标题: In Vitro Cultured Islet-Derived Progenitor Cells of Human Origin Express Human A [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:35 标题: In Vitro Cultured Islet-Derived Progenitor Cells of Human Origin Express Human A
a II. Medical Department, University of Mainz, Mainz, Germany; 5 w6 w8 p! M, g: l! S ( v/ f+ h% K) U7 {! ]b Center for Toxicology, Institute of Legal Medicine and Rudolf-Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany;% e8 a; o) v" C- |. D7 H
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c Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital, Basel, Switzerland;, U. U9 o0 m9 m2 y% ]7 M" L3 t
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d Molecular Genetics Laboratory, University Children’s Hospital, University of Mainz, Mainz, Germany;, f: ?: L: ~/ H
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e Paul-Flechsig Institute, University of Leipzig, Leipzig, Germany ! q+ O. W- h: R $ T% t# p: f5 T% v, pKey Words. Pancreas ? Islet of Langerhans ? Liver ? Stem cell3 U5 y' d0 q" _
" @: P" ^: T% E2 }/ fCorrespondence: 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$ {# D" g, x9 y7 I# g
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ABSTRACT4 ^8 N4 |. a1 j' X
$ \( e5 `: \- |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. ' A9 R7 x$ D9 p. n/ e9 K' f5 v8 k6 d- _* R% M. G+ R R. k
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.$ U1 @% n9 A0 u7 k" T) ~& e1 c9 ^' R
; Y" O+ p2 N3 ?9 I3 M6 wMATERIALS AND METHODS 8 q8 Z. K6 a+ D" R% b) l# ^0 L' r/ U7 s) H0 ?* C- c. g
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). G3 T; ]' Q: T8 g- n6 N
+ P* E% H2 j( CFigure 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.0 y& l& {8 ?4 l* U% @
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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. - Z p8 V& ^# u% _% K. ^ j/ |, `* c+ J) ^ m1 I% K
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.4 M K, I7 h5 b' S" A
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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. # K* U' k" Q) K k* g' n5 H% C$ S
DISCUSSION : q- W+ t1 l6 z. z' ]8 p & H% K+ g7 V& w+ N- C. NHuman 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.5 T7 s+ T$ D/ V% Z* c
5 D; l8 i8 p6 _+ xACKNOWLEDGMENTS1 T0 j- V/ w1 V
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