Collagen matrix physical properties modulate endothelial colony forming cell-derived vessels in vivo.

Developing tissue engineering approaches to generate functional vascular networks is important for improving treatments of peripheral and cardiovascular disease. Endothelial colony forming cells (ECFCs) are an endothelial progenitor cell (EPC) population defined by high proliferative potential and an ability to vascularize collagen-based matrices in vivo. Little is known regarding how physical properties of the local cell microenvironment guide vessel formation following EPC transplantation. In vitro evidence suggests that collagen matrix stiffness may modulate EPC vessel formation. The present study determined the ability of 3D collagen matrix physical properties, varied by changing collagen concentration, to influence ECFC vasculogenesis in vivo. Human umbilical cord blood ECFCs were cultured within matrices for 18 h in vitro and then fixed for in vitro analysis or implanted subcutaneously into the flank of immunodeficient mice for 14 days. We report that increasing collagen concentration significantly decreased ECFC derived vessels per area (density), but significantly increased vessel sizes (total cross sectional area). These results demonstrate that the physical properties of collagen matrices influence ECFC vasculogenesis in vivo and that by modulating these properties, one can guide vascularization.

Cleaved high molecular weight kininogen inhibits tube formation of endothelial progenitor cells via suppression of matrix metalloproteinase 2.

BACKGROUND AND OBJECTIVE: Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, thus promoting wide interest in their therapeutic potential in vascular injury and prevention of their dysfunction in cardiovascular diseases. Cleaved high molecular weight kininogen (HKa), an activation product of the plasma kallikrein-kinin system (KKS), inhibits the functions of differentiated endothelial cells including in vitro and in vivo angiogenesis. In this study, our results provided the first evidence that HKa is able to target EPCs and inhibits their tube forming capacity. METHODS AND RESULTS: We determined the effect of HKa on EPCs using a three-dimensional vasculogenesis assay. Upon stimulation with vascular endothelial growth factor (VEGF) alone, EPCs formed vacuoles and tubes, and differentiated into capillary-like networks. As detected by gelatinolytic activity assay, VEGF stimulated secretion and activation of matrix metallopeptidase 2 (MMP-2), but not MMP-9, in the conditioned medium of 3D culture of EPCs. Specific inhibition or gene ablation of MMP-2, but not MMP-9, blocked the vacuole and tube formation by EPCs. Thus, MMP-2 is selectively required for EPC vasculogenesis. In a concentration-dependent manner, HKa significantly inhibited tube formation by EPCs and the conversion of pro-MMP-2 to MMP-2. Moreover, HKa completely blocked the association between pro-MMP-2 and alphavbeta3 integrin, and its inhibition of MMP-2 activation was dependent on the presence of alphavbeta3 integrin. In a purified system, HKa did not directly inhibit MMP-2 activity. CONCLUSIONS: HKa inhibits tube forming capacity of EPCs by suppression of MMP-2 activation, which may constitute a novel link between activation of the KKS and EPC dysfunction.

Future of cord blood for non-oncology uses.

For the last 5 years cord blood (CB) has been under intense experimental investigation in in vitro differentiation models and in preclinical animal models ranging from bone to muscle regeneration, cardiovascular diseases including myocardial and peripheral arterial disease, stroke and Parkinson's disease. On the basis of its biological advantages, CB can be an ideal source for tissue regeneration. However, in the hype of the so-called 'plasticity', many cell types have been characterized either on cell surface Ag expression alone or by RNA expression only, and without detailed characterization of genetic pathways; frequently, cells are defined without analysis of cellular function in vitro and in vivo, and the definition of the lineage of origin and cells have not been defined in preclinical studies. Here, we explore not only the most consistent data with regard to differentiation of CB cells in vitro and in vivo, but also show technical limitations, such as why in contrast to cell populations isolated from fresh CB, cryopreserved CB is not the ideal source for tissue regeneration. By taking advantage of numerous CB units discarded due to lack of sufficient hematopoietic cells for clinical transplantation, new concepts to produce off-the-shelf products are presented as well.

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation.

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH-oxidase activity caused by defects in the gene-encoding gp91(phox). Here, we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a gamma-retroviral vector for gp91(phox) expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared with lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared with fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that the intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.

Defining human endothelial progenitor cells.

There is no specific marker to identify an endothelial progenitor cell (EPC) and this deficiency is restricting the ability of an entire field of research in defining these cells. We will review current methods to define EPC in the human system and suggest approaches to define better the cell populations involved in neoangiogenesis. PubMed was used to identify articles via the search term 'endothelial progenitor cell' and those articles focused on defining the term were evaluated. The only human cells expressing the characteristics of an EPC, as originally proposed, are endothelial colony forming cells. A variety of hematopoietic cells including stem and progenitors, participate in initiating and modulating neoangiogenesis. Future studies must focus on defining the specific hematopoietic subsets that are involved in activating, recruiting, and remodeling the vascular networks formed by the endothelial colony forming cells.

Working hypothesis to redefine endothelial progenitor cells.

Since 1997, postnatal vasculogenesis has been purported to be an important mechanism for neoangiogenesis via bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs). Based on this paradigm, EPCs have been extensively studied as biomarkers to assess severity of cardiovascular disease and as a cell-based therapy for several human cardiovascular disorders. In the majority of studies to date, EPCs were identified and enumerated by two primary methodologies; EPCs were obtained and quantified following in vitro cell culture, or EPCs were identified and enumerated by flow cytometry. Both methods have proven controversial. This review will attempt to outline the definition of EPCs from some of the most widely cited published reports in an effort to provide a framework for understanding subsequent studies in this rapidly evolving field. We will focus this review on studies that used cell culture techniques to define EPCs.

Low-molecular-weight peptides derived from extracellular matrix as chemoattractants for primary endothelial cells.

The development of synthetic and naturally occurring scaffolds for tissue engineering applications has included strategies to promote attachment of specific cell types, control the rate of scaffold degradation, encourage angiogenesis, or otherwise modulate the host response. We have reported that bioscaffolds developed from porcine small intestinal submucosa (SIS) facilitate the constructive remodeling of tissues and recruit marrow-derived cells that persist long after the acute inflammatory stages have resolved. We have not yet determined which cells are recruited, the eventual fate of these cells, or via what mechanisms the events occur. We now have analyzed various molecular weight fractions of acid-hydrolyzed SIS by both functional and morphologic methods and have determined that fraction 4 (5 to 16 kDa) possesses chemoattractant activity for primary murine adult liver, heart, and kidney endothelial cells in vitro. Addition of fraction 4 to Matrigel plugs promoted in vivo vascularization when the plugs were implanted subcutaneously in mice. These results indicate that small-molecular-weight peptides derived from the degradation of porcine SIS are biologically active in the recruitment of murine endothelial cells in vitro and in vivo.

Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo.

Adeno-associated virus 2 (AAV) vectors are currently in use in Phase I/II clinical trials for gene therapy of cystic fibrosis and hemophilia B. Although 100% of murine hepatocytes can be targeted by AAV vectors, the transgene expression is limited to approximately 5% of hepatocytes. Since the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strand-annealing accounts for the lack of efficient transgene expression. We and others, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis attributes to the observed low efficiency of transgene expression. We have previously documented that a cellular protein, designated FKBP52, when present in phosphorylated forms, inhibits the viral second-strand DNA synthesis, and consequently, limits transgene expression in nonhepatic cells, whereas unphosphorylated forms of FKBP52 have no effect. To further evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice overexpressing the cellular T-cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. We demonstrate here that dephosphorylation of FKBP52 in TC-PTP transgenic (TC-PTP-TG) mice, and removal of FKBP52 in FKBP52-knockout (FKBP52-KO) mice results in efficient transduction of murine hepatocytes following tail-vein injection of recombinant AAV vectors. We also document efficient viral second-strand DNA synthesis in hepatocytes from both TC-PTP-TG and FKBP52-KO mice. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction of hepatocytes, which should have implications in the optimal use of recombinant AAV vectors in human gene therapy.

Embryonic hematopoiesis in mice and humans.

AIM: The aim of this report is to review briefly the ontogeny of hematopoiesis in mice and humans and to discuss recent evidence for an intraembryonic source of hematopoietic stem cells. METHODS: The hematopoiesis overview summarizes information present in the PubMed online database and from experiments conducted in our laboratories. RESULTS: The major sites of hematopoiesis change throughout development in mice and humans. Recent evidence suggests that hematopoietic cells may emerge from mesoderm precursors within the embryo as well as in the yolk sac. CONCLUSION: The ontogeny of hematopoiesis is similar in mice and humans. The murine system is a useful model to study the earliest events involved in forming hematopoietic stem cells.

Marrow-derived cells populate scaffolds composed of xenogeneic extracellular matrix.

INTRODUCTION: The source of cells that participate in wound repair directly affects outcome. The extracellular matrix (ECM) and other acellular biomaterials have been used as therapeutic scaffolds for cell attachment and proliferation and as templates for tissue repair. The ECM consists of structural and functional proteins that influence cell attachment, gene expression patterns, and the differentiation of cells. OBJECTIVE: The objective of this study was to determine if the composition of acellular matrix scaffolds affects the recruitment of bone marrow-derived cellular elements that populate the scaffolds in vivo. METHODS: Scaffolds composed of porcine tissue ECM, purified Type I collagen, poly(L)lactic coglycolic acid (PLGA), or a mixture of porcine ECM and PLGA were implanted into subcutaneous pouches on the dorsum of mice. The origin of cells that populated the matrices was determined by first performing bone marrow transplantation to convert the marrow of glucose phosphate isomerase 1b (Gpi-1(b)) mice to cells expressing glucose phosphate isomerase 1a (Gpi-1(a)). RESULTS: A significant increase in Gpi-1(a) expressing cells was present in sites implanted with the porcine ECM compared to sites implanted with either Type I collagen or PLGA. Use of recipient mice transplanted with marrow cells that expressed beta-galactosidase confirmed that the majority of cells that populated and remodeled the naturally occurring porcine ECM were marrow derived. Addition of porcine ECM to the PLGA scaffold caused a significant increase in the number of marrow-derived cells that became part of the remodeled implant site. CONCLUSION: The composition of bioscaffolds affects the cellular recruitment pattern during tissue repair. ECM scaffolds facilitate the recruitment of marrow-derived cells into sites of remodeling.

Homing, cell cycle kinetics and fate of transplanted hematopoietic stem cells.

Homing of transplanted hematopoietic stem cells to recipient bone marrow is a critical step in engraftment and initiation of marrow reconstitution. At present, only partial understanding of the cellular and molecular mechanisms governing homing exists. Likewise, only an incomplete list of adhesion molecules implicated in directing the trafficking of stem cells to the marrow microenvironment is available. Opposing hypotheses that attribute homing to an orderly and orchestrated cascade of events or to random migration of circulating cells find ample experimental support. Also unsettled is the fate of marrow-homed cells shortly after transplantation and the rapidity at which they begin to proliferate in their new marrow microenvironment. The limited number of studies in this field and disparities in their experimental design intensifies the confusion surrounding these critical aspects of stem cell biology. However, this area of research is moving forward rapidly and results capable of clarifying many of these issues are forthcoming.

Yolk-sac hematopoiesis: the first blood cells of mouse and man.

OBJECTIVE: To review the process of blood-cell formation in the murine and human yolk sac. DATA SOURCES: Most articles were selected from the PubMed database. DATA SYNTHESIS: The yolk sac is the first site of blood-cell production during murine and human ontogeny. Primitive erythroid cells originate in the yolk sac and complete their maturation, including enucleation, in the bloodstream. Though species differences exist, the pattern of hematopoietic progenitor cell emergence in the yolk sac is similar in mouse and man. In both species, there is a stage of development where both primitive red blood cells and definitive erythroid progenitors are produced in the yolk sac. An "embryonic" hematopoietic stem cell that engrafts in myeloablated newborn but not adult mice can be detected in the murine yolk sac and embryo. Stem-cell activity in the human yolk sac has not been reported. CONCLUSIONS: The yolk sac is the sole site of embryonic erythropoiesis. However, definitive erythroid, myeloid, and multipotential progenitors also originate in the yolk sac. The relationship between these progenitors and the "embryonic" hematopoietic stem cell has not been elucidated. Yolk sac-derived progenitor cells may seed the developing liver via the circulation and serve as the immediate source of the mature blood cells that are required to meet the metabolic needs of the rapidly growing fetus.

Adeno-associated virus 2-mediated transduction and erythroid lineage-restricted long-term expression of the human beta-globin gene in hematopoietic cells from homozygous beta-thalassemic mice.

Adeno-associated virus 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. Here, we report successful AAV-mediated stable transduction and high-efficiency, long-term, erythroid lineage-restricted expression of a human beta-globin gene in primary murine hematopoietic stem cells in vivo. Bone marrow-derived primitive Sca-1(+), lin(-) hematopoietic stem cells from homozygous beta-thalassemic mice were transduced ex vivo with a recombinant AAV vector containing a normal human beta-globin gene followed by transplantation into low-dose-irradiated B6.c-kitW(41/41) anemic recipient mice. Six months posttransplantation, tail-vein blood samples were analyzed by PCR amplification to document the presence of the transduced human beta-globin gene sequences in the peripheral blood cells. Semiquantitative PCR analyses revealed that the transduced human beta-globin gene sequences were present at approximately 1 copy per cell. The efficiency of the human beta-globin gene expression was determined to be up to 35% compared with the murine endogenous beta-globin gene by semiquantitative RT-PCR analyses. Peripheral blood samples from several positive recipient mice obtained 10 months posttransplantation were fractionated to obtain enriched populations of granulocytes, lymphocytes, and erythroid cells. PCR analyses revealed the presence of the human beta-globin gene sequences in granulocytes and lymphocytes, indicating multilineage reconstitution. However, only the erythroid population was positive following RT-PCR analyses, suggesting lineage-restricted expression of the transduced human beta-globin gene. Southern blot analyses of total genomic DNA samples isolated from bone marrow cells from transplanted mice also documented proviral integration. These results provide further support for the potential use of recombinant AAV vectors in gene therapy of beta-thalassemia and sickle-cell disease.

Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis.

During mouse embryogenesis, two waves of hematopoietic progenitors originate in the yolk sac. The first wave consists of primitive erythroid progenitors that arise at embryonic day 7.0 (E7.0), whereas the second wave consists of definitive erythroid progenitors that arise at E8.25. To determine whether these unilineage hematopoietic progenitors arise from multipotential precursors, we investigated the kinetics of high proliferative potential colony-forming cells (HPP-CFC), multipotent precursors that give rise to macroscopic colonies when cultured in vitro. No HPP-CFC were found at presomite stages (E6.5-E7.5). Rather, HPP-CFC were detected first at early somite stages (E8.25), exclusively in the yolk sac. HPP-CFC were found subsequently in the bloodstream at higher levels than the remainder of the embryo proper. However, the yolk sac remains the predominant site of HPP-CFC expansion (>100-fold) until the liver begins to serve as the major hematopoietic organ at E11.5. On secondary replating, embryonic HPP-CFC give rise to definitive erythroid and macrophage (but not primitive erythroid) progenitors. Our findings support the hypothesis that definitive but not primitive hematopoietic progenitors originate from yolk sac-derived HPP-CFC during late gastrulation.

Recombinant human parvovirus B19 vectors: erythrocyte P antigen is necessary but not sufficient for successful transduction of human hematopoietic cells.

The blood group P antigen, known to be abundantly expressed on erythroid cells, has been reported to be the cellular receptor for parvovirus B19. We have described the development of recombinant parvovirus B19 vectors with which high-efficiency, erythroid lineage-restricted transduction can be achieved (S. Ponnazhagan, K. A. Weigel, S. P. Raikwar, P. Mukherjee, M. C. Yoder, and A. Srivastava, J. Virol. 72:5224-5230, 1998). However, since a low-level transduction of nonerythroid cells could also be detected and since P antigen is expressed in nonerythroid cells, we reevaluated the role of P antigen in the viral binding and entry into cells. Cell surface expression analyses revealed that approximately 75% of primary human bone marrow mononuclear erythroid cells and approximately 31% of cells in the nonerythroid population were positive for P antigen. Two human erythroleukemia cell lines, HEL and K562, and a human promyelocytic leukemia cell line, HL-60, were also examined for P antigen expression and binding and entry of the vector. HEL and K562 cells showed intermediate levels, whereas HL-60 cells demonstrated high levels of expression of P antigen. However, the efficiency of vector binding to these cells did not correlate with P antigen expression. Moreover, despite P antigen positivity and efficient viral binding, HEL, K562, and HL-60 cells could not be transduced with the vector. Low levels of P antigen expression could also be detected in two primary cell types, human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblasts (NHLF). In addition, vector binding occurred in both cell types and was inhibited by globoside, indicating the involvement of P antigen in virus binding to these cells. These primary cells could be efficiently transduced with the recombinant vector. These data suggest that (i) P antigen is expressed on a variety of cell types and is involved in binding of parvovirus B19 to human cells, (ii) the level of P antigen expression does not correlate with the efficiency of viral binding, (iii) P antigen is necessary but not sufficient for parvovirus B19 entry into cells, and (iv) parvovirus B19 vectors can be used to transduce HUVEC and NHLF. These studies further suggest the existence of a putative cellular coreceptor for efficient entry of parvovirus B19 into human cells.

mDYRK3 kinase is expressed selectively in late erythroid progenitor cells and attenuates colony-forming unit-erythroid development.

DYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1, an inhibitor of cell cycle progression in yeast. At present, mDYRK-3 and mDYRK-2 have been cloned, and mDYRK-3 has been characterized with respect to kinase activity, expression among tissues and hematopoietic cells, and possible function during erythropoiesis. In sequence, mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a, but is 91.3% identical overall to hDYRK-3. Catalytically, mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro. Expression of mDYRK-1a, mDYRK-2, and mDYRK-3 was high in testes, but unlike mDYRK1a and mDYRK 2, mDYRK-3 was not expressed at appreciable levels in other tissues examined. Among hematopoietic cells, however, mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells. In developmentally synchronized erythroid progenitor cells, expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone), and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver. Interestingly, antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit-erythroid colony formation. Thus, it is proposed that mDYRK-3 kinase functions as a lineage-restricted, stage-specific suppressor of red cell development. (Blood. 2001;97:901-910)