In Vitro Cultured Islet-Derived Progenitor Cells of Human Origin Express Human Albumin in Severe Combined Immunodeficiency Mouse Liver In Vi
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《干细胞学杂志》
a II. Medical Department, University of Mainz, Mainz, Germany;
b Center for Toxicology, Institute of Legal Medicine and Rudolf-Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany;
c Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital, Basel, Switzerland;
d Molecular Genetics Laboratory, University Children’s Hospital, University of Mainz, Mainz, Germany;
e Paul-Flechsig Institute, University of Leipzig, Leipzig, Germany
Key Words. Pancreas ? Islet of Langerhans ? Liver ? Stem cell
Correspondence: Marc-Alexander von Mach, M.D., II. Medical Department, Langenbeckstr. 1, 55131 Mainz, Germany. Telephone: 49-6131-174154; Fax: 49-6131-176605; e-mail: marcm@giftinfo.uni-mainz.de
ABSTRACT
During embryogenesis, progenitor cells of pancreas and liver emerge from neighboring areas of the gut endoderm . There is a large body of evidence suggesting that such progenitors with the potential to generate liver cells from pancreatic cells and vice versa may still exist in adult life . Using the model of mice with a knockout of the tyrosine catabolic enzyme fumarylacetoacetate hydrolase (FAH), which, without treatment, results in liver cirrhosis, Wang et al. succeeded in correction of liver function by transplantation of cell suspensions from adult pancreas of wild-type animals. With this unique repopulation assay, the authors clearly demonstrated that progenitor cells with the ability not only to replace FAH knockout cells in the liver but also to correct the liver function exist in the pancreas, although the exact nature of these cells remains unknown. Interestingly, cells from cultured pancreatic ducts were not able to rescue the failing liver as did the crude pancreatic cell suspension , indicating that the presumed hepatopancreatic stem cells reside in areas outside the pancreatic ducts.
Numerous studies have shown the transdifferentiation potential of pancreatic cells of rodents into hepatocytes in vivo. Some very rare cases of human pancreatic cancer with hepatoid phenotype indicated the existence of similar cells in human pancreas . Recently, progenitor cells have been described in rodent and human islets of Langerhans that express the neural stem cell marker nestin and the side-population phenotype marker ABCG2 . The side-population cells in bone marrow represent a particularly potent stem cell population . Interestingly, the human nestin-expressing islet-derived progenitor (NIP) cells were able to adopt a hepatic phenotype in vitro with expression of markers like alpha feto protein and the transcription factor XBP . In contrast to animal data, however, no in vivo studies have been published so far demonstrating transdifferentiation of human pancreatic cells into a hepatic phenotype. Recently, in vivo models for transplantation of human cord blood cells into severe combined immunodeficiency (SCID) mouse liver have been established . Using the model with direct injection of cells into the liver, we demonstrate in the present report that human cells from cultured pancreatic islets of Langerhans engraft into SCID mouse liver and form cells expressing human albumin in vivo.
MATERIALS AND METHODS
NIP cells expressed beside nestin also the side-population marker ABCG2 as well as SCF, c-Kit, and Thy-1, another potential marker for hepatic stem/progenitor cells (Fig. 1A). These cells were negative for expression of the transcription factor IPF-1 and insulin but also the specific marker for hematopoietic cells CD45 (Fig. 1B). Before transplantation, no albumin expression was found in cultured NIP cells (Fig. 1C). To exclude numerical or structural chromosomal aberration due to prolonged growth stimulation in vitro, a karyotyping was performed and revealed a normal 46, XX karyotype (Fig. 2).
Figure 1. (A): NIP cells not only express the neural stem cell marker nestin but also the side-population marker ABCG2, SCF, c-Kit, and the hepatic stem cell marker Thy-1. NIP cells did not express IPF-1 or insulin. The human housekeeping gene APRT was used as positive control for RT-PCR. (B): Lack of CD45 expression in NIP cells with positive signal in human cord blood cells. (C): Detection of human albumin in SCID mouse liver after transplantation of NIP cells. RT-PCR analysis shows the expression of human albumin mRNA in transplanted SCID mouse number 18. Human albumin is not expressed by cultured NIP cells. HepG2 cells were used as positive control. SCID mouse liver was used as negative control. The origin of all PCR products was confirmed by sequencing. Abbreviations: APRT, adenine phosphoribosyltransferase; NIP, nestin-expressing islet-derived progenitor; RT-PCR, reverse transcription–polymerase chain reaction; SCID, severe combined immunodeficiency.
Transplantation of 1.5 x 104 human NIP cells failed to result in detectable red fluorescent cells that became detectable only after transplantation of 1.5 x 105 cells in three of four animals (Table 1). We next evaluated the impact of time after transplantation on engraftment frequency and found similar results 3 and 12 weeks after transplantation. Cells expressing human albumin were found in liver sections of 8 out of 11 animals. The cells were well integrated into the liver tissue and were predominantly found adjacent to vascular structures (Fig. 3). Transplantation of NIP cells without prior tagging with PKH26 seemed to be more successful and resulted in detection of human albumin-positive cells in all four grafted animals. To analyze fusion as a possible mechanism underlying this phenomenon, immunohistochemistry studies were performed using mouse-specific and human-specific anti-albumin antibodies. In case of fusion, we expected both types of albumin to be expressed in the grafted cell. Using confocal microscopy, expression of human and mouse albumin was found in distinct cells, suggesting that NIP cells did adopt a hepatic phenotype by differentiation induced by surrounding liver tissue rather than fusion (Fig. 4). Additionally, RT-PCR was performed, demonstrating the presence of human albumin mRNA in 4 of 10 animals (Fig. 1C). In the four human albumin–positive livers, the human APRT was also amplified using RT-PCR, although the signal was less abundant than albumin (data not shown). No mononuclear cell infiltration associated with human albumin–positive cells and no neoplasm were observed 3 and 12 weeks after transplantation. In our model, the occurrence of human albumin–positive cells in general, however, was a rare event, with detection of one to three positive cells on every second slice.
Figure 3. Immunohistochemistry 3 weeks after transplantation of human islet-derived progenitor cells into SCID mouse liver using a monoclonal antibody specific for human albumin and diaminobenzidine (brown) for staining (A), nontransplanted SCID mouse liver as negative control (B), and human liver (C) as positive control (bar = 20 μm). Note the proximity of grafted cells to vascular structures.
Figure 4. Fluorescence-immunohistochemistry with human and mouse specific antibodies against albumin using confocal microscopy with 630-fold magnification. (A): One cell stained with antibodies against human albumin. (B): The same cell with additional 4',6'-diamidino-2-phenylindole staining for cell nuclei. (C):Albumin staining with antibodies against mouse albumin. (D): Digital overlay of human and mouse albumin staining showing no costaining for mouse albumin in the human albumin-positive cell.
DISCUSSION
Human islet-derived stem cells are capable of adopting a hepatic phenotype in a SCID mouse liver in vivo, suggesting the presence of a hepatopancreatic stem/progenitor cell within or adjacent to the islets of Langerhans. The mechanism underlying this phenomenon seems to be trans-differentiation, although fusion with host hepatocyte cannot be completely ruled out. In the context of these recent findings, one could envision new therapeutic avenues for the treatment of liver cirrhosis using human pancreatic stem/ progenitor cells in which such cells could be isolated from pancreatic biopsies and expanded in vitro before trans plantation.
ACKNOWLEDGMENTS
Deutsch G, Jung J, Zheng M et al. A bipotential precursor population for pancreas and liver within the embryonic endoderm. Development 2001;128:871–881.
Grompe M. Pancreatic-hepatic switches in vivo. Mech Dev 2003;120:99–106.
Shen CN, Horb ME, Slack JM et al. Transdifferentiation of pancreas to liver. Mech Dev 2003;120:107–116.
Horb ME, Shen CN, Tosh D et al. Experimental conversion of liver to pancreas. Curr Biol 2003;13:105–115.
Zalzman M, Gupta S, Giri RK et al. Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc Natl Acad Sci U S A 2003;100:7253–7258.
Wang X, Al-Dhalimy M, Lagasse E et al. Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells. Am J Pathol 2001;158:571–579.
Paner GP, Thompson KS, Reyes CV. Hepatoid carcinoma of the pancreas. Cancer 2000;88:1582–1589.
Zulewski H, Abraham EJ, Gerlach MJ et al. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001;50:521–533.
Lechner A, Leech CA, Abraham EJ 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. Biochem Biophys Res Commun 2002;293: 670–674.
Zhou S, Schuetz JD, Bunting KD et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028–1034.
Beerheide W, von Mach MA, Ringel M et al. Downregulation of beta2-microglobulin in human cord blood stem cells after transplantation into livers of SCID-mice: an escape mechanism of stem cells? Biochem Biophys Res Commun 2002;294:1052–1063.
Newsome PN, Johannessen I, Boyle S et al. Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion. Gastroenterology 2003;124:1891–1900.
Oh DJ, Lee GM, Francis K et al. Phototoxicity of the fluorescent membrane dyes PKH2 and PKH26 on the human hematopoietic KG1a progenitor cell line. Cytometry 1999;36:312–318.
Eferl J, Sibilia M, Hilberg F et al. Functions of c-jun in liver and heart development. J Cell Biol 1999;145:1049–1061.
Wang X, Willenbring H, Akkari Y et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 2003;422:897–901.
Camargo FD, Finegold M, Goodell MA. Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners. J Clin Invest 2004;113:1266–1270.
Weimann JM, Johansson CB, Trejo A et al. Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant. Nat Cell Biol 2003;5:959–966.
Ogle BM, Butters KA, Plummer TB et al. Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo. FASEB J 2004;18:548–550.
Abraham EJ, Kodama S, Lin JC et al. Human pancreatic progenitor cell engraftment in immunocompetent mice. Am J Pathol 2004;164:817–830.(Marc-Alexander von Macha,)
b Center for Toxicology, Institute of Legal Medicine and Rudolf-Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany;
c Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital, Basel, Switzerland;
d Molecular Genetics Laboratory, University Children’s Hospital, University of Mainz, Mainz, Germany;
e Paul-Flechsig Institute, University of Leipzig, Leipzig, Germany
Key Words. Pancreas ? Islet of Langerhans ? Liver ? Stem cell
Correspondence: Marc-Alexander von Mach, M.D., II. Medical Department, Langenbeckstr. 1, 55131 Mainz, Germany. Telephone: 49-6131-174154; Fax: 49-6131-176605; e-mail: marcm@giftinfo.uni-mainz.de
ABSTRACT
During embryogenesis, progenitor cells of pancreas and liver emerge from neighboring areas of the gut endoderm . There is a large body of evidence suggesting that such progenitors with the potential to generate liver cells from pancreatic cells and vice versa may still exist in adult life . Using the model of mice with a knockout of the tyrosine catabolic enzyme fumarylacetoacetate hydrolase (FAH), which, without treatment, results in liver cirrhosis, Wang et al. succeeded in correction of liver function by transplantation of cell suspensions from adult pancreas of wild-type animals. With this unique repopulation assay, the authors clearly demonstrated that progenitor cells with the ability not only to replace FAH knockout cells in the liver but also to correct the liver function exist in the pancreas, although the exact nature of these cells remains unknown. Interestingly, cells from cultured pancreatic ducts were not able to rescue the failing liver as did the crude pancreatic cell suspension , indicating that the presumed hepatopancreatic stem cells reside in areas outside the pancreatic ducts.
Numerous studies have shown the transdifferentiation potential of pancreatic cells of rodents into hepatocytes in vivo. Some very rare cases of human pancreatic cancer with hepatoid phenotype indicated the existence of similar cells in human pancreas . Recently, progenitor cells have been described in rodent and human islets of Langerhans that express the neural stem cell marker nestin and the side-population phenotype marker ABCG2 . The side-population cells in bone marrow represent a particularly potent stem cell population . Interestingly, the human nestin-expressing islet-derived progenitor (NIP) cells were able to adopt a hepatic phenotype in vitro with expression of markers like alpha feto protein and the transcription factor XBP . In contrast to animal data, however, no in vivo studies have been published so far demonstrating transdifferentiation of human pancreatic cells into a hepatic phenotype. Recently, in vivo models for transplantation of human cord blood cells into severe combined immunodeficiency (SCID) mouse liver have been established . Using the model with direct injection of cells into the liver, we demonstrate in the present report that human cells from cultured pancreatic islets of Langerhans engraft into SCID mouse liver and form cells expressing human albumin in vivo.
MATERIALS AND METHODS
NIP cells expressed beside nestin also the side-population marker ABCG2 as well as SCF, c-Kit, and Thy-1, another potential marker for hepatic stem/progenitor cells (Fig. 1A). These cells were negative for expression of the transcription factor IPF-1 and insulin but also the specific marker for hematopoietic cells CD45 (Fig. 1B). Before transplantation, no albumin expression was found in cultured NIP cells (Fig. 1C). To exclude numerical or structural chromosomal aberration due to prolonged growth stimulation in vitro, a karyotyping was performed and revealed a normal 46, XX karyotype (Fig. 2).
Figure 1. (A): NIP cells not only express the neural stem cell marker nestin but also the side-population marker ABCG2, SCF, c-Kit, and the hepatic stem cell marker Thy-1. NIP cells did not express IPF-1 or insulin. The human housekeeping gene APRT was used as positive control for RT-PCR. (B): Lack of CD45 expression in NIP cells with positive signal in human cord blood cells. (C): Detection of human albumin in SCID mouse liver after transplantation of NIP cells. RT-PCR analysis shows the expression of human albumin mRNA in transplanted SCID mouse number 18. Human albumin is not expressed by cultured NIP cells. HepG2 cells were used as positive control. SCID mouse liver was used as negative control. The origin of all PCR products was confirmed by sequencing. Abbreviations: APRT, adenine phosphoribosyltransferase; NIP, nestin-expressing islet-derived progenitor; RT-PCR, reverse transcription–polymerase chain reaction; SCID, severe combined immunodeficiency.
Transplantation of 1.5 x 104 human NIP cells failed to result in detectable red fluorescent cells that became detectable only after transplantation of 1.5 x 105 cells in three of four animals (Table 1). We next evaluated the impact of time after transplantation on engraftment frequency and found similar results 3 and 12 weeks after transplantation. Cells expressing human albumin were found in liver sections of 8 out of 11 animals. The cells were well integrated into the liver tissue and were predominantly found adjacent to vascular structures (Fig. 3). Transplantation of NIP cells without prior tagging with PKH26 seemed to be more successful and resulted in detection of human albumin-positive cells in all four grafted animals. To analyze fusion as a possible mechanism underlying this phenomenon, immunohistochemistry studies were performed using mouse-specific and human-specific anti-albumin antibodies. In case of fusion, we expected both types of albumin to be expressed in the grafted cell. Using confocal microscopy, expression of human and mouse albumin was found in distinct cells, suggesting that NIP cells did adopt a hepatic phenotype by differentiation induced by surrounding liver tissue rather than fusion (Fig. 4). Additionally, RT-PCR was performed, demonstrating the presence of human albumin mRNA in 4 of 10 animals (Fig. 1C). In the four human albumin–positive livers, the human APRT was also amplified using RT-PCR, although the signal was less abundant than albumin (data not shown). No mononuclear cell infiltration associated with human albumin–positive cells and no neoplasm were observed 3 and 12 weeks after transplantation. In our model, the occurrence of human albumin–positive cells in general, however, was a rare event, with detection of one to three positive cells on every second slice.
Figure 3. Immunohistochemistry 3 weeks after transplantation of human islet-derived progenitor cells into SCID mouse liver using a monoclonal antibody specific for human albumin and diaminobenzidine (brown) for staining (A), nontransplanted SCID mouse liver as negative control (B), and human liver (C) as positive control (bar = 20 μm). Note the proximity of grafted cells to vascular structures.
Figure 4. Fluorescence-immunohistochemistry with human and mouse specific antibodies against albumin using confocal microscopy with 630-fold magnification. (A): One cell stained with antibodies against human albumin. (B): The same cell with additional 4',6'-diamidino-2-phenylindole staining for cell nuclei. (C):Albumin staining with antibodies against mouse albumin. (D): Digital overlay of human and mouse albumin staining showing no costaining for mouse albumin in the human albumin-positive cell.
DISCUSSION
Human islet-derived stem cells are capable of adopting a hepatic phenotype in a SCID mouse liver in vivo, suggesting the presence of a hepatopancreatic stem/progenitor cell within or adjacent to the islets of Langerhans. The mechanism underlying this phenomenon seems to be trans-differentiation, although fusion with host hepatocyte cannot be completely ruled out. In the context of these recent findings, one could envision new therapeutic avenues for the treatment of liver cirrhosis using human pancreatic stem/ progenitor cells in which such cells could be isolated from pancreatic biopsies and expanded in vitro before trans plantation.
ACKNOWLEDGMENTS
Deutsch G, Jung J, Zheng M et al. A bipotential precursor population for pancreas and liver within the embryonic endoderm. Development 2001;128:871–881.
Grompe M. Pancreatic-hepatic switches in vivo. Mech Dev 2003;120:99–106.
Shen CN, Horb ME, Slack JM et al. Transdifferentiation of pancreas to liver. Mech Dev 2003;120:107–116.
Horb ME, Shen CN, Tosh D et al. Experimental conversion of liver to pancreas. Curr Biol 2003;13:105–115.
Zalzman M, Gupta S, Giri RK et al. Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc Natl Acad Sci U S A 2003;100:7253–7258.
Wang X, Al-Dhalimy M, Lagasse E et al. Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells. Am J Pathol 2001;158:571–579.
Paner GP, Thompson KS, Reyes CV. Hepatoid carcinoma of the pancreas. Cancer 2000;88:1582–1589.
Zulewski H, Abraham EJ, Gerlach MJ et al. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001;50:521–533.
Lechner A, Leech CA, Abraham EJ 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. Biochem Biophys Res Commun 2002;293: 670–674.
Zhou S, Schuetz JD, Bunting KD et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028–1034.
Beerheide W, von Mach MA, Ringel M et al. Downregulation of beta2-microglobulin in human cord blood stem cells after transplantation into livers of SCID-mice: an escape mechanism of stem cells? Biochem Biophys Res Commun 2002;294:1052–1063.
Newsome PN, Johannessen I, Boyle S et al. Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion. Gastroenterology 2003;124:1891–1900.
Oh DJ, Lee GM, Francis K et al. Phototoxicity of the fluorescent membrane dyes PKH2 and PKH26 on the human hematopoietic KG1a progenitor cell line. Cytometry 1999;36:312–318.
Eferl J, Sibilia M, Hilberg F et al. Functions of c-jun in liver and heart development. J Cell Biol 1999;145:1049–1061.
Wang X, Willenbring H, Akkari Y et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 2003;422:897–901.
Camargo FD, Finegold M, Goodell MA. Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners. J Clin Invest 2004;113:1266–1270.
Weimann JM, Johansson CB, Trejo A et al. Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant. Nat Cell Biol 2003;5:959–966.
Ogle BM, Butters KA, Plummer TB et al. Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo. FASEB J 2004;18:548–550.
Abraham EJ, Kodama S, Lin JC et al. Human pancreatic progenitor cell engraftment in immunocompetent mice. Am J Pathol 2004;164:817–830.(Marc-Alexander von Macha,)