Major histocompatibility complex-I expression on embryonic stem cell-derived vascular progenitor cells is critical for syngeneic transplant survival.

Donor-recipient cell interactions are essential for functional engraftment after nonautologous cell transplantation. During this process, transplant engraftment is characterized and defined by interactions between transplanted cells with local and recruited inflammatory cells. The outcome of these interactions determines donor cell fate. Here, we provide evidence that lineage-committed embryonic stem cell (ESC)-derived vascular progenitor cells are the target of major histocompatibility complex (MHC) class I-dependent, natural killer (NK) cell-mediated elimination in vitro and in vivo. Treatment with interferon γ was found to significantly upregulate MHC class I expression on ESC-derived vascular progenitor cells, rendering them less susceptible to syngeneic NK cell-mediated killing in vitro and enhancing their survival and differentiation potential in vivo. Furthermore, in vivo ablation of NK cells led to enhanced progenitor cell survival after transplantation into a syngeneic murine ischemic hindlimb model, providing additional evidence that NK cells mediate ESC-derived progenitor cell transplant rejection. These data highlight the importance of recipient immune-donor cell interactions, and indicate a functional role for MHC-I antigen expression during successful ESC-derived syngeneic transplant engraftment.

Neuropilin-1 identifies endothelial precursors in human and murine embryonic stem cells before CD34 expression.

BACKGROUND: In murine embryonic stem cells, the onset of vascular endothelial growth factor receptor 2 (VEGFR-2) expression identifies endothelial precursors. Undifferentiated human embryonic stem cells express VEGFR-2, and VEGFR-2 expression persists on differentiation. The objective of our study was to identify a single population of endothelial precursors with common identifying features from both human and murine embryonic stem cells. METHODS AND RESULTS: We report that expression of the VEGF coreceptor neuropilin-1 (NRP-1) coincides with expression of Brachyury and VEGFR-2 and identifies endothelial precursors in murine and human embryonic stem cells before CD31 or CD34 expression. When sorted and differentiated, VEGFR-2(+)NRP-1(+) cells form endothelial-like colonies that express CD31 and CD34 7-fold more efficiently than NRP-1 cells. Finally, antagonism of both the VEGF and Semaphorin binding functions of NRP-1 impairs the differentiation of vascular precursors to endothelial cells. CONCLUSIONS: The onset of NRP-1 expression identifies endothelial precursors in murine and human stem cells. The findings define the origin of a single population of endothelial precursors from human and murine stem cells to endothelial cells. Additionally, the function of both the VEGF and Semaphorin binding activities of NRP-1 has important roles in the differentiation of stem cells to endothelial cells, providing novel insights into the role of NRP-1 in a model of vasculogenesis.

Cross-talk between the VEGF-A and HGF signalling pathways in endothelial cells.

BACKGROUND INFORMATION: Endothelial cells play a major role in angiogenesis, the process by which new blood vessels arise from a pre-existing vascular bed. VEGF-A (vascular endothelial growth factor-A) is a key regulator of angiogenesis during both development and in adults. HGF (hepatocyte growth factor) is a pleiotropic cytokine that may promote VEGF-A-driven angiogenesis, although the signalling mechanisms underlying this co-operation are not completely understood. RESULTS: We analysed the effects of the combination of VEGF-A and HGF on the activation of VEGFR-2 (VEGF receptor-2) and c-met receptors, and on the stimulation of downstream signalling pathways in endothelial cells. We found that VEGFR-2 and c-met do not physically associate and do not transphosphorylate each other, suggesting that co-operation involves signalling events more distal from receptor activation. We demonstrate that the VEGF isoform VEGF-A(165) and HGF stimulate a similar set of MAPKs (mitogen-activated protein kinases), although the kinetics and strengths of the activation differ depending on the growth factor and pathway. An enhanced activation of the signalling was observed when endothelial cells were stimulated by the combination of VEGF-A(165) and HGF. Moreover, the combination of VEGF-A and HGF results in a statistically significant synergistic activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 kinases. We demonstrated that VEGF-A(165) and HGF activate FAK (focal adhesion kinase) with different kinetics and stimulate the recruitment of phosphorylated FAK to different subsets of focal adhesions. VEGF-A(165) and HGF regulate distinct morphogenic aspects of the cytoskeletal remodelling that are associated with the preferential activation of Rho or Rac respectively, and induce structurally distinct vascular-like patterns in vitro in a Rho- or Rac-dependent manner. CONCLUSIONS: Under angiogenic conditions, combining VEGF-A with HGF can promote neovascularization by enhancing intracellular signalling and allowing more finely regulated control of the signalling molecules involved in the regulation of the cytoskeleton and cellular migration and morphogenesis.

Improved Protocol for Cardiac Differentiation and Maturation of Pluripotent Stem Cells.

Pluripotent stem cells feature the capacity to differentiate into any somatic cell types including cardiomyocytes. We report a cost-effective and simple protocol for the differentiation of specific ventricular cardiomyocytes. These cells are elongated, do not spontaneously beat, and do not feature any Ca2+-transient, an index of their stage of maturation toward adult cardiac cells. They represent a suitable model to screen both the efficiency and toxicology of drugs.

[Activation of auto-mesenchymal stem cells of skeletal muscle by bone morphogenetic protein for rescuing bone marrow failure].

OBJECTIVE: To explore the effect of bone morphogenetic protein (BMP) to activate mesenchymal stem cells of skeletal muscle for rescuing bone marrow failure. METHODS: The study was performed on lethal rat acute aplastic anemia model induced by combined 5-fluorouracil (5-FU) and busulfan. The rh-BMP-2 was implanted into the thigh muscle of the rats at 3 days before aplastic anemia was induced. In the control group the rats were implanted with agar into the thigh muscle. The blood picture, pathologic changes and the mortality in two groups were observed. At the same time, rh-BMP-2 were implanted into the thigh muscle of normal Kun-min mice for dynamic control observation of the implantation local morphological changes, colony forming units-spleen (CFU-S) and stem cell growth factor (SCF) expression of the stroma cells of ectopic ossicles induced by BMP. RESULTS: At 7 days after BMP implantation in the mice the mesenchymal cells around BMP in muscle proliferated, and appeared in bone marrow to form an ectopic ossicles. The SCF expression of stroma cells in ectopic ossicles were higher than that of self-bone marrow. 56.3% of BMP-treated aplastic rats were survived over 3 months and its hematopoiesis was completely reconstituted and the histo-morphological picture of the spleen and bone marrow were recovered to normal. But in the control group only one of 23 rats was survived, the remainder died of hematopoietic failure. CONCLUSIONS: BMP-implantation into the skeletal muscle could rescue the bone marrow hematopoietic failure. The mechanism might be related to the BMP activated auto-mesenchymal cells of skeletal muscles to direct hematopoietic cell differentiation. In our hands it might create a new pathway for utilization of auto-muscle derived mesenchymal cells to reconstitute hematopoiesis.

HGF receptor up-regulation contributes to the angiogenic phenotype of human endothelial cells and promotes angiogenesis in vitro.

Hepatocyte growth factor (HGF) is a mesenchyme-derived pleiotropic growth factor and a powerful stimulator of angiogenesis, which acts on cells by binding to the c-met receptor. The exact role of the endogenous HGF/c-met system in one or more steps of the angiogenic process is not completely understood. To contribute to this question we used immunocytochemical analysis, Western blotting, and reverse transcription-polymerase chain reaction to study the expression of c-met in endothelial cells cultured in different growth conditions. We found that c-met is not colocalized with vascular endothelial (VE)-cadherin in cell-cell junctions. c-met and VE-cadherin were shown to be inversely regulated by cell density, at both the protein and the mRNA levels. We established that c-met is up-regulated during the in vitro recapitulation of several steps of angiogenesis. The c-met expression was increased shortly after switching to angiogenic growth conditions and remained high during the very first steps of angiogenesis, including cell migration, and cell proliferation. The endothelial cells in which the expression of c-met was up-regulated were more responsive to HGF and exhibited a higher rate of morphogenesis. Moreover, the antibody directed against the extracellular domain of the c-met inhibited angiogenesis in vitro. Our results suggest that c-met is a marker of angiogenic phenotype for endothelial cells and represents an attractive target for the development of new antiangiogenic therapies.

The N-terminal domain of hepatocyte growth factor inhibits the angiogenic behavior of endothelial cells independently from binding to the c-met receptor.

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an important role in complex biological processes such as embryogenesis, tissue regeneration, cancerogenesis, and angiogenesis. HGF promotes cell proliferation, survival, motility, and morphogenesis through binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene (c-met). Structurally speaking, HGF is a polypeptide related to the enzymes of the blood coagulation cascade. Thus, it comprises kringle domains that in some other proteins have been shown to be responsible for the anti-angiogenic activity. To check whether the isolated kringles of HGF were able to inhibit angiogenesis, we produced them as recombinant proteins and compared their biological activity with that of the recombinant HGF N-terminal domain (N). We showed that (i) none of the isolated HGF kringle exhibits an anti-angiogenic activity; (ii) N is a new anti-angiogenic polypeptide; (iii) the inhibitory action of N is not specific toward HGF, because it antagonized the angiogenic activity of other growth factors, such as fibroblast growth factor-2 and vascular endothelial growth factor; and (iv) in contrast with full-length HGF, N does not bind to the c-met receptor in vitro, but fully retains its heparin-binding capacity. Our results suggest that N inhibits angiogenesis not by disrupting the HGF/c-met interaction but rather by interfering with the endothelial glycosaminoglycans, which are the secondary binding sites of HGF.

[Biological characterization of mouse erythroblastic leukemia cells in haploiden tical mice].

Using transplantable erythroblastic leukemia cells of EL9611(H-2d), the cells were inoculated to CB6F(1)(H-2d/b) generation of BALB/c x C57BL/6 mouse, the biological characterization of erythroblastic leukemia in haploidentical mouse was studied, that provides an experimental model for the study of graft-versus leukemia (GVL) with bone marrow or stem cell transplantation. When 10(3) - 10(8) of the spleen cells of EL9611(H-2d) had been intravenously inoculated to CB6F(1) mouse, the erythroblastic leukemia cells were transplanted successively and the F(1) generation of erythroblastic leukemia model in mice was established with 100% incidence of erythroblastic leukemia. There was a linear relationship between the survival time and the number of leukemic cell. The survival time of the mice was (9.6 +/- 0.8) days when 10(6) cells were inoculated. If the CB6F(1) mouse was transplanted successively for four generations, the incidence was 100%. The main targets for the leukemic EL9611(H-2d) cells were liver, spleen and marrow. The reaction of the erythroblastic leukemia cells for hemoglobin staining was positive, while the peroxidase reaction was negative. These cells were sensitive to some chemotherapeutic drugs, such as cytosine arabinoside and cyclophosphamide. This study presents the convenience for the studies on the GVL with haplo-allogeneic transplantation, in the F(1) generation of erythroblastic leukemia model of the commonly-used CD57BL/6 x BALB/c mouse.

[Study of Low Dose Irradiation Enhancing the Effect of Chemotherapeutic Agents against Leukemia and Its Mechanism]

For effectively enhancing the anti-leukemia effect of chemotherapeutic agents, and meanwhile decreasing the side effect of these agents, the study has been made to explore the synergistic effect of low dose irradiation (LDI) combined with Ara-C on murine leukemia and its mechanism. Firstly, an optimal scheme of low dose total body irradiation combined with Ara-C was established in L615 leukemia (T lymphocytic leukemia) mouse model. The machanism of the enhancing effect was explored by patho-morphological observation, examination of residual leukemia cells, the expression of GM-CSF on the surface of marrow stromal cells and in the bone marrow cultural supernatants. The results showed that the optimal scheme was 300 cGy irradiation at 4 days after inoculation of leukemic cells followed by Ara-C 30 mg/kg x 3 days in an interval of 1, 2 or 3 days after irradiation. The mean survival time of the L615 leukemia mice in LDI + Ara-C combined treatment groups was longer than that of control groups. The percentage of long-term survival mice (> 30 days) was the highest (58% - 72%), too. 17% of the mice were be cured. The numbers of blood leukocytes and marrow nucleated cells were transiently decreased in combined treatment group, and then recovered rapidly. Slight myelosuppression and marrow sinus dilation and congestion were seen after 300 cGy irradiation. The expression of GM-CSF either on the stromal cells or in marrow cultural supernatant after irradiation increased strikingly (P < 0.05). Therefore, LDI combined with Ara-C possesses synergistic effect. The mechanism is possibly related to three facts: LDI could increase the permeability of bone marrow sinus; LDI could promote marrow stromal cells to produce some cytokines (such as GM-CSF, etc.) which drive leukemia cells into cell cycle to make the cells more sensitive to chemotherapeutic agents; and LDI could augment Ara-C-induced cytotoxicity through the mechanism of apoptosis.