Stem Cells http://www.stemcellsportal.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Stem Cells Vol. 23 No. 9 October 2005, pp. 1409 -1416
doi:10.1634/stemcells.2005-0079; www.StemCells.com
© 2005 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoshida, S.
Right arrow Articles by Harada, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshida, S.
Right arrow Articles by Harada, M.

Human Cord Blood–Derived Cells Generate Insulin-Producing Cells In Vivo

Shuro Yoshidaa, Fumihiko Ishikawaa, Noriaki Kawanoa, Kazuya Shimodaa, Seiho Nagafuchia, Shinji Shimodaa, Masaki Yasukawab, Takaaki Kanemaruc, Hiromi Ishibashid, Leonard D. Shultze, Mine Haradaa

a Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medicine, Fukuoka, Japan;
b First Department of Internal Medicine, Ehime University School of Medicine, Toon, Japan;
c Morphology Core, Kyushu University, Fukuoka, Japan;
d Clinical Research Center, National Hospital Organization Nagasaki Medical Center, Ohmura, Japan;
e The Jackson Laboratory, Bar Harbor, Maine, USA

Key Words. Human cord blood • Neonate • Insulin • Pancreas

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{at}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{at}jax.org


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Here we report the capacity of human cord blood (CB)–derived cells to generate insulin-producing cells. To investigate in vivo capacity of human CB–derived cells, T cell–depleted mononuclear cells were intravenously transplanted into nonobese diabetic/severe combined immunodeficient/ß2-microglobulinnull mice within 48 hours of birth. At 1–2 months post-transplantation, immunofluorescence staining for insulin and fluorescence in situ hybridization (FISH) analysis using a human chromosome probe indicated that human CB–derived cells generated insulin-producing cells at a frequency of 0.65% ± 0.64% in xenogeneic hosts. Reverse transcription–polymerase chain reaction analysis confirmed the transcription of human insulin in the pancreatic tissue of the recipient mice. To clarify the mechanism underlying CB-derived insulin-producing cells, double FISH analysis using species-specific probes was performed. Almost equal proportions of human chromosome+ murine chromosome– insulin+ cells and human chromosome+ murine chromosome+ insulin+ cells were present in recipient pancreatic islets. Taken together, human CB contains progenitor cells, which can generate insulin-producing cells in recipient pancreatic tissues across a xenogeneic histocompatibility barrier by fusion-dependent and -independent mechanisms.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The regeneration of pancreatic beta cells from progenitor cells has long been awaited for treatment of insulin-dependent and noninsulin-dependent diabetes mellitus [1]. 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 [2]. Pancreas and kidney organ transplantations have been performed for patients with diabetes-induced renal insufficiency [3, 4]. For less invasive surgical modalities, pancreatic islet transplantation has been developed [5–7]. 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 [8]. 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 [9, 10] and human [11, 12] 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 [9, 13, 14]. However, the analytical methods for in vitro production of insulin-producing cells from ESCs have recently been questioned [15, 16], 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 [17].

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 [18, 19] or even reconstitute diseased function in type-I tyrosinemia model mice [20]. 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. [21] first suggested the contribution of bone marrow–derived cells to generate insulin-producing cells. Hess et al. [22] 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. [21] 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 [23–27].

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/ß2-microglobulinnull (NOD/SCID/ß2mnull) mice, which lacked mature T and B cells and showed extremely low activity of natural killer (NK) cells [28, 29]. The severe deficiency of adaptive and innate immunity in the NOD/SCID/ß2mnull 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.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
NOD/LtSz-Prkdcscid/PrkdcscidB2mnull (NOD/SCID/ß2mnull) mice were developed at the Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). Mice were bred and maintained under defined flora with irradiated food and acidified water at the animal facility of Kyushu University. All experiments were performed according to the guidelines established by the Institutional Animal Committee and Institutional Review Board of Kyushu University.

Transplantation
Human CB MNCs were purchased from Cambrex (Walkersville, MD, http://www.cambrex.com). CB MNCs were depleted of T cells, using mouse anti-human CD3 (BD Biosciences – Immunocytometry Systems, San Jose, CA, http://www.bdbiosciences.com/immunocytometry_systems), CD4, and CD8 antibodies (BD Biosciences Pharmingen, San Jose, CA, http://www.bdbiosciences.com/pharmingen) to prevent xenogeneic graft-versus-host reaction. Antibody-bound human T cells were depleted with sheep anti-mouse immunomagnetic beads (Dynal Biotech, Brown Deer, WI, http://www.dynalbiotech.com). 107 CD3–CD4–CD8– cells derived from CBMNCs were injected intravenously into newborn NOD/SCID/ß2mnull mice (within 2 days of birth) after conditioning with 100 cGy total body irradiation.

Flow Cytometric Analysis
Bone marrow cells were harvested from the femurs and tibias of recipient mice. To analyze the engraftment levels and lineage expression of human CB–derived cells, bone marrow cells were stained with phycoerythrin-conjugated mouse anti-human CD33, CD19, and CD3 monoclonal antibodies along with fluorescein isothiocyanate (FITC)–conjugated mouse anti-human CD45 monoclonal antibody (BD Biosciences – Immunocytometry Systems).

FISH and Immunofluorescence Analyses
After the pancreatic tissues were harvested from the recipient mice, the tissues were fixed with 3% paraformaldehyde (Sigma, St. Louis, http://www.sigmaaldrich.com) for 1 hour at room temperature. The tissues were embedded in paraffin after dehydration with graded ethyl alcohol. The 5-µm sections were subjected to FISH and immunofluorescence analyses.

For FISH analysis, paraffin-embedded samples were incubated with paraffin pretreatment solution (Vysis, Downers Grove, IL, http://www.vysis.com) at 85°C for 30 minutes and with protease at 37°C for 10 minutes. After DNA denaturation in 70% formamide at 75°C for 5 minutes, the specimens were incubated with species-specific probes for 14–18 hours at 37°C. A Spectrum Orange– or Spectrum Green–conjugated human X-chromosome probe (Vysis) or a Cy-3–conjugated human centromere probe (Cambio Ltd., Cambridge, U.K., http://www.cambio.co.uk) was used for detecting human cells, and a FITC-conjugated murine centromere probe (Cambio Ltd.) was used for detecting murine cells. To count total numbers of islet cells and to exclude erythrocyte autofluorescence, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com).

To perform immunofluorescence studies for beta cells, guinea pig anti-insulin antibody (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.dk) or rabbit anti-human c-peptide antibody (Linco Research, Inc., St. Charles, MO, http://www.lincoresearch.com) was used as primary antibody and visualized with FITC- or Cy-5–conjugated donkey anti-guinea pig IgG or donkey anti-rabbit IgG antibodies, respectively (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com). To distinguish hematopoietic cells from insulin-producing cells, anti-CD45 antibody (Dako-Cytomation) was used and visualized with FITC-conjugated donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc.). Anti-insulin and anti-human c-peptide antibodies reacted with both human and mouse epitopes. Stained specimens were observed with laser-scanning confocal microscopy (LSM510META; Carl Zeiss, Jena, Germany, http://www.zeiss.com) or light microscopy (Olympus, Tokyo, http://www.olympus-global.com). To remove autofluorescence in FISH and immunofluorescence analyses, only the signals with an emission wavelength specific for each fluorescence were identified as positive by confocal microscopy (Spectrum Green or FITC: 515–540 nm, Spectrum Orange: 575–590 nm, Cy-3: 585–600 nm). To determine the localization of hybridization signals, serial X-Y images were obtained from different depths of a specimen. The incidence of human CB–derived insulin+ cells was defined as the number of human chromosome+ insulin+ cells out of total number of insulin+ cells in pancreatic islets.

Reverse Transcription–Polymerase Chain Reaction
RNA was isolated from fresh or frozen pancreatic tissues of the recipient mice, using Isogen (Nippon Gene, Tokyo, http://www.nippongene.com). cDNA was prepared from pancreas-derived RNA, using oligo dT primer and reverse transcriptase (Takara, Otsu, Japan, http://www.takara.co.jp). The homology between human insulin and mouse insulin I or insulin II cDNA was 78% and 82% according to the complete cDNA sequences (human insulin cDNA GI 30582454; mouse insulin I cDNA GI 31982248; mouse insulin II cDNA GI 31982250). According to the sequence differences, we designed primers as follows: The forward primer for human insulin was designed as 5'-AAC ACC TGT GCG GCT CAC A-3'. The reverse primer for human insulin was designed as 5'-CGT TCC CCG CAC ACT AGG TA-3'. The forward primer for mouse insulin was designed as 5'-CCA CCC AGG CTT TTG TCA A-3'. The reverse primer for mouse insulin was designed as 5'-ACT TGT GGG TCC TCC ACT TCA-3'. The forward primer for mouse glyseraldehyde-3-phosphate dehydrogenase (GAPDH) was designed as 5'-GCA GTG GCA AAG TGG AGA TTG-3'. The reverse primer for mouse GAPDH was designed as 5'-ATT TGC CGT GAG TGG AGT CAT-3'. After initial denaturation at 50°C for 2 minutes and 95°C for 10 minutes, amplification was performed by 50 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The amplified product by reverse transcription–polymerase chain reaction (RT-PCR) was incorporated into pCR 2.1 TOPO vector (Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com) and further subjected to sequence analysis using an ABI Prism 310 (PerkinElmer, Wellesley, CA, http://www.perkinelmer.com).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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/ß2mnull 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. 1Go). Successful human adaptive immunity after human CB engraftment should result in tolerance of developing human cells in pancreatic tissues in recipient mice.



View larger version (26K):
[in this window]
[in a new window]
  		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/ß2mnull 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/ß2mnull, nonobese diabetic/severe combined immunodeficient/ß2-microglobulinnull; PE, phycoerythrin.

 
Generation of Human Insulin+ Cells in Pancreatic Islet of Xenogeneic Hosts
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. 2AGo). In contrast, more than 60% of the nucleated islet cells expressed insulin. CD45+ hematopoietic cells observed in recipient pancreas did not express insulin (Fig. 2BGo).



View larger version (75K):
[in this window]
[in a new window]
  		Figure 2. Discrimination of hematopoietic cells and pancreatic beta cells. (A): Lymph nodes of the engrafted NOD/SCID/ß2mnull 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/ß2mnull 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/ß2mnull, nonobese diabetic/severe combined immunodeficient/ß2-microglobulinnull.

 
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 3Go. 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 1Go). As we reported previously [30], 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, 3FGo).



View larger version (61K):
[in this window]
[in a new window]
  		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.

 

View this table:
[in this window]
[in a new window]
  		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. 4Go). 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.



View larger version (53K):
[in this window]
[in a new window]
  		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.

 
The Mechanism Underlying Generation of Human Insulin+ Cells in Xenogeneic Hosts
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 [23–27]. 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. 5Go), and 53% of the cells possessed murine chromosomes (Fig. 6Go). 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.



View larger version (93K):
[in this window]
[in a new window]
  		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.

 


View larger version (52K):
[in this window]
[in a new window]
  		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.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have demonstrated that insulin-producing cells are generated from human CB in vivo as evidenced by the presence of human insulin at the RNA level and human chromosome-containing insulin-positive cells in situ. The findings from double FISH analysis suggest that the generation of human CB–derived insulin-producing cells can be mediated through both fusion-dependent and -independent mechanisms.

Stem cell plasticity has been investigated in various organs, using rodent bone marrow cells [31]. A recent report demonstrated that human CB MNCs expressed pancreas-related genes such as nestin, neurogenin-3 (Ngn3), and paired box gene 4 (Pax4) at the RNA level [32]. The primary purpose of the present study was to determine whether "human" hematopoietic tissue–derived cells possessed the capacity to regenerate the insulin-producing cells. To examine the capacity of human CB cells, we developed a xeno-geneic transplantation assay using newborn NOD/SCID/ß2mnull mice [33, 34]. Extremely low levels of NK cell activity along with the lack of mature murine T cells and B cells of the specific strain supported the significantly higher levels of engraftment by human cells for a longer term in bone marrow [33, 35] compared with the previous assays using adult NOD/SCID mice [36]. The presence of human myeloid and lymphoid cells in recipient marrow indicated that transplanted CB stem/progenitor cells successfully homed to niches, as we previously showed [35]. We also presumed that human T cells that developed in recipient mice would be tolerant to the human CB–derived insulin-producing cells. In the engrafted mice, we succeeded in detecting human CB–derived insulin-producing cells in situ and human insulin transcript at the RNA level by RT-PCR. Four-color staining and three-dimensional analysis with laser-scanning confocal microscopy [30] circumvented the analytical problems of cell overlay [31]. Based on the presence of human chromosome-containing c-peptide+ cells and human insulin transcript in recipient pancreas, it is unlikely that human phagocytes such as macrophages or dendritic cells took up murine insulin secreted by host beta cells. The presence of c-peptide as well as insulin on human CB–derived cells implies that human insulin is generated through physiological degeneration of proinsulin. As a recent study showed the regeneration of host-derived beta cells after the transplantation of bone marrow cells [22], the regeneration of host-derived beta cells could be compared with that of donor CB–derived insulin-producing cells in future studies. The finding of human insulin transcript suggested that human CB included the progenitor cells capable of generating human insulin-producing cells at low levels, not just fostering the regeneration of host-derived beta cells. We identified cells dually labeled for human chromosomes and insulin, but these cells might be different from resident beta cells. To address this possibility, we compared the intensities of insulin staining between human chromosome+ cells and murine chromosome+ cells. Although the exact insulin content in the cytoplasm of individual cells could best be determined by electron microscopy, human CB–derived insulin+ cells possess similar levels of insulin compared with host beta cells as evaluated by laser scanning confocal microscopy.

Furthermore, we investigated the mechanisms underlying the generation of human CB–derived insulin-producing cells. Terada et al. [23] and Ying et al. [24] questioned the differentiation from bone marrow cells into nonhematopoietic cells by showing that the cell fusion between ESCs and hematopoietic cells or neural cells could occur in vitro. The cell fusion between bone marrow cells and hepatocytes, cardiomyocytes, or Purkinje neurons was further confirmed in vivo by Vassilopoulos et al. [25], Wang et al. [26], and Alvarez-Dolado et al. [27]. In contrast, in the pancreatic endocrine system, Ianus et al. [21] and Kodama et al. [37] did not find any evidence for cell fusion as a mechanism underlying the generation of bone marrow–derived insulin+ cells. Using the xenogeneic transplantation assay, we first clarified that human CB–derived cells gave rise to insulin-producing cells through both fusion-dependent and -independent mechanisms in vivo.

The capacity of hematopoietic tissue–derived cells to generate insulin-producing cells [21, 22, 37] has been questioned by recent reports [38–40]. In vitro studies demonstrated that bone marrow–derived cells recapitulated pancreatic lineage cells and synthesized varying degrees of insulin in response to glucose challenge [41–43]. In our study, the incidence of human CB–derived insulin+ cells was 0.65%. The incidence of bone marrow– or spleen–derived insulin+ cells in previous studies was higher using syngeneic or allogeneic transplantation models. The lower incidence of hematopoietic tissue–derived insulin+ cells in our study compared with their study could be explained by the xenogeneic histocompatibility barrier and by the difference of experimental designs, including strains of mice, transplanted cell sources and numbers, and observation time.

The identification of stem cell populations needs to be carried out in future studies. Kodama et al. [37] suggested the potency of CD45– nonhematopoietic cells to generate insulin+ cells, whereas Hess et al. [22] described the potency of c-kit+ hematopoietic progenitor cells to initiate regeneration of pancreatic beta cells. Although clonal studies by single-cell transplantation are difficult to achieve in a xenogeneic transplantation system, highly enriched populations such as CD34+CD38– hematopoietic stem cells or mesenchymal stem cells could be transplanted using our neonatal transplantation system to determine which bone marrow–derived stem cell population can generate insulin-producing cells in pancreatic tissues. For development of regenerative medicine for pancreatic beta cells, it needs to be elucidated whether the use of enriched stem cells can increase the incidence of hematopoietic tissue–derived insulin+ cells.

Altogether, our neonatal NOD/SCID/ß2mnull mouse assay provides a helpful tool for examining the multipotential capacity of human cells, and the findings would encourage the further investigation in regeneration of insulin-producing cells.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.

DISCLOSURES
The authors indicate no potential conflicts of interest.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zimmet P, Alberti KG, Shaw J. Global and societal implications of the diabetes epidemic. Nature 2001;414:782–787.[CrossRef][Medline]

  2. Hussain MA, Theise ND. Stem-cell therapy for diabetes mellitus. Lancet 2004;364:203–205.[CrossRef][Medline]

  3. Kelly WD, Lillehei RC, Merkel FK et al. Allotransplantation of the pancreas and duodenum along with the kidney in diabetic nephropathy. Surgery 1967;61:827–837.[Medline]

  4. Hakim NS. Pancreatic transplantation for patients with type 1 diabetes. Transplant Proc 2003;35:2801–2802.[CrossRef][Medline]

  5. Moskalewski S. Isolation and Culture of the Islets of Langerhans of the Guinea Pig. Gen Comp Endocrinol 1965;44:342–353.[CrossRef][Medline]

  6. Lacy PE, Kostianovsky M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 1967;16:35–39.[Medline]

  7. Weir GC, Bonner-Weir S. Scientific and political impediments to successful islet transplantation. Diabetes 1997;46:1247–1256.[Abstract]

  8. 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.[Abstract/Free Full Text]

  9. 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.[Abstract]

  10. Lumelsky N, Blondel O, Laeng P et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001;292:1389–1394.[Abstract/Free Full Text]

  11. Assady S, Maor G, Amit M et al. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–1697.[Abstract/Free Full Text]

  12. Segev H, Fishman B, Ziskind A et al. Differentiation of human embryonic stem cells into insulin-producing clusters. STEM CELLS 2004;22:265–274.[Abstract/Free Full Text]

  13. Hori Y, Rulifson IC, Tsai BC et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. Proc Natl Acad Sci U S A 2002;99:16105–16110.[Abstract/Free Full Text]

  14. Blyszczuk P, Czyz J, Kania G et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. Proc Natl Acad Sci U S A 2003;100:998–1003.[Abstract/Free Full Text]

  15. Rajagopal J, Anderson WJ, Kume S et al. Insulin staining of ES cell progeny from insulin uptake. Science 2003;299:363.[Free Full Text]

  16. Hansson M, Tonning A, Frandsen U et al. Artifactual insulin release from differentiated embryonic stem cells. Diabetes 2004;53:2603–2609.[Abstract/Free Full Text]

  17. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.[Abstract/Free Full Text]

  18. Petersen BE, Bowen WC, Patrene KD et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168–1170.[Abstract/Free Full Text]

  19. 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.[CrossRef][Medline]

  20. 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.[CrossRef][Medline]

  21. 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.[CrossRef][Medline]

  22. Hess D, Li L, Martin M et al. Bone marrow-derived stem cells initiate pancreatic regeneration. Nat Biotechnol 2003;21:763–770.[CrossRef][Medline]

  23. Terada N, Hamazaki T, Oka M et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542–545.[CrossRef][Medline]

  24. Ying QL, Nichols J, Evans EP et al. Changing potency by spontaneous fusion. Nature 2002;416:545–548.[CrossRef][Medline]

  25. Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901–904.[CrossRef][Medline]

  26. Wang X, Willenbring H, Akkari Y et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 2003;422:897–901.[CrossRef][Medline]

  27. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 2003;425:968–973.[CrossRef][Medline]

  28. Christianson SW, Greiner DL, Hesselton RA et al. Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice. J Immunol 1997;158:3578–3586.[Abstract]

  29. Greiner DL, Hesselton RA, Shultz LD. SCID mouse models of human stem cell engraftment. STEM CELLS 1998;16:166–177.[Abstract/Free Full Text]

  30. Ishikawa F, Yasukawa M, Yoshida S et al. Human cord blood- and bone marrow-derived CD34+ cells regenerate gastrointestinal epithelial cells. FASEB J 2004;18:1958–1960.[Abstract/Free Full Text]

  31. Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood 2003;102:3483–3493.[Abstract/Free Full Text]

  32. Pessina A, Eletti B, Croera C et al. Pancreas developing markers expressed on human mononucleated umbilical cord blood cells. Biochem Biophys Res Commun 2004;323:315–322.[CrossRef][Medline]

  33. Ishikawa F, Livingston AG, Wingard JR et al. An assay for long-term engrafting human hematopoietic cells based on newborn NOD/SCID/beta2-microglobulin (null) mice. Exp Hematol 2002;30:488–494.[CrossRef][Medline]

  34. Ishikawa F, Drake CJ, Yang S et al. Transplanted human cord blood cells give rise to hepatocytes in engrafted mice. Ann N Y Acad Sci 2003;996:174–185.[Abstract/Free Full Text]

  35. Ishikawa F, Livingston AG, Minamiguchi H et al. Human cord blood long-term engrafting cells are CD34+CD38–. Leukemia 2003;17:960–964.[CrossRef][Medline]

  36. Hesselton RM, Greiner DL, Mordes JP et al. High levels of human peripheral blood mononuclear cell engraftment and enhanced susceptibility to human immunodeficiency virus type 1 infection in NOD/LtSz-scid/scid mice. J Infect Dis 1995;172:974–982.[Medline]

  37. Kodama S, Kuhtreiber W, Fujimura S et al. Islet regeneration during the reversal of autoimmune diabetes in NOD mice. Science 2003;302:1223–1227.[Abstract/Free Full Text]

  38. Choi JB, Uchino H, Azuma K et al. Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells. Diabetologia 2003;46:1366–1374.[CrossRef][Medline]

  39. Lechner A, Yang YG, Blacken RA et al. No evidence for significant trans-differentiation of bone marrow into pancreatic beta-cells in vivo. Diabetes 2004;53:616–623.[Abstract/Free Full Text]

  40. 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.[Abstract/Free Full Text]

  41. D’Ippolito G, Diabira S, Howard GA et al. Marrow-isolated adult multi-lineage 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.[Abstract/Free Full Text]

  42. Oh SH, Muzzonigro TM, Bae SH et al. Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes. Lab Invest 2004;84:607–617.[CrossRef][Medline]

  43. Tang DQ, Cao LZ, Burkhardt BR et al. In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes 2004;53:1721–1732.[Abstract/Free Full Text]

Received February 24, 2005; accepted for publication May 3, 2005.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoshida, S.
Right arrow Articles by Harada, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshida, S.
Right arrow Articles by Harada, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
http://www.epitomics.com