Human Bone Marrow Mesenchymal Stem Cells Can Express Insulin and Key Transcription Factors of the Endocrine Pancreas Developmental Pathway u
http://www.100md.com
《干细胞学杂志》
a Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 578, Grenoble, France;
b Centre d’Investigation Biologique, Centre Hospitalier Universitaire, Grenoble, France;
c Institut National de la Santé et de la Recherche Médicale (Inserm), Unité 371, Institut Fédératif des Neurosciences de Lyon, Bron, France;
d Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5161, Ecole Normale Supérieure de Lyon, France;
e Cell Isolation and Transplantation Center, University of Geneva Medical Center, Geneva, Switzerland;
f Département d’Endocrinologie, Centre Hospitalier Universitaire, Grenoble, France
Key Words. Bone marrow ? Mesenchymal stem cell ? Pancreatic beta cell ? Cell differentiation ? Insulin ? Transcription factors
Correspondence: Pierre-Yves Benhamou, M.D., Ph.D, Centre d’Investigation Biologique, Pavillon B, Centre Hospitalier Universitaire, 38043 Grenoble Cedex 9, France. Telephone: 33-4-76-76-88-56; Fax: 33-4-76-76-50-42; e-mail: pierre-yves.benhamou@wanadoo.fr
ABSTRACT
An important application for cell therapy is diabetes mellitus. By restoring normal endogenous insulin secretion in patients with diabetes, cell therapy may challenge the actual treatment by exogenous insulin. Transplantation of pancreatic islet cells as a potential cure for diabetes has become the subject of intense interest and activity over the past two decades . However, the limited supply of human islet tissue available for transplantation prevents this therapy from being used to treat the thousands of patients with type 1 diabetes. In vitro expansion of human beta cells or genetic engineering of human insulin-secreting cells may represent one approach, but the clinical use is limited by the difficulties in achieving prolonged or physiologically regulated insulin secretion . One way to overcome these problems and obtain functional glucose-sensitive insulin-secreting cells for transplantation is to derive islet cells from other sources such as embryonic stem cells and intestinal , hepatic , ductal, or pancreatic stem cells . These studies have opened fascinating perspectives. An easily accessible, expandable, glucose-responsive, and autologous stem cell would offer obvious advantages in a clinical setting.
Recent work suggests that adult stem cells from one tissue or organ can differentiate into cells of other organs, either in vitro or in vivo . Among them, bone marrow–derived stem cells (hematopoietic or mesenchymal) carry the more significant implications for possible clinical development, because they are easily accessible for an autograft and routinely collected from adults without ethical concern inherent to fetal embryonic tissues .
Based on their ability to adhere to plastic support , multipotential stem cells can be isolated from the bone marrow, expanded, and cultured. Under appropriate experimental conditions, they differentiate into multiple mesenchymal cell types, including cartilage, bone, adipose and fibrous tissues, and myelosupportive stroma . Moreover, treatment with growth factors such as epithelial growth factor and brain-derived neurotrophic factor or chemical products such as dimethyl sulfoxide and butylated hydroxyanisole induced the bone marrow stroma cells to exhibit a neuronal phenotype .
A more likely candidate may be the multipotential adult progenitor cells (MAPCs) derived from adult bone marrow . These cells exhibit a remarkable plasticity, with the ability to differentiate into cells with mesodermal, neuroectodermal, and endodermal characteristics in vitro . Furthermore, upon transplantation, MAPCs can differentiate into epithelium of the liver, lung, and gut. MAPCs express the OCT4 and REX1 transcription factors, two specific markers of undifferentiated embryonic stem (ES) cells. Recently, the marrow-isolated adult multilineage-inducible (MIAMI) cells capable of differentiating in vitro into cell lineages from all three germ layers have been described .
During development, the formation of the pancreas and its subsequent differentiation into the different exocrine and endocrine cell types and mature adult beta cells result from the orderly activation and extinction of a large number of genes. Experiments with transgenic mice have identified a hierarchy in the transcription key factors, such as HLXB9, FOXA2 (formerly named HNF3?), IPF1 (PDX1), NEUROG3 (NGN3), NEUROD1, NKX2-2, PAX4, NKX6-1, and PAX6, that control embryonic formation of pancreatic islets . Among these factors, FOXA2 and IPF1 play a central role in initiating the differentiation of the islet cells.
If human mesenchymal stem cells (hMSCs) could form new beta cells, they would become a particularly useful target for therapies that aim at beta-cell replacement in diabetic patients, because they are abundantly available in the human bone marrow. In this study, we first confirm that hMSCs express the phenotypic surface marker characteristics of multipotent cells. We second provide evidence, by reverse transcription–polymerase chain reaction (RT-PCR), for the presence of some factors implicated in pancreatic development and function. Their phenotype as well as their adipogenic differentiation ability were not modified by adenoviral infection. Finally, we show that genetic manipulations or appropriate culture conditions allow hMSCs to express insulin mRNA.
MATERIALS AND METHODS
Phenotype Characteristics of Expanded Undifferentiated hMSCs
After plastic adherence selection, hMSCs were cultured over four passages. Growth was exponential over the studied period (60 days) with slight differences between donors (Fig. 1A). FACS analysis of hMSCs showed that these cells were negative for CD11b, CD31, CD34, CD45, CD49b, and CD117. They expressed high levels of CD44, CD73, CD90, CD105, and CD147 and low levels of CD10. CD34 and CD45 represent two of the major hematopoietic markers, whereas CD73, CD90, CD105, CD147, and CD10 are cell-surface markers characteristic of MSCs (Fig. 1B). The same phenotype was maintained for passages 0 through 3 and for all of the hMSCs analyzed. We showed that hMSCs express OCT4, a transcription factor important in maintaining undifferentiated ES cells (Fig. 2B). Furthermore, average telomere length of hMSCs cultured during 46 days (three passages) was at least as long as the high telomere length control of 10.2 kbp (Fig. 2A), even though no telomerase activity was detected by quantitative RT-PCR (data not shown).
Figure 1. Culture of human mesenchymal stem cells (hMSCs). (A): Growth curves of hMSCs isolated from two donors, hMSC 54 (filled square) and hMSC 56 (filled circle), were determined by numeration at each passage. Cell number was plotted against time in days. (B): Phenotype of hMSCs. Cells were harvested and labeled with antibodies against CD10, CD11b, CD31, CD34, CD44, CD45, CD49b, CD73, CD90, CD105, CD117, and CD147 or control immunoglobulin G, as indicated and analyzed by fluorescence-activated cell sorting. Plots show isotype control immunoglobulin G staining profile (dotted line) versus specific antibody staining profile (thick line). A representative example of more than 20 hMSCs is shown.
Figure 2. Characteristics of undifferentiated human mesenchymal stem cells (hMSCs). (A): Cells from two donors (hMSC 54 and hMSC 56) were expanded and harvested at each passage (0, 1, and 2) and telomere lengths were evaluated. (B): Reverse transcription (RT)–polymerase chain reaction for OCT4 and ACTB (used as RNA quality and RT efficiency control). RNA obtained at passages 0, 1, 2, and 3 for hMSC 56 was analyzed.
Expression of Islet-Associated Transcription Factors in hMSCs
To evaluate the potential of hMSCs to differentiate into beta cells, we first examined the expression of islet-related transcription factors. As shown in Figure 3A, RT-PCR analysis revealed that hMSCs express NKX6-1 at a low level but lacked all the other transcription factors involved in beta-cell differentiation without variation from passage 0 through 3. Additional experiments on different hMSCs (n = 4) confirmed this pattern of expression. hMSCs express the marker CD90 (Fig. 3B) and present some similarities with pancreatic beta cells, as we detected mRNA of epithelial markers such as cytokeratin 18 and 19 as well as proconvertase 1/3. Cytokeratins 18 and 19 were expressed in all of the samples analyzed, whereas proconvertase expression seems to vary from one passage to the other and between donors. In addition, we failed to detect any gene implicated in glucose metabolism (glucose transporter 2, glucokinase, or insulin).
Figure 3. Endogenous expression of (A) key pancreatic developmental transcription factors and (B) pancreatic genes and epithelial or surface MSC markers in hMSCs. Reverse transcription–polymerase chain reaction (RT-PCR) amplifications of RNAs extracted from hMSCs harvested at different passages (0, 1, 2, and 3) were analyzed by agarose gel electrophoresis. RNA isolated from human pancreatic islets or fetal pancreas was used as a positive control. For NEUROG3 RT-PCR analysis, human fetal pancreas was used because adult pancreatic islets do not express this gene. Abbreviation: hMSC, human mesenchymal stem cell.
Effect of Adenoviral Infection on hMSC Differentiation and Transformation
To check that adenoviral infection had no effect on hMSC differentiation ability, we infected hMSCs with AdNull, and 7 days after infection, we cultured them in adipogenic differentiation medium. Nearly all of the cells, infected or not infected, showed adipose tissue-forming capacity and accumulated large amounts of triglycerides in their cytoplasm (Fig. 4). Moreover, soft agar assay did not show any colonies with either control or infected cells (data not shown).
Figure 4. In vitro differentiation of human mesenchymal stem cells into adipocytes. Oil Red-O staining of the lipid vesicles performed 3 weeks after adipogenic stimulation demonstrates an ongoing adipogenesis (magnification, x20). (A): Control cells, (B) control cells cultured in adipogenic medium, (C) infected cells, and (D) infected cells cultured in adipogenic medium.
hMSCs Express Insulin After Adenoviral Infection with Genes Coding for Transcription Factors of the Beta Endocrine Pathway or Specific Culture Conditions
Based on the hypothesis that hMSCs’ ectopic expression of transcription factors involved in beta-endocrine pathway might favor their differentiation into insulin-expressing cells, we infected hMSCs 4 days after plating with adenoviruses coding for mouse IPF1, mouse HLXB9, or mouse FOXA2 using various MOI ratios. hMSCs were concomitantly cultured either alone, in the presence of islet-conditioned medium, or in the presence of human islets placed in a culture insert. Cells were harvested and RNAs were extracted 7 days after infection and analyzed by RT-PCR (Fig. 5).
Figure 5. Effect of adenovirus-mediated ectopic expression of pancreatic transcription factors (AdmIPF1, AdmHLXB9, and AdmFOXA2) and islet environment on insulin and key transcription factor expression in hMSCs. (A): Model of the cascade of transcription factors controlling beta-cell differentiation as described by Schwitzgebel et al. . (B, C): RT-PCR analysis of infected hMSCs cultured with or without (B) islet environment and (C) control human islet. ACTB PCR amplification was used as RNA quality and RT efficiency control. Abbreviations: hMSC, human mesenchymal stem cell; MCo, islet-conditioned medium; MOI, multiplicity of infection; ND, nondetermined; Neg, nonexpressed; RT-PCR, reverse transcription–polymerase chain reaction.
In the first experiment, hMSCs were infected with AdmIPF1 using a high MOI ratio (100:1). We detected expression of insulin and expression of three transcription factors involved in beta-cell differentiation, FOXA2, PAX4, and ISL1. In the second experiment, we lowered the MOI ratio (40:1), and hMSCs were concomitantly cultured either in islet-conditioned medium or in control medium. Insulin expression and only one transcription factor expression, PAX4, could be detected in hMSCs cultured with islet-conditioned medium. In the third experiment, a MOI ratio of 20:1 was used either in the presence or in the absence of pancreatic human islets. Infected hMSCs cocultured with human islets expressed insulin gene at a very low level, but no expression of transcription factors was detected. In the fourth experiment, coinfection of hMSCs with AdmHLXB9 (MOI 50:1) and AdmFOXA2 (MOI 50:1) was performed with or without islets. In the presence of human islets, insulin as well as NEUROD1 and ISL1 expressions were detected.
DISCUSSION
We are grateful to J. Méo, V. Konik-Mathevet, M. Samuel, O. Vermeulen, and L. Ydoux for technical assistance. We thank the Laboratoire de Thérapie Génique de Nantes and Généthon III for generation adenoviruses and Dr. T. Berney for the generous islet gift. This work was supported by grants from AGIRaDom (to P.-Y.B.), AFM/Inserm (to P.S.), Region Rhone-Alpes Emergence (to P.S.), ARC (to P.S.), and AVENIR program (to P.S.).
REFERENCES
Shapiro AM, Lakey JR, Ryan EA et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343:230–238.
Benhamou PY, Oberholzer J, Toso C et al. Human islet transplantation network for the treatment of type I diabetes: first data from the Swiss-French GRAGIL consortium (1999–2000). Groupe de Recherche Rhin Rh?ne Alpes Geneve pour la transplantation d’Ilots de Langerhans. Diabetologia 2001;44:859–864.
Soria B, Skoudy A, Martin F. From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus. Diabetologia 2001;44:407–415.
Serup P, Madsen OD, Mandrup-Poulsen T. Islet and stem cell transplantation for treating diabetes. BMJ 2001;322:29–32.
Rosenberg L. In vivo cell transformation: neogenesis of beta cells from pancreatic ductal cells. Cell Transplant 1995;4:371–383.
Assady S, Maor G, Amit M et al. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–1697.
Castaing M, Peault B, Basmaciogullari A et al. Blood glucose normalization upon transplantation of human embryonic pancreas into beta-cell-deficient SCID mice. Diabetologia 2001;44:2066–2076.
Soria B, Roche E, Berna G et al. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000;49:157–162.
Kojima H, Nakamura T, Fujita Y et al. Combined expression of pancreatic duodenal homeobox 1 and islet factor 1 induces immature enterocytes to produce insulin. Diabetes 2002;51:1398–1408.
Yang L, Li S, Hatch H et al. In vitro transdifferentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci U S A 2002;99:8078–8083.
Ferber S, Halkin A, Cohen H et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia. Nat Med 2000;6:568–572.
Bonner-Weir S, Taneja M, Weir GC et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci U S A 2000;97:7999–8004.
Ramiya VK, Maraist M, Arfors KE et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med 2000;6:278–282.
Anderson DJ, Gage FH, Weissman IL. Can stem cells cross lineage boundaries? Nat Med 2001;7:393–395.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–1234.
Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.
Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000;100:157–168.
Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol 2000;28:875–884.
Sanchez-Ramos J, Song S, Cardozo-Pelaez F et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247–256.
Woodbury D, Schwarz EJ, Prockop DJ et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61:364–370.
Reyes M, Lund T, Lenvik T et al. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001;98:2615–2625.
Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
D’Ippolito G, Diabira S, Howard GA et al. Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci 2004;117:2971–2981.
Schwitzgebel VM, Scheel DW, Conners JR et al. Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development 2000;127:3533–3542.
Wu KL, Gannon M, Peshavaria M et al. Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene. Mol Cell Biol 1997;17:6002–6013.
Marshak S, Benshushan E, Shoshkes M et al. Functional conservation of regulatory elements in the pdx-1 gene: PDX-1 and hepatocyte nuclear factor 3beta transcription factors mediate beta-cell-specific expression. Mol Cell Biol 2000;20:7583–7590.
Rooman I, Heremans Y, Heimberg H et al. Modulation of rat pancreatic acinoductal transdifferentiation and expression of PDX-1 in vitro. Diabetologia 2000;43:907–914.
Beattie GM, Itkin-Ansari P, Cirulli V et al. Sustained proliferation of PDX-1+ cells derived from human islets. Diabetes 1999;48:1013–1019.
Sharma A, Zangen DH, Reitz P et al. The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 1999;48:507–513.
Colter DC, Class R, DiGirolamo CM et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A 2000;97:3213–3218.
Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 1976;4:267–274.
Kobune M, Kawano Y, Ito Y et al. Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells. Exp Hematol 2003;31:715–722.
Zimmermann S, Voss M, Kaiser S et al. Lack of telomerase activity in human mesenchymal stem cells. Leukemia 2003;17:1146–1149.
Parsch D, Fellenberg J, Brummendorf TH et al. Telomere length and telomerase activity during expansion and differentiation of human mesenchymal stem cells and chondrocytes. J Mol Med 2004;82:49–55.
Okamoto T, Aoyama T, Nakayama T et al. Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells. Biochem Biophys Res Commun 2002;295:354–361.
Kogler G, Sensken S, Airey JA et al. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 2004;200:123–135.
Pochampally RR, Smith JR, Ylostalo J et al. Serum deprivation of human marrow stromal cells (hMSCs) selects for a sub-population of early progenitor cells with enhanced expression of Oct-4 and other embryonic genes. Blood 2004;103:1647–1652.
Bouwens L, Wang RN, De Blay E et al. Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neonatal rat pancreas. Diabetes 1994;43:1279–1283.
Schwartz RE, Reyes M, Koodie L et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002;109:1291–1302.
Sander M, Sussel L, Conners J et al. Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas. Development 2000;127:5533–5540.
Watada H, Mirmira RG, Leung J et al. Transcriptional and translational regulation of beta-cell differentiation factor Nkx6.1. J Biol Chem 2000;275:34224–34230.
Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res 2002;69:908–917.
Scopsi L, Gullo M, Rilke F et al. Proprotein convertases (PC1/PC3 and PC2) in normal and neoplastic human tissues: their use as markers of neuroendocrine differentiation. J Clin Endocrinol Metab 1995;80:294–301.
Schwarz EJ, Alexander GM, Prockop DJ et al. Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease. Hum Gene Ther 1999;10:2539–2549.
Lee K, Majumdar MK, Buyaner D et al. Human mesenchymal stem cells maintain transgene expression during expansion and differentiation. Mol Ther 2001;3:857–866.
Allay JA, Dennis JE, Haynesworth SE et al. LacZ and interleukin-3 expression in vivo after retroviral transduction of marrow-derived human osteogenic mesenchymal progenitors. Hum Gene Ther 1997;8:1417–1427.
Li H, Arber S, Jessell TM et al. Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9. Nat Genet 1999;23:67–70.
Harrison KA, Thaler J, Pfaff SL et al. Pancreas dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9-deficient mice. Nat Genet 1999;23:71–75.
Lee CS, Sund NJ, Vatamaniuk MZ et al. Foxa2 controls Pdx1 gene expression in pancreatic beta-cells in vivo. Diabetes 2002;51:2546–2551.
McKinnon CM, Docherty K. Pancreatic duodenal homeobox-1, PDX-1, a major regulator of beta cell identity and function. Diabetologia 2001;44:1203–1214.
Scharfmann R. Control of early development of the pancreas in rodents and humans: implications of signals from the mesenchyme. Diabetologia 2000;43:1083–1092.
Sander M, German MS. The beta cell transcription factors and development of the pancreas. J Mol Med 1997;75:327–340.
Sosa-Pineda B, Chowdhury K, Torres M et al. The Pax4 gene is essential for differentiation of insulin-producing beta cells in the mammalian pancreas. Nature 1997;386:399–402.
Ahlgren U, Pfaff SL, Jessell TM et al. Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells. Nature 1997;385:257–260.
Naya FJ, Huang HP, Qiu Y et al. Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neu-roD-deficient mice. Genes Dev 1997;11:2323–2334.
Heremans Y, Van De Casteele M, in’t Veld P et al. Recapitulation of embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3. J Cell Biol 2002;159:303–312.
Hess D, Li L, Martin M et al. Bone marrow-derived stem cells initiate pancreatic regeneration. Nat Biotechnol 2003;21:763–770.
Ianus A, Holz GG, Theise ND et al. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest 2003;111:843–850.
Mathews V, Hanson PT, Ford E et al. Recruitment of bone marrow-derived endothelial cells to sites of pancreatic beta-cell injury. Diabetes 2004;53:91–98.(Christine Moriscota,b, Fl)
b Centre d’Investigation Biologique, Centre Hospitalier Universitaire, Grenoble, France;
c Institut National de la Santé et de la Recherche Médicale (Inserm), Unité 371, Institut Fédératif des Neurosciences de Lyon, Bron, France;
d Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5161, Ecole Normale Supérieure de Lyon, France;
e Cell Isolation and Transplantation Center, University of Geneva Medical Center, Geneva, Switzerland;
f Département d’Endocrinologie, Centre Hospitalier Universitaire, Grenoble, France
Key Words. Bone marrow ? Mesenchymal stem cell ? Pancreatic beta cell ? Cell differentiation ? Insulin ? Transcription factors
Correspondence: Pierre-Yves Benhamou, M.D., Ph.D, Centre d’Investigation Biologique, Pavillon B, Centre Hospitalier Universitaire, 38043 Grenoble Cedex 9, France. Telephone: 33-4-76-76-88-56; Fax: 33-4-76-76-50-42; e-mail: pierre-yves.benhamou@wanadoo.fr
ABSTRACT
An important application for cell therapy is diabetes mellitus. By restoring normal endogenous insulin secretion in patients with diabetes, cell therapy may challenge the actual treatment by exogenous insulin. Transplantation of pancreatic islet cells as a potential cure for diabetes has become the subject of intense interest and activity over the past two decades . However, the limited supply of human islet tissue available for transplantation prevents this therapy from being used to treat the thousands of patients with type 1 diabetes. In vitro expansion of human beta cells or genetic engineering of human insulin-secreting cells may represent one approach, but the clinical use is limited by the difficulties in achieving prolonged or physiologically regulated insulin secretion . One way to overcome these problems and obtain functional glucose-sensitive insulin-secreting cells for transplantation is to derive islet cells from other sources such as embryonic stem cells and intestinal , hepatic , ductal, or pancreatic stem cells . These studies have opened fascinating perspectives. An easily accessible, expandable, glucose-responsive, and autologous stem cell would offer obvious advantages in a clinical setting.
Recent work suggests that adult stem cells from one tissue or organ can differentiate into cells of other organs, either in vitro or in vivo . Among them, bone marrow–derived stem cells (hematopoietic or mesenchymal) carry the more significant implications for possible clinical development, because they are easily accessible for an autograft and routinely collected from adults without ethical concern inherent to fetal embryonic tissues .
Based on their ability to adhere to plastic support , multipotential stem cells can be isolated from the bone marrow, expanded, and cultured. Under appropriate experimental conditions, they differentiate into multiple mesenchymal cell types, including cartilage, bone, adipose and fibrous tissues, and myelosupportive stroma . Moreover, treatment with growth factors such as epithelial growth factor and brain-derived neurotrophic factor or chemical products such as dimethyl sulfoxide and butylated hydroxyanisole induced the bone marrow stroma cells to exhibit a neuronal phenotype .
A more likely candidate may be the multipotential adult progenitor cells (MAPCs) derived from adult bone marrow . These cells exhibit a remarkable plasticity, with the ability to differentiate into cells with mesodermal, neuroectodermal, and endodermal characteristics in vitro . Furthermore, upon transplantation, MAPCs can differentiate into epithelium of the liver, lung, and gut. MAPCs express the OCT4 and REX1 transcription factors, two specific markers of undifferentiated embryonic stem (ES) cells. Recently, the marrow-isolated adult multilineage-inducible (MIAMI) cells capable of differentiating in vitro into cell lineages from all three germ layers have been described .
During development, the formation of the pancreas and its subsequent differentiation into the different exocrine and endocrine cell types and mature adult beta cells result from the orderly activation and extinction of a large number of genes. Experiments with transgenic mice have identified a hierarchy in the transcription key factors, such as HLXB9, FOXA2 (formerly named HNF3?), IPF1 (PDX1), NEUROG3 (NGN3), NEUROD1, NKX2-2, PAX4, NKX6-1, and PAX6, that control embryonic formation of pancreatic islets . Among these factors, FOXA2 and IPF1 play a central role in initiating the differentiation of the islet cells.
If human mesenchymal stem cells (hMSCs) could form new beta cells, they would become a particularly useful target for therapies that aim at beta-cell replacement in diabetic patients, because they are abundantly available in the human bone marrow. In this study, we first confirm that hMSCs express the phenotypic surface marker characteristics of multipotent cells. We second provide evidence, by reverse transcription–polymerase chain reaction (RT-PCR), for the presence of some factors implicated in pancreatic development and function. Their phenotype as well as their adipogenic differentiation ability were not modified by adenoviral infection. Finally, we show that genetic manipulations or appropriate culture conditions allow hMSCs to express insulin mRNA.
MATERIALS AND METHODS
Phenotype Characteristics of Expanded Undifferentiated hMSCs
After plastic adherence selection, hMSCs were cultured over four passages. Growth was exponential over the studied period (60 days) with slight differences between donors (Fig. 1A). FACS analysis of hMSCs showed that these cells were negative for CD11b, CD31, CD34, CD45, CD49b, and CD117. They expressed high levels of CD44, CD73, CD90, CD105, and CD147 and low levels of CD10. CD34 and CD45 represent two of the major hematopoietic markers, whereas CD73, CD90, CD105, CD147, and CD10 are cell-surface markers characteristic of MSCs (Fig. 1B). The same phenotype was maintained for passages 0 through 3 and for all of the hMSCs analyzed. We showed that hMSCs express OCT4, a transcription factor important in maintaining undifferentiated ES cells (Fig. 2B). Furthermore, average telomere length of hMSCs cultured during 46 days (three passages) was at least as long as the high telomere length control of 10.2 kbp (Fig. 2A), even though no telomerase activity was detected by quantitative RT-PCR (data not shown).
Figure 1. Culture of human mesenchymal stem cells (hMSCs). (A): Growth curves of hMSCs isolated from two donors, hMSC 54 (filled square) and hMSC 56 (filled circle), were determined by numeration at each passage. Cell number was plotted against time in days. (B): Phenotype of hMSCs. Cells were harvested and labeled with antibodies against CD10, CD11b, CD31, CD34, CD44, CD45, CD49b, CD73, CD90, CD105, CD117, and CD147 or control immunoglobulin G, as indicated and analyzed by fluorescence-activated cell sorting. Plots show isotype control immunoglobulin G staining profile (dotted line) versus specific antibody staining profile (thick line). A representative example of more than 20 hMSCs is shown.
Figure 2. Characteristics of undifferentiated human mesenchymal stem cells (hMSCs). (A): Cells from two donors (hMSC 54 and hMSC 56) were expanded and harvested at each passage (0, 1, and 2) and telomere lengths were evaluated. (B): Reverse transcription (RT)–polymerase chain reaction for OCT4 and ACTB (used as RNA quality and RT efficiency control). RNA obtained at passages 0, 1, 2, and 3 for hMSC 56 was analyzed.
Expression of Islet-Associated Transcription Factors in hMSCs
To evaluate the potential of hMSCs to differentiate into beta cells, we first examined the expression of islet-related transcription factors. As shown in Figure 3A, RT-PCR analysis revealed that hMSCs express NKX6-1 at a low level but lacked all the other transcription factors involved in beta-cell differentiation without variation from passage 0 through 3. Additional experiments on different hMSCs (n = 4) confirmed this pattern of expression. hMSCs express the marker CD90 (Fig. 3B) and present some similarities with pancreatic beta cells, as we detected mRNA of epithelial markers such as cytokeratin 18 and 19 as well as proconvertase 1/3. Cytokeratins 18 and 19 were expressed in all of the samples analyzed, whereas proconvertase expression seems to vary from one passage to the other and between donors. In addition, we failed to detect any gene implicated in glucose metabolism (glucose transporter 2, glucokinase, or insulin).
Figure 3. Endogenous expression of (A) key pancreatic developmental transcription factors and (B) pancreatic genes and epithelial or surface MSC markers in hMSCs. Reverse transcription–polymerase chain reaction (RT-PCR) amplifications of RNAs extracted from hMSCs harvested at different passages (0, 1, 2, and 3) were analyzed by agarose gel electrophoresis. RNA isolated from human pancreatic islets or fetal pancreas was used as a positive control. For NEUROG3 RT-PCR analysis, human fetal pancreas was used because adult pancreatic islets do not express this gene. Abbreviation: hMSC, human mesenchymal stem cell.
Effect of Adenoviral Infection on hMSC Differentiation and Transformation
To check that adenoviral infection had no effect on hMSC differentiation ability, we infected hMSCs with AdNull, and 7 days after infection, we cultured them in adipogenic differentiation medium. Nearly all of the cells, infected or not infected, showed adipose tissue-forming capacity and accumulated large amounts of triglycerides in their cytoplasm (Fig. 4). Moreover, soft agar assay did not show any colonies with either control or infected cells (data not shown).
Figure 4. In vitro differentiation of human mesenchymal stem cells into adipocytes. Oil Red-O staining of the lipid vesicles performed 3 weeks after adipogenic stimulation demonstrates an ongoing adipogenesis (magnification, x20). (A): Control cells, (B) control cells cultured in adipogenic medium, (C) infected cells, and (D) infected cells cultured in adipogenic medium.
hMSCs Express Insulin After Adenoviral Infection with Genes Coding for Transcription Factors of the Beta Endocrine Pathway or Specific Culture Conditions
Based on the hypothesis that hMSCs’ ectopic expression of transcription factors involved in beta-endocrine pathway might favor their differentiation into insulin-expressing cells, we infected hMSCs 4 days after plating with adenoviruses coding for mouse IPF1, mouse HLXB9, or mouse FOXA2 using various MOI ratios. hMSCs were concomitantly cultured either alone, in the presence of islet-conditioned medium, or in the presence of human islets placed in a culture insert. Cells were harvested and RNAs were extracted 7 days after infection and analyzed by RT-PCR (Fig. 5).
Figure 5. Effect of adenovirus-mediated ectopic expression of pancreatic transcription factors (AdmIPF1, AdmHLXB9, and AdmFOXA2) and islet environment on insulin and key transcription factor expression in hMSCs. (A): Model of the cascade of transcription factors controlling beta-cell differentiation as described by Schwitzgebel et al. . (B, C): RT-PCR analysis of infected hMSCs cultured with or without (B) islet environment and (C) control human islet. ACTB PCR amplification was used as RNA quality and RT efficiency control. Abbreviations: hMSC, human mesenchymal stem cell; MCo, islet-conditioned medium; MOI, multiplicity of infection; ND, nondetermined; Neg, nonexpressed; RT-PCR, reverse transcription–polymerase chain reaction.
In the first experiment, hMSCs were infected with AdmIPF1 using a high MOI ratio (100:1). We detected expression of insulin and expression of three transcription factors involved in beta-cell differentiation, FOXA2, PAX4, and ISL1. In the second experiment, we lowered the MOI ratio (40:1), and hMSCs were concomitantly cultured either in islet-conditioned medium or in control medium. Insulin expression and only one transcription factor expression, PAX4, could be detected in hMSCs cultured with islet-conditioned medium. In the third experiment, a MOI ratio of 20:1 was used either in the presence or in the absence of pancreatic human islets. Infected hMSCs cocultured with human islets expressed insulin gene at a very low level, but no expression of transcription factors was detected. In the fourth experiment, coinfection of hMSCs with AdmHLXB9 (MOI 50:1) and AdmFOXA2 (MOI 50:1) was performed with or without islets. In the presence of human islets, insulin as well as NEUROD1 and ISL1 expressions were detected.
DISCUSSION
We are grateful to J. Méo, V. Konik-Mathevet, M. Samuel, O. Vermeulen, and L. Ydoux for technical assistance. We thank the Laboratoire de Thérapie Génique de Nantes and Généthon III for generation adenoviruses and Dr. T. Berney for the generous islet gift. This work was supported by grants from AGIRaDom (to P.-Y.B.), AFM/Inserm (to P.S.), Region Rhone-Alpes Emergence (to P.S.), ARC (to P.S.), and AVENIR program (to P.S.).
REFERENCES
Shapiro AM, Lakey JR, Ryan EA et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343:230–238.
Benhamou PY, Oberholzer J, Toso C et al. Human islet transplantation network for the treatment of type I diabetes: first data from the Swiss-French GRAGIL consortium (1999–2000). Groupe de Recherche Rhin Rh?ne Alpes Geneve pour la transplantation d’Ilots de Langerhans. Diabetologia 2001;44:859–864.
Soria B, Skoudy A, Martin F. From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus. Diabetologia 2001;44:407–415.
Serup P, Madsen OD, Mandrup-Poulsen T. Islet and stem cell transplantation for treating diabetes. BMJ 2001;322:29–32.
Rosenberg L. In vivo cell transformation: neogenesis of beta cells from pancreatic ductal cells. Cell Transplant 1995;4:371–383.
Assady S, Maor G, Amit M et al. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–1697.
Castaing M, Peault B, Basmaciogullari A et al. Blood glucose normalization upon transplantation of human embryonic pancreas into beta-cell-deficient SCID mice. Diabetologia 2001;44:2066–2076.
Soria B, Roche E, Berna G et al. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000;49:157–162.
Kojima H, Nakamura T, Fujita Y et al. Combined expression of pancreatic duodenal homeobox 1 and islet factor 1 induces immature enterocytes to produce insulin. Diabetes 2002;51:1398–1408.
Yang L, Li S, Hatch H et al. In vitro transdifferentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci U S A 2002;99:8078–8083.
Ferber S, Halkin A, Cohen H et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia. Nat Med 2000;6:568–572.
Bonner-Weir S, Taneja M, Weir GC et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci U S A 2000;97:7999–8004.
Ramiya VK, Maraist M, Arfors KE et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med 2000;6:278–282.
Anderson DJ, Gage FH, Weissman IL. Can stem cells cross lineage boundaries? Nat Med 2001;7:393–395.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–1234.
Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.
Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000;100:157–168.
Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol 2000;28:875–884.
Sanchez-Ramos J, Song S, Cardozo-Pelaez F et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247–256.
Woodbury D, Schwarz EJ, Prockop DJ et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61:364–370.
Reyes M, Lund T, Lenvik T et al. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001;98:2615–2625.
Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
D’Ippolito G, Diabira S, Howard GA et al. Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci 2004;117:2971–2981.
Schwitzgebel VM, Scheel DW, Conners JR et al. Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development 2000;127:3533–3542.
Wu KL, Gannon M, Peshavaria M et al. Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene. Mol Cell Biol 1997;17:6002–6013.
Marshak S, Benshushan E, Shoshkes M et al. Functional conservation of regulatory elements in the pdx-1 gene: PDX-1 and hepatocyte nuclear factor 3beta transcription factors mediate beta-cell-specific expression. Mol Cell Biol 2000;20:7583–7590.
Rooman I, Heremans Y, Heimberg H et al. Modulation of rat pancreatic acinoductal transdifferentiation and expression of PDX-1 in vitro. Diabetologia 2000;43:907–914.
Beattie GM, Itkin-Ansari P, Cirulli V et al. Sustained proliferation of PDX-1+ cells derived from human islets. Diabetes 1999;48:1013–1019.
Sharma A, Zangen DH, Reitz P et al. The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 1999;48:507–513.
Colter DC, Class R, DiGirolamo CM et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A 2000;97:3213–3218.
Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 1976;4:267–274.
Kobune M, Kawano Y, Ito Y et al. Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells. Exp Hematol 2003;31:715–722.
Zimmermann S, Voss M, Kaiser S et al. Lack of telomerase activity in human mesenchymal stem cells. Leukemia 2003;17:1146–1149.
Parsch D, Fellenberg J, Brummendorf TH et al. Telomere length and telomerase activity during expansion and differentiation of human mesenchymal stem cells and chondrocytes. J Mol Med 2004;82:49–55.
Okamoto T, Aoyama T, Nakayama T et al. Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells. Biochem Biophys Res Commun 2002;295:354–361.
Kogler G, Sensken S, Airey JA et al. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 2004;200:123–135.
Pochampally RR, Smith JR, Ylostalo J et al. Serum deprivation of human marrow stromal cells (hMSCs) selects for a sub-population of early progenitor cells with enhanced expression of Oct-4 and other embryonic genes. Blood 2004;103:1647–1652.
Bouwens L, Wang RN, De Blay E et al. Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neonatal rat pancreas. Diabetes 1994;43:1279–1283.
Schwartz RE, Reyes M, Koodie L et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002;109:1291–1302.
Sander M, Sussel L, Conners J et al. Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas. Development 2000;127:5533–5540.
Watada H, Mirmira RG, Leung J et al. Transcriptional and translational regulation of beta-cell differentiation factor Nkx6.1. J Biol Chem 2000;275:34224–34230.
Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res 2002;69:908–917.
Scopsi L, Gullo M, Rilke F et al. Proprotein convertases (PC1/PC3 and PC2) in normal and neoplastic human tissues: their use as markers of neuroendocrine differentiation. J Clin Endocrinol Metab 1995;80:294–301.
Schwarz EJ, Alexander GM, Prockop DJ et al. Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease. Hum Gene Ther 1999;10:2539–2549.
Lee K, Majumdar MK, Buyaner D et al. Human mesenchymal stem cells maintain transgene expression during expansion and differentiation. Mol Ther 2001;3:857–866.
Allay JA, Dennis JE, Haynesworth SE et al. LacZ and interleukin-3 expression in vivo after retroviral transduction of marrow-derived human osteogenic mesenchymal progenitors. Hum Gene Ther 1997;8:1417–1427.
Li H, Arber S, Jessell TM et al. Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9. Nat Genet 1999;23:67–70.
Harrison KA, Thaler J, Pfaff SL et al. Pancreas dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9-deficient mice. Nat Genet 1999;23:71–75.
Lee CS, Sund NJ, Vatamaniuk MZ et al. Foxa2 controls Pdx1 gene expression in pancreatic beta-cells in vivo. Diabetes 2002;51:2546–2551.
McKinnon CM, Docherty K. Pancreatic duodenal homeobox-1, PDX-1, a major regulator of beta cell identity and function. Diabetologia 2001;44:1203–1214.
Scharfmann R. Control of early development of the pancreas in rodents and humans: implications of signals from the mesenchyme. Diabetologia 2000;43:1083–1092.
Sander M, German MS. The beta cell transcription factors and development of the pancreas. J Mol Med 1997;75:327–340.
Sosa-Pineda B, Chowdhury K, Torres M et al. The Pax4 gene is essential for differentiation of insulin-producing beta cells in the mammalian pancreas. Nature 1997;386:399–402.
Ahlgren U, Pfaff SL, Jessell TM et al. Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells. Nature 1997;385:257–260.
Naya FJ, Huang HP, Qiu Y et al. Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neu-roD-deficient mice. Genes Dev 1997;11:2323–2334.
Heremans Y, Van De Casteele M, in’t Veld P et al. Recapitulation of embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3. J Cell Biol 2002;159:303–312.
Hess D, Li L, Martin M et al. Bone marrow-derived stem cells initiate pancreatic regeneration. Nat Biotechnol 2003;21:763–770.
Ianus A, Holz GG, Theise ND et al. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest 2003;111:843–850.
Mathews V, Hanson PT, Ford E et al. Recruitment of bone marrow-derived endothelial cells to sites of pancreatic beta-cell injury. Diabetes 2004;53:91–98.(Christine Moriscota,b, Fl)