Isolation and expansion of cytokeratin positive progenitor cells from adult murine pancreatic ducts expressing Pdx-1, Nestin, Sox9, MafA and hepatic nuclear factors.

BACKGROUND: Recent studies suggest that stem cells may represent a putative source for the generation of beta cells. However, the identity and characteristics of stem cells from adult pancreas and conditions for their large scale expansion are still poorly defined. METHODS: DPC were isolated from adult pancreatic ducts of C57Bl/6 mice. Expression profile was investigated by PCR, FACS and immunohistochemistry. RESULTS: DPC express a panel of stem cell associated markers such as Pdx-1, cytokeratin-19 (CK19), nestin, Sox9 together with the transcription factor MafA and hepatic nuclear factors HNF1ß, HNF3ß, HNF4α und HNF6. This gene expression profile is suggesting that DPC might be a promising tool for endocrine differentiation. After stimulation with picolinic acid and hypoxia, DPC expressed the endocrine differentiation marker Ngn3. Nevertheless, insulin production was not observed. CONCLUSIONS: We here describe a protocol for the isolation end expansion of murine pancreatic ductal progenitor cells (DPC) displaying high self-renewal, spheroid- and colony-forming capacity. Further studies are required to elucidate the conditions for differentiation into mature pancreatic endocrine cell lineages.

Xenografted islet cell clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice.

Islet transplantation is a potential treatment for type 1 diabetes, but the shortage of donor organs limits its routine application. As potential donor animals, we generated transgenic pigs expressing LEA29Y, a high-affinity variant of the T-cell costimulation inhibitor CTLA-4Ig, under the control of the porcine insulin gene promoter. Neonatal islet cell clusters (ICCs) from INSLEA29Y transgenic (LEA-tg) pigs and wild-type controls were transplanted into streptozotocin-induced hyperglycemic NOD-scid IL2Rγ(null) mice. Cloned LEA-tg pigs are healthy and exhibit a strong ß-cell-specific transgene expression. LEA-tg ICCs displayed the same potential to normalize glucose homeostasis as wild-type ICCs after transplantation. After adoptive transfer of human peripheral blood mononuclear cells, transplanted LEA-tg ICCs were completely protected from rejection, whereas reoccurrence of hyperglycemia was observed in 80% of mice transplanted with wild-type ICCs. In the current study, we provide the first proof-of-principle report on transgenic pigs with ß-cell-specific expression of LEA29Y and their successful application as donors in a xenotransplantation model. This approach may represent a major step toward the development of a novel strategy for pig-to-human islet transplantation without side effects of systemic immunosuppression.

Isolation and in vitro expansion of Lgr6-positive multipotent hair follicle stem cells.

Hair follicles (HFs) are a well-known niche of multipotent stem cells. Recently, HF cells expressing leucine-rich orphan G protein-coupled receptors (Lgr) have been described as multipotent adult stem cells with a high potential for regenerative therapies. We have now established the conditions for the isolation and long-term expansion of stem cells from HFs (HFSCs) and analyzed their characteristics (reverse transcription with the polymerase chain reaction, immunohistochemistry) and multipotent capacity (differentiation assays). HFSCs possess a high self-renewal capacity and share characteristics of putative HF epithelial stem cells, such as the expression of Lgr6, cytokeratins (Ck18, Ck19), and multipotent stem cell markers (Sca-1, Bcrp1, nestin, P75NTR). Under defined cell culture conditions, HFSCs are able to differentiate into mesenchymal lineages (adipocytes, chondrocytes, muscle cells) or neurons (ßIII-tubulin). We report, for the first time, an in vitro culture method to isolate Lgr6-positive stem cells from HFs. HFSCs represent a unique tool for studying the cell characteristics of Lgr6-positive cells and should provide a novel easily accessible source for regenerative therapies.

Inhibition of T-cell proliferation by murine multipotent mesenchymal stromal cells is mediated by CD39 expression and adenosine generation.

Multipotent mesenchymal stromal cells (MSCs) are bone marrow-derived cells of nonhematopoietic origin with immunoregulatory properties. Although some functions of MSCs have been identified, there are still features that are not explained thus far. The aim of the present study was to identify novel factors involved in MSC-mediated inhibition of T-cell proliferation. We here demonstrate that the surface molecule CD39 is coexpressed in concert with CD73 on murine MSCs catalyzing the generation of adenosine, which can directly act on activated T cells via the adenosine A2A receptor. Blocking of the adenosine pathway either by the A2A receptor antagonist SCH58261 or the specific CD39 inhibitor polyoxotungstate 1 (POM-1) blocked MSC-mediated suppression of T-cell proliferation almost completely. We conclude that CD39/CD73 coexpression is a novel important component of the immunoregulatory functions of murine MSCs. Our findings may both be important to improve our understanding of MSC function and for the development of immunomodulatory cellular therapies.