TEMPORARY REMOVAL: CD271 identifies functional human hepatic stellate cells, which localize in peri-sinusoidal and portal areas in liver after partial hepatectomy.

BACKGROUND AIMS: Hepatic stellate cells (HSCs) are liver-resident mesenchymal cells involved in essential processes in the liver. However, knowledge concerning these cells in human livers is limited because of the lack of a simple isolation method. METHODS: We isolated fetal and adult human liver cells by immunomagnetic beads coated with antibodies to a mesenchymal stromal cell marker (CD271) to enrich a population of HSCs. The cells were characterized by cell cultivation, immunocytochemistry, flow cytometry, reverse-transcription polymerase chain reaction and immunohistochemistry. Cells were injected into nude mice after partial hepatectomy to study in vivo localization of the cells. RESULTS: In vitro, CD271(+) cells were lipid-containing cells expressing several HSC markers: the glial fibrillary acidic protein, desmin, vimentin and α-smooth muscle actin but negative for CK8, albumin and hepatocyte antigen. The cells produced several inflammatory cytokines such as interleukin (IL)-6, IL-1A, IL-1B and IL-8 and matrix metalloproteinases MMP-1 and MMP-3 and inhibitors TIMP-1 and TIMP-2. In vivo, fetal CD271(+) cells were found in the peri-sinusoidal space and around portal vessels, whereas adult CD271(+) cells were found mainly in the portal connective tissue and in the walls of the portal vessels, which co-localized with α-smooth muscle actin or desmin. CD271(-) cells did not show this pattern of distribution in the liver parenchyma. CONCLUSIONS: The described protocol establishes a method for isolation of mesenchymal cell precursors for hepatic stellate cells, portal fibroblasts and vascular smooth muscle cells. These cells provide a novel culture system to study human hepatic fibrogenesis, gene expression and transcription factors controlling HSC regulation.

Phenotypic and in vivo functional characterization of immortalized human fetal liver cells.

We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.

Transplantation of an allogeneic vein bioengineered with autologous stem cells: a proof-of-concept study.

BACKGROUND: Extrahepatic portal vein obstruction can have severe health consequences. Variceal bleeding associated with this disorder causes upper gastrointestinal bleeding, leading to substantial morbidity and mortality. We report the clinical transplantation of a deceased donor iliac vein graft repopulated with recipient autologous stem cells in a patient with extrahepatic portal vein obstruction. METHODS: A 10 year old girl with extrahepatic portal vein obstruction was admitted to the Sahlgrenska University Hospital in Gothenburg, Sweden, for a bypass procedure between the superior mesenteric vein and the intrahepatic left portal vein (meso Rex bypass). A 9 cm segment of allogeneic donor iliac vein was decellularised and subsequently recellularised with endothelial and smooth muscle cells differentiated from stem cells obtained from the bone marrow of the recipient. This graft was used because the patient's umbilical vein was not suitable and other strategies (eg, liver transplantation) require lifelong immunosuppression. FINDINGS: The graft immediately provided the recipient with a functional blood supply (25-30 cm/s in the portal vein and 40 mL/s in the artery was measured intraoperatively and confirmed with ultrasound). The patient had normal laboratory values for 9 months. However, at 1 year the blood flow was low and, on exploration, the shunt was patent but too narrow due to mechanical obstruction of tissue in the mesocolon. Once the tissue causing the compression was removed the graft dilated. We therefore used a second stem-cell populated vein graft to lengthen the previous graft. After this second operation, the portal pressure was reduced from 20 mm Hg to 13 mm Hg and blood flow was 25-40 cm/s in the portal vein. With restored portal circulation the patient has substantially improved physical and mental function and growth. The patient has no anti-endothelial cell antibodies and is receiving no immunosuppressive drugs. INTERPRETATION: An acellularised deceased donor vein graft recellularised with autologous stem cells can be considered for patients in need of vascular vein shunts without the need for immunosuppression. FUNDING: Swedish Government.

Antibodies to kidney endothelial cells contribute to a "leaky" glomerular barrier in patients with chronic kidney diseases.

Anti-endothelial cell antibodies (AECA) have been reported to cause endothelial dysfunction, but their clinical importance for tissue-specific endothelial cells is not clear. We hypothesized that AECA reactive with human kidney endothelial cells (HKEC) may cause renal endothelial dysfunction in patients with chronic kidney diseases. We report that a higher fraction (56%) of end-stage renal disease (ESRD) patients than healthy controls (5%) have AECA reactive against kidney endothelial cells (P <0.001). The presence of antibodies was associated with female gender (P < 0.001), systolic hypertension (P < 0.01), and elevated TNF-α (P < 0.05). These antibodies markedly decrease expression of both adherens and tight junction proteins VE-cadherin, claudin-1, and zonula occludens-1 and provoked a rapid increase in cytosolic free Ca(2+) and rearrangement of actin filaments in HKEC compared with controls. This was followed by an enhancement in protein flux and phosphorylation of VE-cadherin, events associated with augmented endothelial cell permeability. Additionally, kidney biopsies from ESRD patients with AECA but not controls demonstrated a marked decrease in adherens and tight junctions in glomerular endothelium, confirming our in vitro data. In summary, our data demonstrate a causal link between AECA and their capacity to induce alterations in glomerular vascular permeability.

Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes.

BACKGROUND AIMS: One important problem commonly encountered after hepatocyte transplantation is the low numbers of transplanted cells found in the graft. If hepatocyte transplantation is to be a viable therapeutic approach, significant liver parenchyma repopulation is required. Mesenchymal stromal cells (MSC) produce high levels of various growth factors, cytokines and metalloproteinases, and have immunomodulatory effects. We therefore hypothesized that co-transplantation of MSC with human fetal hepatocytes (hFH) could augment in vivo expansion after transplantation. We investigated the ability of human fetal liver MSC (hFLMSC) to augment expansion of phenotypically and functionally well-characterized hFH. METHODS: Two million hFH (passage 6) were either transplanted alone or together (1:1 ratio) with green fluorescence protein-expressing hFLMSC into the spleen of C57BL/6 nude mice with retrorsine-induced liver injury. RESULTS: After 4 weeks, engraftment of cells was detected by fluorescence in situ hybridization using a human-specific DNA probe. Significantly higher numbers of cells expressing human cytokeratin (CK)8, CK18, CK19, Cysteine-rich MNNG HOS Transforming gene (c-Met), alpha-fetoprotein (AFP), human nuclear antigen, mitochondrial antigen, hepatocyte-specific antigen and albumin (ALB) were present in the livers of recipient animals co-transplanted with hFLMSC compared with those without. Furthermore, expression of human hepatocyte nuclear factor (HNF)-4α and HNF-1ß, and cytochrome P450 (CYP) 3A7 mRNA was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) in these animals. In addition, significantly increased amounts of human ALB were detected. Importantly, hFLMSC did not transdifferentiate into hepatocytes. CONCLUSIONS: Our study reports the use of a novel strategy for enhanced liver repopulation and thereby advances this experimental procedure closer to clinical liver cell therapy.

Isolation and characterization of human primary enterocytes from small intestine using a novel method.

Cell culture studies of enterocytes are important in many fields. However, there are difficulties in obtaining cell lines from adult human intestine, such as microbial contamination of cultures from the tissue samples, short life span of enterocytes, overgrowth of mesenchymal cells, etc. Various model used to obtain adult intestinal cell lines are very complex requiring use of feeder layer or gel matrices. The aim of this study was to establish a novel method for the simple and reproducible isolation of human enterocytes. Enterocytes were isolated from SI samples (n = 5) obtained from cadaveric donors using a mechanical procedure, and separation with immunomagnetic beads coated with anti-EpCAM antibodies. Light and electron microscopy, flow cytometry and immunocytochemistry techniques were used to characterize the isolated cells. Immunohistochemical staining of normal SB biopsies confirmed that the cell cultures maintained an in vivo phenotype as reflected in cytokeratin expression CK18, CK20 and expression of intestine-specific markers such as sucrase isomaltase and maltase glucoamylase. Furthermore, the cells strongly expressed TLR-5, 6, 7, 8 and 10 and several molecules such as CD40, CD86, CD44, ICAM-1 and HLA-DR which are important in triggering cell-mediated immune responses. This novel technique provides a unique in vitro system to study the biology of enterocytes in normal conditions as well as to study inflammatory processes in various small bowel disorders.

Relevance of MICA and other non-HLA antibodies in clinical transplantation.

The clinical importance of HLA-specific antibodies for organ allograft outcome is well established. In the past few years, there has been an increasing interest in non-HLA antigens as targets of injury in organ transplant recipients. This increased interest has been spurred by the fact that HLA-identical kidney transplants also undergo immunological rejections. Polymorphisms within non-HLA genes associated with evoking an immune response to alloantigens are currently being studied for their association with transplant outcome. Non-HLA antigens, such as the polymorphic MHC class I-related chain A (MICA), expressed on endothelial cells have been implicated in the pathogenesis of hyperacute, acute and chronic organ allograft rejections. Use of endothelial cells as targets may clarify the specificities of other clinically relevant non-HLA antibodies in graft rejections. This review summarizes past and current knowledge of the clinical importance and specificities of non-HLA antibodies, and mechanisms by which these antibodies may contribute to graft destruction in clinical transplantation. The aims of current research into the role of non-HLA antigens and their genetics in predicting outcome are to develop an improved insight into the basic science of transplantation and to develop a risk or prognostic index for use in the clinical setting. Non-HLA antibody responses are receiving increasing interest in acute and chronic rejection and specificity, affinity, and pathogenicity need to be investigated to estimate their contribution. Undoubtedly, this will continue to be an area of interest in terms of fully understanding the role of non-HLA antigens as targets of immune-mediated injury and the potential for clinical intervention.

Wnt ligands 3a and 5a regulate proliferation and migration in human fetal liver progenitor cells.

BACKGROUND: Since human fetal liver progenitor cells (hFLPC) can differentiate into multiple liver cell types in vitro and in vivo, hFLPC may be a suitable source for cell therapy and regeneration strategies. Imperative for effective clinical applications of hFLPC is the enhanced knowledge of growth factors that mediate and improve migration and proliferation. The canonical wingless/int-1 (Wnt) signal transduction pathway is known to play a key role in proliferation and migration of stem cells. So, we investigated a role for Wnt3a and Wnt5a ligands in regulating the proliferation and migration of hFLPC. METHODS: We used alamarBlue assay and transwell migration assay and examined proliferation and migration of hFLPC to Wnt3a and Wnt5a. In addition, the target genes of Wnt signal transduction pathway was identified using microarray analysis and validated by quantitative real-time polymerase chain reaction (qPCR). RESULTS: We found that Wnt3a or Wnt5a independently significantly increased migration and proliferation in a dose-dependent manner which was significantly inhibited by Wnt inhibitors Wnt-C59 or KN-62. Addition of Wnt3a to hFLPC resulted in increased mRNA expression of the known Wnt target genes Axin-2, DKK2, while Wnt5a increased CXCR7, all of which are closely associated with an enhanced proliferation capacity of stem cells. CONCLUSIONS: Thus, we report that Wnt3a and Wnt5a may play an important role in the proliferation and migration of hFLPC by possibly regulating key target genes-involved in these processes. Incorporating recombinant human Wnt3a and Wnt5a in regenerative strategies using liver stem/progenitor cells might improve the process of liver regeneration.

Characterization and engraftment of long-term serum-free human fetal liver cell cultures.

BACKGROUND AIMS: Cultured human hepatocytes have extensive diagnostic and clinical applications. However, the setting-up of new in vitro culture techniques allowing the long-term survival and functional maintenance of adult human hepatocytes represents a formidable challenge. Fetal liver cells (FLC) are attractive candidate donor cells because of their high proliferative capacity. METHODS: Using cell culture and molecular techniques, we studied the in vitro and in vivo characteristics of FLC grown long-term in serum-free conditions. RESULTS: Serum-free FLC obtained from 6-10-week-old human fetal livers grew as multiple clusters in suspension and could be subcultured for at least six passages. These cells maintained stable hepatocyte phenotypes and gene expression patterns in culture for up to 6 months. When a cluster of these cells in various passages was placed on collagen-coated plates, they formed a monolayer and morphologically resembled hepatocytes. The cells expressed alpha -fetoprotein, cytokeratin (CK) 8, CK18 and CK19 and albumin (ALB). Hepatocyte nuclear factor 4alpha and 1beta and cytochrome P450 (CYP) 3A4 and CYP3A7 mRNA expression was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). Cells at different passages, when transplanted into nude mice with liver injury, engrafted successfully, as detected by in situ hybridization using a human-specific DNA probe. Colonies of human-specific CK8, CK18, c-Met nuclear antigen (Ag), mitochondrial Ag, hepatocyte-specific Ag and ALB-expressing cells were present in the livers of recipient animals. CONCLUSIONS: Primary human FLC can be kept in culture consistently over a long time period and are potential candidates for cell therapy and in vitro diagnostics.

Generation of hepatocyte-like cells from in vitro transdifferentiated human fetal pancreas.

Although the appearance of hepatic foci in the pancreas has been described in animal experiments and in human pathology, evidence for the conversion of human pancreatic cells to liver cells is still lacking. We therefore investigated the developmental plasticity between human embryonic pancreatic cells and liver cells. Cells were isolated and expanded from 7-8-week-old human fetal pancreata (HFP) and were characterized for the absence and presence of pancreatic and hepatic markers. In vitro expanded HFP were treated with fibroblast growth factor 2 (FGF2) and dexamethasone (DX) to induce a liver phenotye in the cells. These treated cells in various passages were further studied for their capacity to be functional in hepatic parenchyma following retrorsine-induced injury in nude C57 black mice. Amylase- and EPCAM-positive-enriched cells isolated from HFP and treated with FGF2 and DX lost expression of pancreatic markers and gained a liver phenotype. Hepatic differentiation was based on the expression (both at the mRNA and protein level) of liver markers albumin and cytokeratin 19. When transplanted in vivo into nude mice treated with retrorsine, both cell types successfully engrafted and functionally differentiated into hepatic cells expressing human albumin, glycogen, dipeptidyl peptidase, and gamma-glutamyltranspeptidase. These data indicate that human fetal pancreatic cells have a capacity to alter their gene expression profile in response to exogenous treatment with FGF2 and DX. It may be possible to generate an unlimited supply of hepatocytes in vitro for cell therapy.

Notice of Retraction: Inappropriate Image Duplication in "Recellularization of Acellular Human Small Intestine Using Bone Marrow Stem Cells".

STEM CELLS TRANSLATIONAL MEDICINE 2013;2:307-315; http://dx.doi.org/10.5966/sctm.2012-0108 The above-referenced article published on March 13, 2013 in Stem Cells Translational Medicine has been retracted by agreement between the Journal Editors and co-publishers, AlphaMed Press and Wiley Periodicals, Inc. The retraction has been agreed to with acknowledgment of problems with Figure 3, which we believe make some of the data unreliable.

Evidence for no relevance of anti-major histocompatibility complex class I-related chain a antibodies in liver transplantation.

The polymorphic major histocompatibility complex class I-related chain A (MICA) antigen is being increasingly recognized as a potential target molecule for immune cells during allograft rejection. Here we studied whether MICA is a target antigen for antibodies in liver transplant patients. Eighty-four patients were investigated for the presence of MICA antibodies before and after liver transplantation with MICA-transfected cells and flow cytometry. MICA typing was performed by polymerase chain reaction. Expression of MICA in liver cells was determined by reverse-transcription polymerase chain reaction, Western blotting, and flow cytometry. Liver biopsy specimens from liver transplant patients were examined for MICA expression. A total of 22 of 84 (26%) patients had MICA antibodies either pre-transplant (8/84, 9.5%) or post-transplant (14/84, 17%). No correlation between rejection frequencies (14/22, 63%) or other clinical parameters was observed in patients with MICA antibody versus those without MICA antibody (29/62, 47% P = not significant). We found weak messenger RNA expression for MICA in liver cells but no protein or cell surface expression. In addition, no MICA expression in liver biopsy sections from liver transplant patients was observed at any time point, including rejections. Thus, our preliminary results demonstrate no causal relationship between the presence of MICA antibodies and liver allograft rejections. Therefore, it is likely that MICA may not be an important target antigen during liver allograft rejections.

Beyond ABO and human histocompatibility antigen: other histocompatibility antigens with a role in transplantation.

PURPOSE OF REVIEW: In the past few years, there has been an increasing interest in nonhuman histocompatibility antigens as targets of injury in organ-transplant recipients. This increased interest has been spurred by the fact that human-histocompatibility-antigen-identical kidney transplants also undergo immunological rejections. RECENT FINDINGS: Polymorphisms within nonhuman histocompatibility antigen genes associated with evoking an immune response to alloantigens are currently being studied for their association with transplant outcome. Studies identify the polymorphic major histocompatibility complex class I-related chain A as one of the important nonhuman histocompatibility antigen antibody targets in kidney allograft rejections. Use of endothelial cells as targets may clarify the specificities of other clinically relevant nonhuman histocompatibility antigen antibodies in graft rejections. SUMMARY: This review summarizes current knowledge of the specificities and associations of nonhuman histocompatibility antigens with graft outcome after transplantation. The aims of current research into the role of nonhuman histocompatibility antigens and their genetics in predicting outcome are to develop an improved insight into the basic science of transplantation and to develop a risk or prognostic index for use in the clinical setting. Undoubtedly, this will continue to be an area of interest in terms of fully understanding the role of nonhuman histocompatibility antigens as targets of immune-mediated injury and the potential for clinical intervention.

Decellularization and Recellularization Methodology for Human Saphenous Veins.

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient's cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 °C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 µL/mL), and acetyl salicylic acid (5 µg/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin & eosin (H&E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&E-staining showed the presence of cells on the luminal surface of the vein.

Cold-perfusion decellularization of whole-organ porcine pancreas supports human fetal pancreatic cell attachment and expression of endocrine and exocrine markers.

Despite progress in the field of decellularization and recellularization, the outcome for pancreas has not been adequate. This might be due to the challenging dual nature of pancreas with both endocrine and exocrine tissues. We aimed to develop a novel and efficient cold-perfusion method for decellularization of porcine pancreas and recellularize acellular scaffolds with human fetal pancreatic stem cells. Decellularization of whole porcine pancreas at 4°C with sodium deoxycholate, Triton X-100 and DNase efficiently removed cellular material, while preserving the extracellular matrix structure. Furthermore, recellularization of acellular pieces with human fetal pancreatic stem cells for 14 days showed attached and proliferating cells. Both endocrine (C-peptide and PDX1) and exocrine (glucagon and α-amylase) markers were expressed in recellularized tissues. Thus, cold-perfusion can successfully decellularize porcine pancreas, which when recellularized with human fetal pancreatic stem cells shows relevant endocrine and exocrine phenotypes. Decellularized pancreas is a promising biomaterial and might translate to clinical relevance for treatment of diabetes.

Significantly Accelerated Wound Healing of Full-Thickness Skin Using a Novel Composite Gel of Porcine Acellular Dermal Matrix and Human Peripheral Blood Cells.

Here we report the fabrication of a novel composite gel from decellularized gal-gal-knockout porcine skin and human peripheral blood mononuclear cells (hPBMCs) for full-thickness skin wound healing. Decellularized skin extracellular matrix (ECM) powder was prepared via chemical treatment, freeze drying, and homogenization. The powder was mixed with culture medium containing hyaluronic acid to generate a pig skin gel (PSG). The effect of the gel in regeneration of full-thickness wounds was studied in nude mice. We found significantly accelerated wound closure already on day 15 in animals treated with PSG only or PSG + hPBMCs compared to untreated and hyaluronic acid-treated controls (p < 0.05). Addition of the hPBMCs to the gel resulted in marked increase of host blood vessels as well as the presence of human blood vessels. At day 25, histologically, the wounds in animals treated with PSG only or PSG + hPBMCs were completely closed compared to those of controls. Thus, the gel facilitated generation of new skin with well-arranged epidermal cells and restored bilayer structure of the epidermis and dermis. These results suggest that porcine skin ECM gel together with human cells may be a novel and promising biomaterial for medical applications especially for patients with acute and chronic skin wounds.

Activation of PERK-Nrf2 oncogenic signaling promotes Mdm2-mediated Rb degradation in persistently infected HCV culture.

The mechanism of how chronic hepatitis C virus (HCV) infection leads to such a high rate of hepatocellular carcinoma (HCC) is unknown. We found that the PERK axis of endoplasmic reticulum (ER) stress elicited prominent nuclear translocation of Nrf2 in 100% of HCV infected hepatocytes. The sustained nuclear translocation of Nrf2 in chronically infected culture induces Mdm2-mediated retinoblastoma protein (Rb) degradation. Silencing PERK and Nrf2 restored Mdm2-mediated Rb degradation, suggesting that sustained activation of PERK/Nrf2 axis creates oncogenic stress in chronically infected HCV culture model. The activation of Nrf2 and its nuclear translocation were prevented by ER-stress and PERK inhibitors, suggesting that PERK axis is involved in the sustained activation of Nrf2 signaling during chronic HCV infection. Furthermore, we show that HCV clearance induced by interferon-α based antiviral normalized the ER-stress response and prevented nuclear translocation of Nrf2, whereas HCV clearance by DAAs combination does neither. In conclusion, we report here a novel mechanism for how sustained activation of PERK axis of ER-stress during chronic HCV infection activates oncogenic Nrf2 signaling that promotes hepatocyte survival and oncogenesis by inducing Mdm2-mediated Rb degradation.

Macroencapsulation protects against sensitization after allogeneic islet transplantation in rats.

BACKGROUND: The aim of this study was to evaluate the risks of sensitization by islet grafts encapsulated in a bilaminar immunoprotective membrane. METHODS: We studied five groups of Lewis rats: one control group (no islets), two groups that received free islets (200 or 1000 s.c.), and two groups that received encapsulated ones (200 or 1000 s.c.) from Dark Agouti (DA) rats. Four weeks later, abdominal heterotopic DA-heart transplantation was performed on the same recipients. The time-to-heart graft rejection was assessed by the cessation of heart contractions. Rejection was confirmed by histological examinations. Antidonor antibodies were determined by fluorescence activated cell sorter (FACS) analysis. RESULTS: The control animals had a mean heart graft survival of 6.4 days. The free islet groups had significantly shorter heart graft survivals-i.e., 4.8 days (200 islets) and 1.0 day (1000 islets) (P < 0.001)-while those of the encapsulated islet groups were about the same as that of the control group-i.e., 6.4 days (200 islets) and 6.0 days (1000 islets). In the free islet groups, anti-DA antibodies developed in 7/10 (200 islets) and 8/8 (1000 islets) animals after the islet transplantation. In the encapsulated groups, 1/10 (200 islets) and 3/8 (1000 islets) animals developed anti-DA antibodies after these transplantations. All animals had anti-DA antibodies at the time of heart graft rejection. On histological examination all grafts showed various features of rejection. CONCLUSIONS: The bilaminar membrane protects against sensitization and prevents accelerated rejection of a subsequent vascularized graft, at least during the first month after the islet transplantation.

Expression of vascular endothelial growth factor receptor-2 or Tie-2 on peripheral blood cells defines functionally competent cell populations capable of reendothelialization.

BACKGROUND: Receptor tyrosine kinases that include vascular endothelial growth factor (VEGFR)-1, VEGFR-2, and Tie-2 regulate cardiovascular development and physiological and pathological angiogenesis. We were interested in the phenotypic and functional characterization of peripheral blood cells expressing these receptors and their therapeutic potential in vascular injury. METHODS AND RESULTS: VEGFR-1+, VEGFR-2+, and Tie-2+ cells constituted approximately 3.0+/-0.2%, 0.8+/-0.5%, and 2.0+/-0.3%, respectively, of the total population of mononuclear cells in blood. Phenotypic analysis demonstrated that all 3 cell populations mainly expressed markers of monocytic/macrophage lineage. Only VEGFR-2+ and Tie-2+ cells phenotypically, morphologically, and functionally differentiated to endothelial cells after culture, whereas VEGFR-1+ cells did not. None of the cell types proliferated in vitro. Only freshly isolated VEGFR-2+ or Tie-2+ cells but not VEGFR-2- or Tie-2- cell populations significantly contributed to efficient endothelialization of balloon-injured femoral arteries of nude mice. Furthermore, these cells also differentiated into -actin-positive smooth muscle cells. Administration of bromodeoxyuridine to animals transplanted with human endothelial progenitor cells showed that VEGFR-2+ and Tie-2+ cells proliferated in vivo. CONCLUSIONS: These data demonstrate that expression of VEGFR-2 and/or Tie-2 on peripheral blood cells defines functionally competent cell populations that proliferate in vivo and that contribute to reendothelialization. These findings may have implications for a cell-based approach in vascular diseases.