must exist a mechanism allowing generation of new beta-cells. The goal of our work is to take advantage of the regenerative capacity of adult pancreas for devising in vitro culturing strategies for expansion of islet cell mass. We hypothesize that by optimizing in vitro culture conditions we could overcome limited regenerative capacity of adult pancreas, and thereby obtain an abundant supply of pancreatic beta-cell for transplantation into diabetic patients. Moreover, we anticipate that the new knowledge gained from this work would facilitate identification of promising strategies for enhancing endogenous islet regeneration in diabetic patients. While our ultimate goal is to expand human beta-cells mass, our current research is mainly focused on mouse system. Mouse pancreas provides us with an easily accessible pancreatic tissue of consistent quality. Additionally, the power of mouse genetics allows us to address mechanistic questions, which are difficult to address with human cells. Several years ago we developed a short-term culture system, which utilizes adult mouse pancreatic tissue to generate glucose responsive hormone-producing islet-like cell clusters (ILCCs). We had shown that the new hormone-producing cells were indeed generated in these cultures albeit at the rate commensurate with the cell loss due to apoptosis. In addition to ILCCs, neural cell types were efficiently generated. Among neural cell types, we detected an abundant population (which we refer to as pancreatic neuroepithelial, PNE cells) with phenotypic characteristics of neuroepithelial/radial glial cells of the embryonic central nervous system (CNS). Surprisingly, we found that an array of pluripotent embryonic stem (ES) cell and multipotent stem cell-specific mRNAs were induced in these adult pancreatic cultures, suggesting that adult pancreas might be capable of giving rise to developmentally primitive stem-like cells. At present we are not certain whether such primitive cells pre-exist in the adult pancreas, or are generated de novo as a result of exposure to the growth factors included in our cultures. Irrespective of the mechanism, however, if these primitive cells could be obtained in large numbers and induced to undergo pancreatic endocrine differentiation, they would provide a valuable source for generation of abundant endocrine cell supply for transplantation into diabetic patients. More recently we extended our original short-term culture system by incorporating into the culture medium bone morphogenetic protein-4 (BMP-4), a growth factor of the transforming growth factor-beta family. This modification allowed us to achieve long-term expansion of primitive pancreatic progenitor-like cells (IPLCs), which we can obtain in virtually unlimited quantities. Importantly, the IPLCs stably express high levels of the ?master? transcription factor of pancreatic development and beta cells function, Pdx1, and several transcription factors found in early pancreatic endoderm. Further, IPLCs express several Notch gene family members known to be responsible for expansion of pancreatic progenitor cells in vivo. All the aforementioned genes are expressed at levels at least as high or higher as in normal pancreas, and their expression is stable over continuous passaging of cells in culture for at least 6 months. Overall, the phenotypic characteristics of the IPLCs are similar to those of developmentally primitive embryonic pancreatic progenitors prior to their commitment to endocrine differentiation. In accordance with their primitive phenotype, the IPLCs express only low level of insulin and other endocrine pancreatic hormones. Upon withdrawal of growth factors from the culture medium, the IPLCs organize into islet-like cell clusters and undergo partial endocrine differentiation manifested by increase in insulin gene expression. However, under our current culture conditions differentiated IPLCs express reduced levels of insulin as compared to that of normal beta-cells. We believe that by virtue of their explicit pancreatic progenitor phenotype, the IPLCs are ?primed? for efficient pancreatic endocrine differentiation. The work is now in progress to augment differentiation of IPLCs into physiologically competent insulin-producing cells. An additional line of investigation in my laboratory is addressing mechanistic aspects of generation of progenitor-like cells in cultures of adult pancreas. Several years ago we proposed that epithelial-mesenchymal transition (EMT) and the reverse process, mesenchymal-epithelial transition (MET), might play functional role in generation, expansion and differentiation of pancreatic progenitor-like cells. We are presently using cre-recombinase/loxP-based genetic lineage tracing approaches to directly test this hypothesis. Further, we are examining whether the EMT and MET could be flexibly controlled to achieve optimal cell expansion and differentiation responses.
| Choi, Yong; Ta, Malancha; Atouf, Fouad et al. (2004) Adult pancreas generates multipotent stem cells and pancreatic and nonpancreatic progeny. Stem Cells 22:1070-84 |
