Redox-Mediated Enrichment of Self-Renewing Adult Human Pancreatic Cells That Possess Endocrine Differentiation Potential

Objectives: The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-l-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue. Methods: Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions. Results: Human pancreatic cell (HPC) cultures coexpressed &agr;-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein. Conclusion: These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation.

[1]  J. Reig,et al.  Bio-engineering insulin-secreting cells from embryonic stem cells: A review of progress , 2003, Medical and Biological Engineering and Computing.

[2]  J. Degen,et al.  Replacement by a lacZ reporter gene assigns mouse connexin36, 45 and 43 to distinct cell types in pancreatic islets. , 2004, Experimental cell research.

[3]  Yuval Dor,et al.  Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. , 2004, Nature.

[4]  A. Trumpp,et al.  Nestin expression in pancreatic exocrine cell lineages , 2004, Mechanisms of Development.

[5]  S. Leach,et al.  Origin of exocrine pancreatic cells from nestin-positive precursors in developing mouse pancreas , 2004, Mechanisms of Development.

[6]  A. Shapiro,et al.  Clinical islet transplant: current and future directions towards tolerance , 2003, Immunological reviews.

[7]  M. Raff.  Adult stem cell plasticity: fact or artifact? , 2003, Annual review of cell and developmental biology.

[8]  Ana D. Lopez,et al.  Characterization and isolation of promoter-defined nestin-positive cells from the human fetal pancreas. , 2003, Diabetes.

[9]  Donna M. Martin,et al.  Nestin-lineage cells contribute to the microvasculature but not endocrine cells of the islet. , 2003, Diabetes.

[10]  J. Haefliger,et al.  Connexin-36 Contributes to Control Function of Insulin-producing Cells* , 2003, Journal of Biological Chemistry.

[11]  I. Barshack,et al.  Functional, Persistent, and Extended Liver to Pancreas Transdifferentiation* , 2003, Journal of Biological Chemistry.

[12]  O. Korsgren,et al.  Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture. , 2003, Diabetes.

[13]  B. Wolf,et al.  Insulin Independence Following Isolated Islet Transplantation and Single Islet Infusions , 2003, Annals of surgery.

[14]  M. Noble,et al.  Redox State as a Central Modulator of Precursor Cell Function , 2003, Annals of the New York Academy of Sciences.

[15]  R. Hruban,et al.  Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis. , 2003, Cancer cell.

[16]  B. Raaka,et al.  Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[17]  B. Wolf,et al.  The use of non-heart-beating donors for isolated pancreatic islet transplantation , 2003, Transplantation.

[18]  Andreas Lechner,et al.  Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus. , 2003, American journal of physiology. Endocrinology and metabolism.

[19]  L. Olson,et al.  Characterization of Gap Junctional Intercellular Communication in Immortalized Human Pancreatic Ductal Epithelial Cells With Stem Cell Characteristics , 2003, Pancreas.

[20]  Seung K. Kim,et al.  Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  I. Koyama,et al.  A restriction endonuclease assay for expression of human α-amylase isozymes , 2002 .

[22]  J. Habener,et al.  Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. , 2002, Endocrinology.

[23]  A. Shapiro,et al.  Successful islet transplantation: continued insulin reserve provides long-term glycemic control. , 2002, Diabetes.

[24]  B. Petersen,et al.  In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[25]  I. Rooman,et al.  Nestin expression in pancreatic stellate cells and angiogenic endothelial cells , 2002, Histochemistry and Cell Biology.

[26]  A. Nolan,et al.  Nestin-positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP-binding cassette transporter. , 2002, Biochemical and biophysical research communications.

[27]  Lars Selander,et al.  Nestin is expressed in mesenchymal and not epithelial cells of the developing mouse pancreas , 2002, Mechanisms of Development.

[28]  E. Major,et al.  Analysis of the temporal expression of nestin in human fetal brain derived neuronal and glial progenitor cells. , 2002, Brain research. Developmental brain research.

[29]  I. Koyama,et al.  A restriction endonuclease assay for expression of human alpha-amylase isozymes. , 2002, Clinica chimica acta; international journal of clinical chemistry.

[30]  C. Wollheim,et al.  Pdx1 Level Defines Pancreatic Gene Expression Pattern and Cell Lineage Differentiation* , 2001, The Journal of Biological Chemistry.

[31]  D. Alpers,et al.  alpha-Amylase expressed in human liver is encoded by the AMY-2B gene identified in tumorous tissues. , 2001, Clinica chimica acta; international journal of clinical chemistry.

[32]  R. McKay,et al.  Differentiation of Embryonic Stem Cells to Insulin-Secreting Structures Similar to Pancreatic Islets , 2001, Science.

[33]  C. Wright,et al.  Glucagon-like peptide 1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells. , 2001, Diabetes.

[34]  J. Habener,et al.  Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. , 2001, Diabetes.

[35]  M. Noble,et al.  Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[36]  S. Bonner-Weir,et al.  In vitro cultivation of human islets from expanded ductal tissue. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  I. Rooman,et al.  Modulation of rat pancreatic acinoductal transdifferentiation and expression of PDX-1 in vitro , 2000, Diabetologia.

[38]  I. Barshack,et al.  Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia , 2000, Nature Medicine.

[39]  E. Hunziker,et al.  Nestin-expressing cells in the pancreatic islets of Langerhans. , 2000, Biochemical and biophysical research communications.

[40]  D. Schatz,et al.  Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells , 2000, Nature Medicine.

[41]  E. Major,et al.  Coexpression of Nestin in Neural and Glial Cells in the Developing Human CNS Defined by a Human-Specific Anti-nestin Antibody , 2000, Experimental Neurology.

[42]  Ames,et al.  Islet Transplantation in Seven Patients with Type 1 Diabetes Mellitus Using a Glucocorticoid-Free Immunosuppressive Regimen , 2000 .

[43]  C. Newgard,et al.  Increase in PDX-1 levels suppresses insulin gene expression in RIN 1046-38 cells. , 1999, Endocrinology.

[44]  N. Sarvetnick,et al.  Pancreatic expression of keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells. , 1999, The American journal of pathology.

[45]  S. Bonner-Weir,et al.  The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. , 1999, Diabetes.

[46]  A. Messing,et al.  Nestin in the liver—lessons from the brain , 1999, Hepatology.

[47]  T. Niki,et al.  Class VI intermediate filament protein nestin is induced during activation of rat hepatic stellate cells , 1999, Hepatology.

[48]  M. Tsao,et al.  Comparative phenotypic studies of duct epithelial cell lines derived from normal human pancreas and pancreatic carcinoma. , 1998, The American journal of pathology.

[49]  H. Edlund,et al.  beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. , 1998, Genes & development.

[50]  V. Tchernev,et al.  In Vitro-Generation of Islets in Long-Term Cultures of Pluripotent Stem Cells From Adult Mouse Pancreas , 1997, Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme.

[51]  S. Thorgeirsson,et al.  Wholesale hepatocytic differentiation in the rat from ductular oval cells, the progeny of biliary stem cells. , 1997, Journal of hepatology.

[52]  Y. Matsuzawa,et al.  PDX-1 Induces Insulin and Glucokinase Gene Expressions in αTC1 Clone 6 Cells in the Presence of Betacellulin , 1996, Diabetes.

[53]  G. Waeber,et al.  Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor. , 1996, Molecular endocrinology.

[54]  M. Tsao,et al.  Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by the E6E7 gene of human papilloma virus 16. , 1996, The American journal of pathology.

[55]  B. Hogan,et al.  PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. , 1996, Development.

[56]  H. Edlund,et al.  IPF1, a homeodomain protein with a dual function in pancreas development. , 1995, The International journal of developmental biology.

[57]  J. Slack.  Developmental biology of the pancreas. , 1995, Development.

[58]  R. Stein,et al.  Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny. , 1995, Development.

[59]  H. Edlund,et al.  Insulin-promoter-factor 1 is required for pancreas development in mice , 1994, Nature.

[60]  J. H. Johnson,et al.  Overexpression of hexokinase I in isolated islets of Langerhans via recombinant adenovirus. Enhancement of glucose metabolism and insulin secretion at basal but not stimulatory glucose levels. , 1994, The Journal of biological chemistry.

[61]  N. Sarvetnick,et al.  Transitional cells in the regenerating pancreas. , 1994, Development.

[62]  C. Newgard,et al.  Use of recombinant adenovirus for metabolic engineering of mammalian cells. , 1994, Methods in cell biology.

[63]  L. Baxter,et al.  A Second Pathway for Regeneration of Adult Exocrine and Endocrine Pancreas: A Possible Recapitulation of Embryonic Development , 1993, Diabetes.

[64]  M. Bendayan,et al.  Differentiation of pancreatic acinar cells into duct-like cells in vitro. , 1993, Laboratory investigation; a journal of technical methods and pathology.

[65]  C. Ricordi,et al.  Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. , 1993, The Journal of clinical investigation.

[66]  N. Sarvetnick,et al.  Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice. , 1993, Development.

[67]  R. Gerard,et al.  Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[68]  G. Merlino,et al.  Cytological changes in the pancreas of transgenic mice overexpressing transforming growth factor alpha. , 1992, Gastroenterology.

[69]  N. Lemoine,et al.  Rapid acinar to ductal transdifferentiation in cultured human exocrine pancreas , 1992, The Journal of pathology.

[70]  S. Bonner-Weir,et al.  Effects of glutamine-enriched parenteral nutrition on the exocrine pancreas. , 1990, JPEN. Journal of parenteral and enteral nutrition.

[71]  G. Merlino,et al.  TGFα overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas , 1990, Cell.

[72]  R. Palmiter,et al.  Overexpression of TGFα in transgenic mice: Induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast , 1990, Cell.

[73]  S. Hootman,et al.  Regulation of pancreatic duct epithelial growth in vitro. , 1990, The American journal of physiology.

[74]  R. McKay,et al.  CNS stem cells express a new class of intermediate filament protein , 1990, Cell.

[75]  T. Makino,et al.  Transdifferentiation of ductular cells into hepatocytes in regenerating hamster pancreas. , 1990, Laboratory investigation; a journal of technical methods and pathology.

[76]  M. Rao,et al.  Role of periductal and ductular epithelial cells of the adult rat pancreas in pancreatic hepatocyte lineage. A change in the differentiation commitment. , 1989, The American journal of pathology.

[77]  M. Rao,et al.  Carcinogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the Syrian golden hamster. , 1988, Carcinogenesis.

[78]  N. Walker.  Ultrastructure of the rat pancreas after experimental duct ligation. I. The role of apoptosis and intraepithelial macrophages in acinar cell deletion. , 1987, The American journal of pathology.

[79]  C. Logsdon.  Stimulation of pancreatic acinar cell growth by CCK, epidermal growth factor, and insulin in vitro. , 1986, The American journal of physiology.

[80]  M. Rao,et al.  Induction and origin of hepatocytes in rat pancreas , 1984, The Journal of cell biology.

[81]  M. Anderson,et al.  Origin of tubular complexes in human chronic pancreatitis. , 1982, American journal of surgery.

[82]  M. Rao,et al.  Differentiation of regenerating pancreatic cells into hepatocyte-like cells. , 1981, Proceedings of the National Academy of Sciences of the United States of America.