Human Cord Blood–Derived Cells Generate Insulin-Producing Cells In Vivo - 《stemcells》2004-2005文献干细胞之家



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江边孤钓

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发表于 2009-3-5 10:48 |只看该作者 |倒序浏览 |打印

a Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medicine, Fukuoka, Japan;
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b First Department of Internal Medicine, Ehime University School of Medicine, Toon, Japan;
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c Morphology Core, Kyushu University, Fukuoka, Japan;
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d Clinical Research Center, National Hospital Organization Nagasaki Medical Center, Ohmura, Japan;$ t1 E) Y: x, Q( h

e The Jackson Laboratory, Bar Harbor, Maine, USA% s, \# J: j2 F/ Y4 F8 ~7 D
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Key Words. Human cord blood ? Neonate ? Insulin ? Pancreas7 s2 K0 W; {9 `% j' c0 p
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Correspondence: Fumihiko Ishikawa, M.D., Ph.D., Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Telephone: 81-92-642-5230; Fax: 81-92-642-5247; e-mail: f_ishika@intmed1.med.kyushu-u.ac.jp; and Leonard D. Shultz, Ph.D., The Jackson Laboratory, Bar Harbor, Maine 04609, USA. Telephone: 207-288-6405; Fax: 207-288-6079; e-mail: lenny.shultz@jax.org& [! ?" H2 ^2 _2 Z$ ^1 A+ m  P6 n6 z

ABSTRACT1 f$ Q9 o5 k# M  R" J' p
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The regeneration of pancreatic beta cells from progenitor cells has long been awaited for treatment of insulin-dependent and noninsulin-dependent diabetes mellitus . The impaired quality of life associated with daily infusion of insulin throughout life and the potentially lethal complications due to macro- and micro-angiopathy requires novel treatment modalities for diabetes . Pancreas and kidney organ transplantations have been performed for patients with diabetes-induced renal insufficiency . For less invasive surgical modalities, pancreatic islet transplantation has been developed . Edmonton protocols, which were characterized by the infusion of multiple, fresh donor islets followed by the host immune suppression with nonsteroidal regimens, have been reported to improve long-term graft acceptance . In both whole pancreas and islet transplantations, however, the lack of donor tissues has yet to be resolved.

To reduce the need for such organ transplantations, investigators have been trying to identify stem/progenitor cells that can physiologically generate insulin in response to glucose. Embryonic stem cells (ESCs) have been studied based on their multipotential capacity. It has been reported that insulin-producing cells could differentiate from murine  and human  ESCs in vitro. The regenerative property of ESCs has been further evidenced by the results that transplantation of ESC-derived cells normalized or ameliorated elevated blood glucose levels in diabetic mice . However, the analytical methods for in vitro production of insulin-producing cells from ESCs have recently been questioned , and the risk of forming teratomas and ethical issues may limit the clinical use of ESCs and their derivatives at least at the present time .0 T9 A2 L: A& T6 ?
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Thus, we attempted to use postnatal cell sources of islet progenitor cells to foster de novo generation of insulin-producing cells. Since the end of the 20th century, bone marrow–derived cells have been reported to give rise to endodermal-origin cells  or even reconstitute diseased function in type-I tyrosinemia model mice . In the pancreatic tissue, several reports described the regeneration of bone marrow–derived pancreatic beta cells based on mouse syngeneic or allogeneic transplantation assay. Ianus et al.  first suggested the contribution of bone marrow–derived cells to generate insulin-producing cells. Hess et al.  further demonstrated the improvement of blood glucose levels following bone marrow transplantation using chemically induced diabetic mice. Although donor bone marrow–derived insulin-producing cells were present in the recipient mice, the authors suggested that the improved glucose levels in diabetic recipient mice were due to the regeneration of host-derived beta cells rather than that of donor bone marrow–derived insulin-producing cells as evidenced by increased numbers of BrdU-labeled green fluorescent protein (GFP)– insulin  cells, not GFP  insulin  cells at 4–7 days after transplantation. Ianus et al.  reported the donor (Ins2-Cre mice) bone marrow–derived insulin-producing cells in recipient (Rosa-lox-GFP mice) pancreatic tissue, which could likely be generated through a fusion-independent mechanism. On the other hand, bone marrow–derived stem cells contributed to the regeneration of other endodermal tissue–derived cells, such as hepatocytes, through cell fusion ., r3 |8 |* V0 y8 a

In the present study, we investigated the regenerative property of "human" hematopoietic tissue–derived cells and obtained insights into mechanisms underlying regeneration of insulin-producing cells. For this purpose, human cord blood (CB)–derived T cell–depleted mononuclear cells (MNCs) were transplanted into newborn nonobese diabetic/severe combined immunodeficient/?2-microglobulinnull (NOD/SCID/?2mnull) mice, which lacked mature T and B cells and showed extremely low activity of natural killer (NK) cells . The severe deficiency of adaptive and innate immunity in the NOD/SCID/?2mnull recipient mice prevents rejection of human progenitors and their progeny by the murine immune system. Newborn mice may provide an optimal environment for transplanted stem/progenitor cells to show their developmental plasticity. In this xenogeneic transplantation assay, we consistently identified the presence of human chromosome-containing insulin-producing cells in xenogeneic pancreatic tissue. Double fluorescence in situ hybridization (FISH) analyses using species-specific probes enabled us to determine that the mechanisms underlying donor CB–derived insulin-producing cells include both cell fusion-dependent and -independent pathways at equivalent levels. The in vivo production of human insulin-producing cells may encourage the future use of regenerative medicine in treatment of diabetes mellitus.( u1 {$ z4 z) d: o  u) y( D

MATERIALS AND METHODS
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Transplantation of Human CB Cells into Xenogeneic Hosts

Because stem cell plasticity or cell fusion occurs at a very low incidence, high levels of human chimerism in recipient mice are essential. For this purpose, we intravenously transplanted 107 human T cell–depleted CB MNCs into newborn NOD/SCID/?2mnull mice, which lacked mature T and B cells and showed an extremely low level of NK cell function. At 1–2 months post-transplantation, bone marrow cells were analyzed for the engraftment of human cells by flow cytometry. Engraftment levels of human CD45  cells were 56.8% ± 25.6% (n = 6) in recipient marrow. Both mature myeloid and lymphoid cells were present in bone marrow (Fig. 1). Successful human adaptive immunity after human CB engraftment should result in tolerance of developing human cells in pancreatic tissues in recipient mice.

Figure 1. Multilineage engraftment of human cells in mouse bone marrow. At 6 weeks after the transplantation of 107 T cell–depleted CB MNCs into NOD/SCID/?2mnull mice, bone marrow cells were analyzed for the presence of human CD33  myeloid cells (A), CD19  B-lineage cells (B), and CD3  T-lineage cells (C). The chimerism of human leukocytes was determined by the percentage of human CD45  cells. Abbreviations: CB, cord blood; FITC, fluorescein isothiocyanate; MNC, mononuclear cell; NOD/SCID/?2mnull, nonobese diabetic/severe combined immunodeficient/?2-microglobulinnull; PE, phycoerythrin.
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Generation of Human Insulin  Cells in Pancreatic Islet of Xenogeneic Hosts
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As we confirmed the reconstitution of the human hemato-lymphoid system in recipient bone marrow, we next evaluated the presence of human-derived cells in pancreatic tissues. To establish analytical methods for detecting human-specific insulin-producing cells in murine pancreatic tissue, we first distinguished insulin  cells and hematopoietic cells by dual immunostaining for insulin and CD45 on the same specimen. In lymph nodes, the vast majority of the cells expressed both human chromosomes and CD45, whereas none of the cells was positively stained for insulin (Fig. 2A). In contrast, more than 60% of the nucleated islet cells expressed insulin. CD45  hematopoietic cells observed in recipient pancreas did not express insulin (Fig. 2B).
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Figure 2. Discrimination of hematopoietic cells and pancreatic beta cells. (A): Lymph nodes of the engrafted NOD/SCID/?2mnull mice were stained with anti-insulin antibody (Cy5, white) and anti-CD45 antibody (FITC) after the FISH analysis, using a Spectrum Orange–conjugated human X-chromosome probe. (B): Pancreatic tissue of the engrafted NOD/SCID/?2mnull mice was stained with anti-insulin antibody (Cy5, white) and anti-CD45 antibody (FITC) after FISH analysis, using a Spectrum Orange–conjugated human X-chromosome probe. The presence of a human CD45  cell is shown (arrowhead). Bars = 20 μm. Abbreviations: FISH, fluorescence in situ hybridization; FITC, fluorescein isothiocyanate; NOD/SCID/?2mnull, nonobese diabetic/severe combined immunodeficient/?2-microglobulinnull.0 I2 o  K* d& ?( E- |
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Next, to detect human CB–derived insulin  cells, we performed FISH and immunofluorescence analyses on the same specimens. FISH analysis determined the origin of the cells using human centromere or X-chromosome probes, whereas the immunofluorescence analysis identified the cell type using anti-insulin antibody, which reacted with both human and murine insulin. A representative result of FISH and immunofluorescence analyses is shown in Figure 3. Human chromosome  insulin  cells were consistently identified in the islets of the recipient pancreatic tissues. Three-color staining for nuclei, human chromosomes, and insulin combined with differential interference contrast (DIC) imaging enabled us to analyze the incidence of human insulin  cells out of total insulin  cells. The incidence of human CB–derived insulin  cells was 0.65% ± 0.64% (n = 6) in our xenogeneic transplantation (Table 1). As we reported previously , we further obtained serial images from different depths of the specimens with laser-scanning confocal microscopy and performed three-dimensional analysis on the FISH signal to rule out the possibility of cell overlay. The X-Z image reconstructed from 10 serial X-Y images demonstrated that the chromosome signals were located inside the nucleus. Human chromosome  c-peptide  cells were detected in recipient islets at a similar frequency as human chromosome  insulin  cells (Figs. 3E, 3F).$ I& s/ {) V. x$ F

Figure 3. Human CB–derived insulin  cells in recipient pancreatic tissues. At 6 weeks post-transplantation, FISH and immunofluorescence studies were performed on the specimens derived from recipient pancreatic tissue. (A): Normarsky image of the specimen is shown. (B): The same specimen was subjected to FISH analysis, using a Cy-3–conjugated human centromere probe. (C): The pancreatic specimen was stained with anti-insulin antibody (FITC). (D): Images (A–C) merged. Nuclei of the islet cells were stained with DAPI. Bar = 20μm. (E): The specimen was subjected to FISH analysis, using a Spectrum Orange–conjugated human X-chromosome probe. Nuclei were stained with DAPI. (F): Staining for c-peptide (FITC), human X chromosome (Spectrum Orange), and nuclei (DAPI) is shown. Bar = 20 μm. Abbreviations: CB, cord blood; DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; FITC, fluorescein isothiocyanate.$ t, J: j7 [* E, c

Table 1. Incidence of human CB–derived insulin  cells in recipient islets

RT-PCR for Human Insulin

To confirm the generation of human insulin-producing cells from CB-derived cells at the RNA level, we performed RT-PCR for human and mouse insulin, using RNA derived from fresh or frozen pancreas of the recipient mice. Considering the homology between human and murine insulin cDNA, we designed the forward and reverse primers that specifically amplified human insulin cDNA, not murine insulin cDNA. The amplified products derived from the recipient pancreas were clearly seen on agarose gel (Fig. 4). The amplified human insulin products were further subjected to sequence analysis. Amplified products were completely matched with an already-known human insulin cDNA sequence. These results indicated that donor CB–derived human insulin was generated in the recipient pancreas at the RNA level and supported the results from FISH and immunofluorescence analyses., [6 d9 t5 c; a" u9 F& v9 ~
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Figure 4. Detection of human insulin RNA reverse transcription–polymerase chain reaction was performed using RNA derived from pancreatic tissue of two independent recipient mice (Rec1 and Rec2). Human insulin (A), mouse insulin (B), and mouse GAPDH (C) were amplified. The amplified products were detected at expected 55 bp (human insulin), at 127 bp (mouse insulin), and at 96 bp (mouse GAPDH). The products without reverse transcriptase (RTase) were used as negative controls. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.  i& P$ J4 y* T; z

The Mechanism Underlying Generation of Human Insulin  Cells in Xenogeneic Hosts) c' q$ C/ ^2 b

Recently, several investigators questioned "transdifferentiation" from hematopoietic tissue–derived stem cells into endodermal or ectodermal cells, and suggested the "cell fusion" between stem cells and mature cells as an alternative mechanism . To determine the mechanism underlying the generation of insulin-producing cells from human CB cells, we performed double FISH analysis using human and murine chromosome probes combined with immunostaining for insulin. Consequently, insulin  cells in pancreatic islets of the recipient mice were classified into human chromosome– murine chromosome  cells, human chromosome  murine chromosome  cells, and human chromosome  murine chromosome– cells. The vast majority of pancreatic beta cells were of mouse origin. Among human chromosome  insulin  cells in the five recipient mice tested, 47% of the cells did not possess murine chromosomes (Fig. 5), and 53% of the cells possessed murine chromosomes (Fig. 6). Almost equal proportions of human chromosome  murine chromosome– cells and human chromosome  murine chromosome  cells indicated that both potential differentiation and cell fusion could contribute to the generation of donor marker  insulin-producing cells after human CB cell transplantation into xenogeneic hosts.2 y+ S' V! F1 J' b# h

Figure 5. Possible differentiation from CB-derived cells to insulin-producing cells. Pancreatic tissues of recipient mice were subjected to double FISH analysis, using species-specific probes and immunofluorescence studies. (A): The majority of islet cells were positively stained with anti-insulin antibody (Cy-5, yellow). (B): Murine cells were labeled with a FITC-conjugated mouse centromere probe. (C): A human cell (arrowhead) was labeled with a Cy-3–conjugated human X-chromosome probe. (D): Merged image for double FISH analysis, immunostaining for insulin, and nuclear staining with DAPI is shown. The cell (arrowhead) was labeled with both a human X-chromosome probe and anti-insulin antibody, but not with mouse centromere probes. Bar = 20 μm. Abbreviations: CB, cord blood; DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; FITC, fluorescein isothiocyanate.

Figure 6. Cell fusion between human CB–derived cells and murine insulin  cells. Pancreatic tissues of recipient mice were subjected to double FISH analysis, using species-specific probes and immunofluorescence studies with anti-insulin antibody. (A): The majority of islet cells were positively stained with anti-insulin antibody (Cy-5, yellow). (B): Murine cells were labeled with a FITC-conjugated mouse centromere probe. (C): Human cells (arrow and arrowhead) were detected by FISH analysis, using a Cy-3–conjugated human X-chromosome probe. (D): Merged image for double FISH analysis, immunostaining for insulin, and nuclear staining with DAPI is shown. The cell (arrowhead) was labeled with both human X chromosome and mouse centromere probes, and positively stained with anti-insulin antibody. The other human chromosome  cell (arrow) was labeled with a human chromosome probe, not with a mouse centromere probe. Bar = 20 μm. Abbreviations: CB, cord blood; DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; FITC, fluorescein isothiocyanate.+ e3 h& m! l7 y7 V, o3 I6 x

DISCUSSION

This research was supported by the National Institutes of Health (A130389), a grant from the Juvenile Diabetes Research Foundation (JDRF), an NIH Diabetes Endocrinology Research Grant (DERG) grant DK52530, and the Ministry of Health, Labor, and Welfare in Japan. F.I. is a recipient of a fellowship and grant from the Japan Society for the Promotion of Science. We are grateful to Hiroshi Fujii for excellent technical assistance.4 q7 [7 ?/ s+ N* ?& t0 q

DISCLOSURES
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The authors indicate no potential conflicts of interest.
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