Evidence Of Cell Fusion Research Articles

Long-Term Fate of Human Fetal Liver Progenitor Cells Transplanted in Injured Mouse Livers.

Liver progenitor cells have the potential to repair and regenerate a diseased liver. The success of any translational efforts, however, hinges on thorough understanding of the fate of these cells after transplant, especially in terms of long-term safety and efficacy. Here, we report transplantation of a liver progenitor population isolated from human fetal livers into immune-permissive mice with follow-up up to 36 weeks after transplant. We found that human progenitor cells engraft and differentiate into functional human hepatocytes in the mouse, producing albumin, alpha-1-antitrypsin, and glycogen. They create tight junctions with mouse hepatocytes, with no evidence of cell fusion. Interestingly, they also differentiate into functional endothelial cell and bile duct cells. Transplantation of progenitor cells abrogated carbon tetrachloride-induced fibrosis in recipient mice, with downregulation of procollagen and anti-smooth muscle actin. Paradoxically, the degree of engraftment of human hepatocytes correlated negatively with the anti-fibrotic effect. Progenitor cell expansion was most prominent in cirrhotic animals, and correlated with transcript levels of pro-fibrotic genes. Animals that had resolution of fibrosis had quiescent native progenitor cells in their livers. No evidence of neoplasia was observed, even up to 9 months after transplantation. Human fetal liver progenitor cells successfully attenuate liver fibrosis in mice. They are activated in the setting of liver injury, but become quiescent when injury resolves, mimicking the behavior of de novo progenitor cells. Our data suggest that liver progenitor cells transplanted into injured livers maintain a functional role in the repair and regeneration of the liver. Stem Cells 2018;36:103-113.

Cellular Origins of Regenerating Nodules and Malignancy in the FAH Model of Liver Injury after Bone Marrow Cell Transplantation

In previous reports, we and other groups have shown that proliferating hepatocytes are formed by the fusion of donor hematopoietic cells with host hepatocytes in the Fah−/− model. Thus, it would be interesting to determine whether cell fusion occurs during malignancy. However, it is difficult to demonstrate such processes using this model. Therefore, we established a new strain to study the processes of regenerating nodules and malignancy and their origins. The FAH−/− mouse model was crossed with the ROSAnZ strain and their offspring was genotyped for FAH−/− and ROSAnZ mutations to create a new strain (Fah−/−-ROSAnZ). Using this strain as recipients, we performed bone marrow transplantation experiments. As a result, we could not demonstrate the presence of any epithelial cells except hepatocytes that were of donor origin in regenerating tissue, and no evidence of cell fusion was found in tumors. The hepatic malignancy was of host origin in these mice. There was higher expression of extracellular matrix proteins and more inflammatory cells in liver tumor nodules than in regenerating normal liver nodules. Hepatocytes generated by fusion with bone marrow cells did not form malignant tumors. Extracellular matrix and inflammatory cells had significantly accumulated in liver tumors.

250. The Cellular Origins of Regenerating Nodules and Malignant Formation in the FAH Model of Liver Injury After Bone Marrow Transplantation

Recent reports have described the ability of donor-derived cells to form unexpected tissue types after transplantation. Exciting results were found in a mouse strain with mutations in the fumarylacetoacetate hydrolase gene (Fah-/-) by transplantation with hematopoietic cells. In the previous reports, we and other groups have shown that the proliferating hepatocytes are formed by the fusion of donor hematopoietic cells with host hepatocytes. To address how proliferated pattern of regenerating nodules and malignant formation and their origin, we created a transgenic mouse strain (ROSAnZ) that expresses a gene encoding nuclear-localized β-galactosidase from the ROSA26 promoter and used this strain to cross FAH strains to generated a new strain Fah-/- – ROSAnZ. Our results revealed both of β-Gal+ hepatocyte nuclei from host and donor-derived hepatocytes expressed Fah or GFP in their cytoplasm from donor were observed in regenerating nodules in Fah-/- – ROSAnZ after bone marrow transplantation.We showed that only hepatocytes were donor-derived, as we did not detect the presence of the other cell types present in regenerating liver nodules. We also demonstrated that hepatocytes created by fusion with bone marrow cells did not form malignant tumors, and the hepatic malignancies found in these mice were host origins and lacking in evidence of cell fusion in tumor formation in transplanted mice. In addition, we demonstrated that mesenchymal stem cells were recruited to the tumor and inflammatory cell infiltration was involved in forming malignant tumors and promoting tumor growth.

Evidence of cell fusion in carcinogen-induced mice gastric carcinoma.

The role of bone marrow-derived cells in gastric cancer formation was not fully understood. In this study, bone marrow from female green fluorescent protein transgenic mice was transplanted into male wild-type mice to generate sex-mismatched chimeric mice. The chimeric mice were treated with carcinogen to induce gastric cancer. At time of sacrifice, 18.2 % (2/11) of mice showed severe dysplasia and 25 % (3/12) of mice successfully induced with cancer. Fluorescence in situ hybridization results showed that bone marrow-derived cells participated in renewal of gastric mucosa and cell fusion was observed in both precancerous lesions and adenocarcinoma, but no sign of fusion was observed in squamous cell carcinoma. Our findings suggest that bone marrow-derived cells participate in renewal of gastric mucosa during chronic damage and might have acquired the phenotype of gastric epithelial cells through cell fusion. Fusion between gastric epithelial cells and bone marrow-derived cells was involved in increased carcinogenesis.

Rat-Derived Amniotic Epithelial Cells Differentiate into Mature Hepatocytes In Vivo with No Evidence of Cell Fusion

Amniotic epithelial cells (AEC) derived from human placenta represent a useful and noncontroversial source for liver-based regenerative medicine. Previous studies suggested that human- and rat-derived AEC differentiate into hepatocyte-like cells upon transplantation. In the retrorsine (RS) model of liver repopulation, clusters of donor-derived cells engrafted in the recipient liver and, importantly, showed characteristics of mature hepatocytes. The aim of the current study was to investigate the possible involvement of cell fusion in the emergence of hepatocyte clusters displaying a donor-specific phenotype. To this end, 4-week-old GFP+/DPP-IV− rats were treated with RS and then transplanted with undifferentiated AEC isolated from the placenta of DPP-IV+ pregnant rats at 16–19 days of gestational age. Results indicated that clusters of donor-derived cells were dipeptidyl peptidase type IV (DPP-IV) positive, but did not express the green fluorescent protein (GFP), suggesting that rat amniotic epithelial cells (rAEC) did not fuse within the host parenchyma, as no colocalization of the two tags was observed. Moreover, rAEC-derived clusters expressed markers of mature hepatocytes (eg, albumin, cytochrome P450), but were negative for the expression of biliary/progenitor markers (eg, epithelial cell adhesion molecule [EpCAM]) and did not express the marker of preneoplastic hepatic nodules glutathione S-transferase P (GST-P). These results extend our previous findings on the potential of AEC to differentiate into mature hepatocytes and suggest that this process can occur in the absence of cell fusion with host-derived cells. These studies support the hypothesis that amnion-derived epithelial cells can be an effective cell source for the correction of liver disease.

Amniotic fluid-derived stem cells demonstrated cardiogenic potential in indirect co-culture with human cardiac cells.

Amniotic fluid-derived stem cells (AFSC) have been shown to be broadly multipotent and non-tumorogenic. Previous studies of direct mixing of AFSC and neonatal rat ventricle myocytes indicated evidence of AFSC cardiogenesis. In this study, we examined human AFSC cardiogenic potential in indirect co-culture with human cardiac cells in conditions that eliminated the possibility of cell fusion. Human AFSC in contact with human cardiac cells showed expression of cardiac troponin T (cTnT) in immunohistochemistry, and no evidence of cell fusion was found through fluorescent in situ hybridization. When indirectly co-cultured with cardiac cells, human AFSC in contact with cardiac cells across a thin porous membrane showed a statistically significant increase in cTnT expression compared to non-contact conditions but lacked upregulation of calcium modulating proteins and did not have functional or morphological characteristics of mature cardiomyocytes. This suggests that contact is a necessary but not sufficient condition for AFSC cardiac differentiation in co-culture with cardiac cells.

Bone Marrow Cells in Murine Colitis: Multi-Signal Analysis Confirms Pericryptal Myofibroblast Engraftment without Epithelial Involvement

BackgroundThe contribution of bone marrow-derived cells to epithelial tissues in the inflamed gut remains controversial. Recent reports have suggested that cell fusion between bone marrow-derived cells and the intestinal epithelium takes place in inflammatory conditions.MethodsIn attempts to confirm this, we have undertaken gender mis-matched bone marrow (BM) transplants from male Swiss Webster (SWR) mice to B and T cell-deficient female Rag2 KO mice which, 4 weeks later, were given 5% dextran sodium sulphate in drinking water to induce acute colitis. A further BM-treated group of animals with a graft versus host-like condition was also studied. We developed a new method to combine up to three brightfield or fluorescent lectin- or immuno-histochemical signals with fluorescent in situ hybridisation for the Y and X chromosomes to enable us unequivocally to identify BM-derived male cells which presented as different cell types in the gastrointestinal tract.Principal FindingsIn rolled preparations of whole intestines we scanned around 1.5 million crypts at many tissue levels. In no instance did we see a Y chromosome-positive cell in the epithelial compartment, which was not also CD45-positive. We saw no evidence of cell fusion, based on combined X and Y chromosome analysis. Levels of CD45-positive stromal and lymphoid cells and pericryptal myfibroblasts (positive for α-smooth muscle actin) increased with time up to a plateau, which resembled the level seen in untreated control grafted animals. We saw very few Y chromosome-positive endothelial cells in intestinal stromal vessels.ConclusionsWe conclude that whole BM transplantation does not result in intestinal epithelial engraftment in this model. Our new methods can usefully assist in multi-signal analyses of cell phenotypes following BM transplant and in models of chimaerism and regenerative medicine.

Tales from the Crypt: Mesenchymal Stem Cells for Replenishing the Intestinal Stem Cell Pool

Studies have shown that Mesenchymal Stem Cells (MSC) contribute to replenishing the radiation-depleted intestinal stem cell (ISC) compartment after abdominal/pelvic radiotherapy. Coupling these findings with other encouraging results showing that MSC promotes healing in inflammatory bowel disease suggests that MSC may represent an attractive source for regeneration of gastrointestinal tissue. Nevertheless, controversy regarding the true contribution of MSC to the cellular pool of an injured organ versus promotion of healing through production of growth factors/cytokines raised questions regarding the true potential of these cells. Since MSC are known to be heterogeneous and some populations may represent tissue-specific stem cells or cells which have already committed to differentiation to a specific phenotype, it is important to find a means of identifying and enriching for subpopulations that are best suited for intestinal cell therapy. Thus, we examined the in vivo ability of clonally-derived MSC populations to contribute to the stem or mature epithelial pool of the intestine, and correlated these results with genomic analysis in an attempt to find a specific marker that enriches for such an MSC population. BM-derived Stro-1+ MSC clones were established by single cell deposition. Phenotypic characterization of the various clones showed that, despite many similarities, there was phenotypic variance between clones for some commonly expressed stem cell proteins, such as CD44 and c-kit. 6 of the clonally-derived MSC were transplanted into 55–60 day-old fetal sheep at a concentration of 106cells/fetus, and evaluated the recipient intestine at 2.5–3 months post-transplant for the presence of human organ-specific cells using fluorescent in situ hybridization and immunofluorescence. We found that MSC localized preferentially to the mucosal layer, distributed equally between the crypts and the villi, with no evidence of cell fusion. While 4 of the 6 clones had a similar level of intestinal engraftment of 1.5±0.4%, 2 of the clones exhibited enhanced engraftment of 5±0.65%. Dual staining with antibodies to cytokeratin and the ISC marker Musashi showed a direct correlation between the levels of donor contribution to the ISC pool and the levels of mature enterocytes within the villi, such that 1–4% of the Musashi+ cells within the crypt were donor-derived with the 4 clones exhibiting overall intestinal engraftment levels of 1.5%, whereas 12–14% of the total Musashi+ ISC population was donor-derived with the 2 clones exhibiting enhanced levels of intestinal engraftment. Since no correlation was found between the enhanced levels of engraftment and any of the phenotypic markers tested prior to transplant, transcriptome analysis using Affymetrix microarrays, and GeneSifter software was done in the hopes of identifying a marker that would allow selection of these cells. MSC did not express Mushashi, cytokeratin 2,18,19,20, or GPR49 prior to transplant, precluding their use for selection for transplant. MSC expressed calmodulin, caldesmon, B-catenin and cytovillin, proteins expressed by intestinal crypt cells. Unfortunately, the cytoplasmic location of these antigens prevents their use for selection. Nevertheless, EphrinB-2, a receptor expressed at high levels on ISC, was also found to be expressed on MSC. Studies are underway to confirm whether levels of EphrinB-2 correlate with the differentiation potential of the clones. Our studies demonstrate that MSC subsets have the ability to repopulate the ISC pool and generate differentiated intestinal cells and that combining in vivo functionality studies with genomic analysis may lead to identification of markers for enrichment of MSC that exhibit desired traits upon transplantation.

Evidence for cell fusion is absent in vascular lesions associated with pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a fatal disease associated with severe remodeling of the large and small pulmonary arteries. Increased accumulation of inflammatory cells and apoptosis-resistant cells are contributing factors. Proliferative apoptosis-resistant cells expressing CD133 are increased in the circulation of PAH patients. Circulating cells can contribute to tissue repair via cell fusion and heterokaryon formation. We therefore hypothesized that in the presence of increased leukocytes and CD133-positive (CD133(pos)) cells in PAH lung tissue, cell fusion and resulting genomic instability could account for abnormal cell proliferation and the genesis of vascular lesions. We performed analyses of CD45/CD133 localization, cell fusion, and proliferation during late-stage PAH in human lung tissue from control subjects and subjects with idiopathic (IPAH) and familial (FPAH) PAH. Localization, proliferation, and quantitation of cell populations in individual patients were performed by immunolocalization. The occurrence of cellular fusion in vascular lesions was analyzed in lung tissue by fluorescence in situ hybridization. We found the accumulation of CD45(pos) leukocytic cells in the tissue parenchyma and perivascular regions in PAH patients and less frequently observed myeloid cells (CD45/CD11b). CD133(pos) cells were detected in occlusive lesions and perivascular areas in those with PAH and were more numerous in those with IPAH lesions than in FPAH lesions. Cells coexpressing CD133 and smooth muscle alpha-actin were occasionally observed in occlusive lesions and perivascular areas. Proliferating cells were more prominent in IPAH lesions and colocalized with CD45 or CD133. We found no evidence of increased ploidy to suggest cell fusion. Taken together, these data suggest that abnormal lesion formation in PAH occurs in the absence of cell fusion.

Distribution of Single-Cell Expanded Marrow Derived Progenitors in a Developing Mouse Model of Osteogenesis Imperfecta Following Systemic Transplantation

We evaluated single-cell-expanded, marrow-derived progenitors for engraftment in a developing mouse model of osteogenesis imperfecta (OI) following systemic transplantation. The present study was initiated to evaluate the potential of mesenchymal stem cells to treat OI. Single-cell-derived progenitors were prepared from marrow stromal cells harvested from normal mice. Selected single-cell-expanded progenitors marked with green fluorescent protein were injected into the neonatal mouse model of OI, and the recipient mice were sacrificed at 2 and 4 weeks following cell transplantation. Examination of the tissues harvested from recipient mice at 2 and 4 weeks after cell transplantation demonstrated that the cells extravasated and engrafted in most of the bones as well as other tissues. Tissue sections made from the tibias and femurs of a selected recipient mouse showed that the cells were distributed in bone marrow, trabecular, and cortical bone as demonstrated by histology and confocal microscopy. The cells that engrafted in the bones of the recipient mouse synthesized and deposited type I collagen composed of alpha1(I) and alpha2(I) collagen heterotrimers. Genotyping and gene expression analysis of the cells retrieved from the bones of the recipient mouse at 2 and 4 weeks demonstrated that the cells expressed osteoblast-specific genes, suggesting that the donor cells differentiated into osteoblasts in vivo with no evidence of cell fusion. These data suggest that progenitors infused in developing mice will engraft in various tissues including bones, undergo differentiation, and deposit matrix and form bone in vivo. Disclosure of potential conflicts of interest is found at the end of this article.

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver

We established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP+ cells expressing ALB were detected in close proximity to "beating" myocytes after 7 days of EB cultures. GFP+ cells increased in number, acquired hepatocyte-like morphology and hepatocyte-specific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP+ cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl(4)-induced liver injury. The transplanted GFP+ cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy.

Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins

Despite extensive studies, the hematopoietic versus hepatic origin of liver progenitor oval cells remains controversial. The aim of this study was to determine the origin of such cells after liver injury and to establish an oval cell line. Rat liver injury was induced by subcutaneous insertion of 2-AAF pellets for 7 days with subsequent injection of CCl 4. Livers were removed 9 to 13 days post-CCl 4 treatment. Immunohistochemistry was performed using anti-c- kit, OV6, Thy1, CK19, AFP, vWF and Rab3b. Isolated non-parenchymal cells were grown on mouse embryonic fibroblast, and their gene expression profile was characterized by RT-PCR. We identified a subpopulation of OV6/CK19/Rab3b-expressing cells that was activated in the periportal region of traumatized livers. We also characterized a second subpopulation that expressed the HSCs marker c- kit but not Thy1. Although we successfully isolated both cell types, OV6/CK19/Rab3b + cells fail to propagate while c- kit +-HSCs appeared to proliferate for up to 7 weeks. Cells formed clusters which expressed c- kit, Thy1 and albumin. Our results indicate that a bona fide oval progenitor cell population resides within the liver and is distinct from c- kit +-HSCs. Oval cells require the hepatic niche to proliferate, while cells mobilized from the circulation proliferate and transdifferentiate into hepatocytes without evidence of cell fusion.

Mesenchymal Stem Cells Spontaneously Express Neural Proteins in Culture and Are Neurogenic after Transplantation

Reports of neural transdifferentiation of mesenchymal stem cells (MSCs) suggest the possibility that these cells may serve as a source for stem cell-based regenerative medicine to treat neurological disorders. However, some recent studies controvert previous reports of MSC neurogenecity. In the current study, we evaluate the neural differentiation potential of mouse bone marrow-derived MSCs. Surprisingly, we found that MSCs spontaneously express certain neuronal phenotype markers in culture, in the absence of specialized induction reagents. A previously published neural induction protocol that elevates cytoplasmic cyclic AMP does not upregulate neuron-specific protein expression significantly in MSCs but does significantly increase expression of the astrocyte-specific glial fibrillary acidic protein. Finally, when grafted into the lateral ventricles of neonatal mouse brain, MSCs migrate extensively and differentiate into olfactory bulb granule cells and periventricular astrocytes, without evidence of cell fusion. These results indicate that MSCs may be "primed" toward a neural fate by the constitutive expression of neuronal antigens and that they seem to respond with an appropriate neural pattern of differentiation when exposed to the environment of the developing brain.

Increase of Bone Marrow–Derived Secretory Lineage Epithelial Cells During Regeneration in the Human Intestine

We have previously reported that bone marrow (BM)-derived cells contribute to the regeneration of the human intestinal epithelium. To analyze further how these cells arise, proliferate, and differentiate as epithelial cells, histologic analysis was conducted using endoscopic specimens. Thirty biopsy specimens from 14 female, sex-mismatched BM-transplantation recipients were examined. BM-derived cells were identified by fluorescent in situ hybridization (FISH) for the Y chromosome and immunohistochemistry. Multicolor FISH was used to exclude cell fusion. These cells were further analyzed for various differentiation or proliferation markers. No evidence of cell fusion was detected. BM-derived cells did not distribute within the crypt as stem cells and rarely expressed Musashi-1. However, BM-derived epithelial cells frequently expressed Ki-67, and some of these cells appeared as pairs of adjacent cells. These cells also expressed markers of all 4 lineages of terminally differentiated cells. During regeneration following graft-vs-host disease, the number of BM-derived cells was substantially increased within Ki-67-positive cells. Interestingly, the number of cells expressing markers for secretory lineage cells was significantly increased within BM-derived cells. This change was unique for BM-derived cells, resulting in a significantly increased proportion of BM-derived cells among secretory lineage cells. BM-derived epithelial cells arise via a mechanism other than cell fusion and rarely give rise to stem cells. However, a small proportion of these cells express proliferation markers, and a majority reside as terminally differentiated cells. During regeneration BM-derived cells increase as secretory lineage cells, thereby contributing to restore epithelial functions.

Bone marrow cells engraft within the epidermis and proliferate in vivo with no evidence of cell fusion

In adults, bone marrow-derived cells (BMDC) can contribute to the structure of various non-haematopoietic tissues, including skin. However, the physiological importance of these cells is unclear. This study establishes that bone marrow-derived epidermal cells are proliferative and, moreover, demonstrates for the first time that BMDC can localize to a known stem cell niche: the CD34-positive bulge region of mouse hair follicles. In addition, engraftment of bone marrow cells into the epidermis is significantly increased in wounded skin, bone marrow-derived keratinocytes can form colonies in the regenerating epidermis in vivo, and the colony-forming capacity of these cells can be recapitulated in vitro. In some tissues this apparent plasticity is attributed to differentiation, and in others to cell fusion. Evidence is also provided that bone marrow cells form epidermal keratinocytes without undergoing cell fusion. These data suggest a functional role for bone marrow cells in epidermal regeneration, entering known epidermal stem cell niches without heterokaryon formation.

Rat adult stem cells (marrow stromal cells) engraft and differentiate in chick embryos without evidence of cell fusion.

Cell fusion was recently reported to account for the plasticity of adult stem cells in vivo. Adult stem cells, referred to as mesenchymal stem cells or marrow stromal cells, from rat marrow, were infused into 1.5- to 2-day-old chick embryos. After 4 days, the rat cells had expanded 1.3- to 33-fold in one-third of surviving embryos. The cells engrafted into many tissues, and no multinuclear cells were detected. The most common site of engraftment was the heart, apparently because the cells were infused just above the dorsal aorta. Some of the cells in the heart expressed cardiotin, and alpha-heavy-chain myosin. GFP(+) cells reisolated from the embryos had a rat karyotype. Therefore, the cells engrafted and partially differentiated without evidence of cell fusion.

Pancreatic Beta Cells – The Sweet Life

A review of: Kodama S, Kuhtreiber W, Fuimura S, Dale EA, Faustman DL 2003 Islet regeneration during the reversal of autoimmune diabetes in NOD mice. Science 320:1223–1227; and Ianus A, Holz GG, Theise ND, Hussain MA 2003 In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest 111:843–850

One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues.

Most recent evidence suggests that the process of tissue repair is driven by stem-like cells that reside in multiple tissues but are replenished by precursor cells from bone marrow. Among the candidates for the reparative cells are the adult stem cells from bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). We recently found that after MSCs were replated at very low densities to generate single-cell-derived colonies, they did not exit a prolonged lag period until they synthesized and secreted considerable quantities of Dickkopf-1, an inhibitor of the canonical Wnt signaling pathway. We also found that when the cells were cocultured with heat-shocked pulmonary epithelial cells, they differentiated into epithelial cells. Most of the MSCs differentiated without evidence of cell fusion but up to one-quarter underwent cell fusion with the epithelial cells. A few also underwent nuclear fusion. The results are consistent with the interesting possibility that MSCs and similar cells repair tissue injury by three different mechanisms: creation of a milieu that enhances regeneration of endogenous cells, transdifferentiation, and perhaps cell fusion.

Little evidence for cell fusion between recipient and Donor-Derived cells

Despite recent advances in immunosuppressive therapy, accelerated coronary atherosclerosis remains a major problem in the long-term survival of cardiac transplant recipients. However, the pathogenesis of the transplant-associated atherosclerosis remains largely unknown. Here, we investigated the origin of the vascular cells that contribute to graft vasculopathy. We performed heterotopic heart transplantation using genetically modified mice that express LacZ or green fluorescent protein (GFP) ubiquitously and constitutively. At 4 weeks after transplantation, the graft coronary arteries developed neointimal hyperplasia, expressing several smooth muscle cell markers. Most of the neointimal cells were composed of recipient cells but not graft medial smooth muscle cells. We seldom detected neointimal cells that were positive for both LacZ and GFP. When we transplanted wild-type cardiac allografts into the chimeric mice whose bone marrow cells had been replaced with those of LacZ-mice or GFP-mice, we observed that most of the neointimal cells were derived from the bone marrow. These findings suggest that recipient bone marrow-derived cells contribute to the pathogenesis of graft arteriosclerosis. Spontaneous cell fusion between recipient and donor-derived cells seems to be a rare event, if it occurs at all.

In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion.

Bone marrow harbors cells that have the capacity to differentiate into cells of nonhematopoietic tissues of neuronal, endothelial, epithelial, and muscular phenotype. Here we demonstrate that bone marrow-derived cells populate pancreatic islets of Langerhans. Bone marrow cells from male mice that express, using a CRE-LoxP system, an enhanced green fluorescent protein (EGFP) if the insulin gene is actively transcribed were transplanted into lethally irradiated recipient female mice. Four to six weeks after transplantation, recipient mice revealed Y chromosome and EGFP double-positive cells in their pancreatic islets. Neither bone marrow cells nor circulating peripheral blood nucleated cells of donor or recipient mice had any detectable EGFP. EGFP-positive cells purified from islets express insulin, glucose transporter 2 (GLUT2), and transcription factors typically found in pancreatic beta cells. Furthermore, in vitro these bone marrow-derived cells exhibit - as do pancreatic beta cells - glucose-dependent and incretin-enhanced insulin secretion. These results indicate that bone marrow harbors cells that have the capacity to differentiate into functionally competent pancreatic endocrine beta cells and that represent a source for cell-based treatment of diabetes mellitus. The results generated with the CRE-LoxP system also suggest that in vivo cell fusion is an unlikely explanation for the "transdifferentiation" of bone marrow-derived cells into differentiated cell phenotypes.