Biomedical subjects

Mervin C Yoder

Publications and source records attributed to Mervin C Yoder.

At least 19 recordsLinked to original sources

PubMed2006

Neurofibromin plays a critical role in modulating osteoblast differentiation of mesenchymal stem/progenitor cells.

Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1, a pandemic autosomal dominant genetic disorder with an incidence of 1:3000. Individuals with NF1 have a variety of malignant and non-malignant manifestations, including skeletal manifestations, such as osteoporosis, scoliosis and short statures. However, the mechanism(s) underlying the osseous manifestations in NF1 are poorly understood. In the present study, utilizing Nf1 haploinsufficient (+/-) mice, we demonstrate that Nf1+/- mesenchymal stem/progenitor cells (MSPC) have increased proliferation and colony forming unit-fibroblast (CFU-F) capacity compared with wild-type (WT) MSPC. Nf1+/- MSPC also have fewer senescent cells and have a significantly higher telomerase activity compared with WT MSPC. Nf1+/- MSPC have impaired osteoblast differentiation as determined by alkaline phosphatase staining, and confirmed by single CFU-F replating assays. The impaired osteoblast differentiation in Nf1+/- MSPC is consistent with the reduced expression of osteoblast markers at the mRNA level, including osteocalcin and osteonectin. Importantly, re-expression of the full-length NF1 GTPase activating related domain (NF1 GAP-related domain) is sufficient to restore the impaired osteoblast differentiation in Nf1+/- MSPC. Taken together, our results suggest that neurofibromin plays a crucial role in modulating MSPC differentiation into osteoblasts, and the defect in osteoblast differentiation may contribute at least in part to the osseous abnormalities seen in individuals with NF1.

Animals↗

PubMed2006

Rac2 concentrations in umbilical cord neutrophils.

BACKGROUND: Human newborn infants display a variety of immunodeficiencies of immaturity, including diminished neutrophil adhesion, chemotaxis, and migration. Rac2, a guanosine triphosphate-binding protein, is an essential regulator of human neutrophil migration and chemotaxis. Since human subjects and mice deficient in Rac2 display deficiencies in neutrophil functions similar to newborn infants, we postulated that newborn neutrophils may be deficient in Rac2. OBJECTIVES: The aim of the study was to measure Rac1 and Rac2 concentrations in neutrophils from umbilical cord blood. METHODS: Neutrophils from cord and adult blood were isolated, total cell lysates extracted, and Rac protein concentrations determined using Western blot analysis. RESULTS: Rac2 concentrations were significantly lower in the neutrophil protein lysates isolated from cord blood compared to adult blood despite similar levels of Rac1. CONCLUSIONS: Diminished Rac2 expression in cord blood neutrophils may contribute to the defects observed in cord blood neutrophil function.

Aging↗

PubMed2006

Extracellular matrix scaffolds are repopulated by bone marrow-derived cells in a mouse model of achilles tendon reconstruction.

The extracellular matrix derived from porcine small intestinal submucosa (SIS-ECM), an FDA-approved material currently used clinically for rotator cuff repair, has been shown to attract bone marrow-derived cells during in vivo remodeling of a subcutaneous implant and produce chemoattractant peptides following chemical degradation in vitro. The purpose of the present study was to determine if bone marrow-derived cells participate in the long-term remodeling of the Achilles tendon in a mouse model when repaired with SIS-ECM. A 2-mm gap was produced in the Achilles tendon of 40 chimeric mice produced to express green fluorescent protein (GFP) in all of their bone marrow-derived cells. Tendons were repaired by replacing the resected section with autologous tendon tissue or with a single layer sheet of lyophilized SIS-ECM. Four animals from each treatment group were sacrificed at 1, 2, 4, 8, and 16 weeks, and sections were harvested for histologic and fluorescence microscopy. Both groups showed accumulation of GFP-expressing marrow-derived cells at the site of tendon remodeling at 1 and 2 weeks that were associated with areas of angiogenesis and inflammation. By 16 weeks, the SIS-ECM-treated group showed GFP expressing cells throughout the remodeled tendon in the absence of any inflammatory response, while the autologous tendon repair group showed no GFP expressing cells within the tendon except for occasional cells in the lumen of blood vessels. An SIS-ECM scaffold used for tendon repair recruits a population of bone marrow-derived cells that participates in the long-term remodeling process. The ability of SIS-ECM to recruit a population of marrow-derived cells to the remodeling site may alter the default mechanism of tendon healing. The involvement of these cells in the remodeling process may explain in part the process of site specific constructive remodeling as opposed to scar tissue formation when the ECM is used as a biologic scaffold for tendon reconstruction.

Achilles Tendon↗

PubMed2006

Endothelial-monocyte-activating polypeptide II induces migration of endothelial progenitor cells via the chemokine receptor CXCR3.

OBJECTIVE: Recruitment of endothelial progenitor cells to the sites of ischemia has recently been suggested as a mechanism of tissue repair. Here we address the hypothesis that the hypoxia-inducible full-length endothelial-monocyte-activating polypeptide II (EMAP II) provides a mechanism to recruit late outgrowth highly proliferating endothelial progenitor cells (EPCs). MATERIALS AND METHODS: We tested in a transwell migration assay EMAP II for its ability to induce migration of EPCs. Furthermore, we measured changes in cellular calcium levels in EPC to assess the ability of EMAP II to induce intracellular signaling. Finally, we employed neutralizing antibodies and binding competition studies in order to identify the receptor mediating these activities of EMAP II in EPCs. RESULTS: EMAP II elicits dose-dependent migration and intracellular calcium mobilization in EPCs. Functional blocking and binding studies with radiolabeled interferon-gamma-induced protein (IP-10) indicate that EMAP II employs the CXCR3 receptor for these activities in EPCs. Indeed, EMAP II-induced migration of EPCs can be abolished by prior treatment of cells with anti-CXCR3 antibodies or with IP-10. CONCLUSIONS: These data suggests a novel function for EMAP II and a hitherto undescribed role of the CXCR3 chemokine receptor in EPC recruitment.

Cell Movement↗

PubMed2006

Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5.

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.

Animals↗

PubMed2006

The role of Hex in hemangioblast and hematopoietic development.

The orphan homeobox gene Hex has been shown to be critical for normal murine liver development using Hex-/- mice. Because of the early lethality of the Hex-/- mice, however, in vitro embryonic stem cell differentiation assays were employed to elucidate the role of Hex in murine hematopoiesis. These studies revealed that Hex is not required for hemangioblast development but is required in a dose-dependent fashion for hemangioblast differentiation to definitive hematopoietic progenitors. Contained here are the methods needed to conduct embryonic stem cell-derived in vitro hematopoietic assays.

Animals↗

PubMed2005

Reduction of Shp-2 expression by small interfering RNA reduces murine embryonic stem cell-derived in vitro hematopoietic differentiation.

Shp-2 is a member of a small family of cytoplasmic Src homology 2 (SH2) domain-containing protein tyrosine phosphatases. Although Shp-2 has been shown to be necessary for hematopoiesis using a mouse model expressing a mutant residual protein (Shp-2(delta/delta)), we used small interfering RNA (siRNA) to reduce Shp-2 expression and examined the consequences on embryonic stem cell (ESC)-derived hemangioblast, primitive, and definitive hematopoietic development. We found that at a concentration of 50 nM, Shp-2 siRNA effectively diminished Shp-2 expression in differentiating embryoid bodies. Hemangioblast, primitive, and definitive hematopoietic progenitor formation was decreased significantly after transfection with Shp-2 siRNA but not with scrambled siRNA. Because Shp-2 is involved in signals emanating from the basic fibroblast growth factor (bFGF) receptor, we asked whether Shp-2 functions in bFGF-mediated hemangioblast development. Reduction of Shp-2 expression using siRNA, but not scrambled siRNA, blocked the bFGF-induced increase in hemangioblast development. Using siRNA as an independent method of reducing Shp-2 function, in contrast to the mutant mouse model (Shp-2(delta/delta)) previously used, we demonstrate that Shp-2 is required in hemangioblast, primitive, and definitive progenitor hematopoietic development and that Shp-2 is integrally necessary for bFGF-mediated hemangioblast production.

Animals↗

PubMed2005

Knockdown of Pu.1 by small interfering RNA in CD34+ embryoid body cells derived from mouse ES cells turns cell fate determination to pro-B cells.

The factors that regulate murine ES cell-derived hematopoietic progenitor cell (HPC) commitment to the B lymphocyte lineage remain unclear. Pu.1 plays an essential role in the development of all lymphoid lineages; however, it also regulates commitment to other blood cell lineages. In this study, we found evidence for early B cell lineage commitment as determined by coexpression of CD19 and CD45R (B220) when Pu.1 expression was knocked down in HPC by specific small interfering RNA (siRNA); moreover, the expression of early B cell factor (Ebf) and paired box protein 5 (Pax-5) transcription factors was induced when cells were treated by Pu.1 siRNA, but not by control siRNA. We also found that siRNA-mediated knockdown of Pu.1 expression was more efficient in generating progenitor B cells (pro-B cells) compared with the more common in vitro method of B lymphoid development by means of coculture of CD34+ embryoid body (EB) cells with OP9 stromal cells. To investigate whether this phenomenon also exists in HPC from other sources, we then knocked down Pu.1 gene expression in CD34+ murine bone marrow cells and found a similar effect of increased production of CD19+CD43+CD45R+ progenitor B cells upon the siRNA-mediated decrease in Pu.1 expression. We conclude that, in early B cell development from ES cell-derived HPC, constitutive Pu.1 expression inhibits the earliest B cell development through repressing early B cell factor and paired box protein 5 expression, although lower levels of Pu.1 expression in HPC play a key role in promoting B cell fate determination.

Animals↗

PubMed2005

Unresolved questions, changing definitions, and novel paradigms for defining endothelial progenitor cells.

The field of vascular biology has been stimulated by the concept that circulating endothelial progenitor cells (EPCs) may play a role in neoangiogenesis (postnatal vasculogenesis). One problem for the field has been the difficulty in accurately defining an EPC. Likewise, circulating endothelial cells (CECs) are not well defined. The lack of a detailed understanding of the proliferative potential of EPCs and CECs has contributed to the controversy in identifying these cells and understanding their biology in vitro or in vivo. A novel paradigm using proliferative potential as one defining aspect of EPC biology suggests that a hierarchy of EPCs exists in human blood and blood vessels. The potential implications of this view in relation to current EPC definitions are discussed.

Cell Proliferation↗

PubMed2005

Sonic hedgehog and retinoic acid synergistically promote sensory fate specification from bone marrow-derived pluripotent stem cells.

Recent studies demonstrated that stromal cells isolated from adult bone marrow have the competence of differentiating into neuronal cells in vitro and in vivo. However, the capacity of marrow stromal cells or mesenchymal stem cells (MSCs) to differentiate into diverse neuronal cell populations and the identity of molecular factors that confer marrow stromal cells with the competence of a neuronal subtype have yet to be elucidated. Here, we show that Sonic hedgehog (Shh) and retinoic acid (RA), signaling molecules secreted from tissues in the vicinity of peripheral sensory ganglia during embryogenesis, exert synergistic effects on neural-competent MSCs to express a comprehensive set of glutamatergic sensory neuron markers. Application of Shh or RA alone had little or no effect on the expression of these neuronal subtype markers. In addition, incubation of MSCs with embryonic hindbrain/somite/otocyst conditioned medium or prenatal cochlea explants promoted up-regulation of additional sensory neuron markers and process outgrowth. These results identify Shh and RA as sensory competence factors for adult pluripotent cells and establish the importance of interactions between adult pluripotent cells and the host microenvironment in neuronal subtype specification.

Animals↗

PubMed2005

NO role in EPC function.

The nitric oxide pathway plays an important role in modulating endothelial progenitor cell function, playing a critical role in blood vessel repair in response to injury.

Animals↗

PubMed2005

Blood island formation: longstanding observations and modern interpretations.

The yolk sac blood islands have long been recognized as the first site for blood cell emergence during embryonic development. Much of our understanding of how blood islands form is derived from studies conducted in chick embryos. Similar processes are thought to be active during murine blood island development. But, how strong is the evidence that the process of blood island formation in the chick coincides with that documented in the mouse? This review will discuss some of the principle studies that led to two widely accepted definitions of blood islands and will examine more recent studies performed in murine embryos to determine whether either of these popular definitions can be directly applied.

Animals↗

PubMed2005

Application of RNA interference to study stem cell function: current status and future perspectives.

RNA interference is a mechanism displayed by most eukaryotic cells to rid themselves of foreign double-stranded RNA molecules. In the six years since the initial report, RNA interference has now been demonstrated to function in mammalian cells to alter gene expression, and has been used as a means for genetic discovery as well as a possible strategy for genetic correction. An equally popular topic over the past six years has been the proposal to utilize embryonic stem cells or adult stem cells as cell-based therapies for human diseases. The aim of this review is to provide a general overview of how RNA interference suppresses gene expression and to examine some published RNA interference approaches that have resulted in changes in stem cell function and suggest the possible clinical relevance of this work.

Animals↗

PubMed2005

Endothelial cells in the early murine yolk sac give rise to CD41-expressing hematopoietic cells.

Hematopoietic and endothelial cells may be derived from a common precursor cell (hemangioblast) during embryogenesis; however, some evidence suggests that hematopoietic cells may emerge from endothelial cells. The onset of definitive hematopoiesis at E8.25 in the murine embryo is marked by high-level CD41 expression. We questioned whether these hematopoietic cells were derived directly from mesoderm cells or emerged from endothelium. At 8.25 days post coitus (dpc), CD41 was coexpressed with CD31, CD34, and Flk1 in some intraluminal round cells that appeared to arise from flattened endothelial cells lining yolk sac capillary vessels. Cell-sorting studies revealed that all subpopulations of cells expressing CD41 possessed hematopoietic activity. Surprisingly, Tie2(+)Flk1(+) cells, a phenotype enriched in adult endothelial progenitors, also displayed some hematopoietic progenitor activity in vitro, but this activity was restricted to the CD41(+) fraction; only endothelial cells were derived from freshly isolated Tie2 (+)Flk1(bright) CD41() cells. Tie2(+)Flk1(dim)CD41() 8.25-dpc yolk sac cells devoid of hematopoietic progenitor activity gave rise to endothelial-like capillary networks in vitro and differentiated upon co-culture with OP9 stromal cells into definitive hematopoietic progenitors. These results demonstrate that CD41-expressing definitive hematopoietic cells appear to arise from endothelial cells lining nascent capillaries in vivo.

Animals↗

PubMed2005

Clonal multilineage differentiation of murine common pluripotent stem cells isolated from skeletal muscle and adipose stromal cells.

Pluripotent stem cells (PSCs) with transdifferentiation capacity may provide useful therapeutic modalities in the areas of cellular restoration and regenerative medicine. The utility of PSCs depends on their ability to respond to different stimuli and to adapt to tissue-specific differentiation conditions. Given that a number of cells possessing characteristics of PSCs have been identified and isolated from several adult murine tissues, we hypothesized that a common PSC may exist in multiple murine tissues and that these cells may either reside permanently in specific sites or continue to circulate and colonize tissues as needed. Previous data from our laboratory suggest that PSCs exhibiting an immunophenotype of CD45(-)Sca-1(+)c-kit(-)Thy-1(+) can be isolated from multiple murine tissues and may represent putative common PSCs (CoPSCs). To investigate whether the multiple tissue differentiation potential observed with these cells resulted from the presence of different tissue-restricted progenitors within CD45(-)Sca-1(+)c-kit(-)Thy-1(+) cells or was the product of clonal differentiation of CoPSCs, clonality studies were performed. Single skeletal muscle (SM)-derived CoPSCs were expanded for 10 days, and progeny cells were split into three culture conditions designed to stimulate myogenic, adipogenic, and neurogenic differentiation. Analysis of 600 clones indicated that 2.16%, 0.83%, and 0.33% of the total number of plated single cells were capable of unipotent, bipotent, and tripotent differentiation, respectively, into combinations of myocytes, adipocytes, and neuronal cells. Given that SM-derived CoPSCs represent 4.78% of the total cells analyzed, tripotent CoPSCs made up 0.016% of the total muscle cells. Similar results were obtained in clonal analyses using adipose stromal cell (ASC)-derived CoPSCs, suggesting that both SM- and ASC-derived CoPSCs may be phenotypically and functionally identical. Taken together, these data demonstrate that a common PSC can be identified in different murine tissues and suggest that a small fraction of these cells are capable of clonal differentiation into multiple cell types.

Adipocytes↗

PubMed2005

Flow cytometric analysis of hematopoietic development.

More than 30 yr ago, a collection of cells isolated from the bone marrow were first demonstrated to repopulate hematopoiesis in a radioablated animal. These cells self- renewed while producing all of the blood products and were named hematopoietic stem cells (HSCs). Since then, HSCs have been a tremendous boon to both basic science in understanding cell biology and as therapy in a cancer transplant setting. More recent work has shown that the HSCs, and possibly populations of cells residing in other tissues, have the properties of stem cells and the ability to repopulate nonrelated organs and tissues. This promiscuous repopulation has been termed plasticity and is the center of much research and even more debate. Our laboratory has recently proven that the HSCs meet the requirements of a stem cell by self-renewing and producing all of the blood lineages while concurrently demonstrating their ability to produce nonhematopoietic endothelial cells of blood vessels. This plasticity of the HSCs demonstrates hemangioblast activity, proven by both single cell and serial HSC transplants, and is the focus of this chapter.

Animals↗

PubMed2005

Reconstitution of hematopoiesis following transplantation into neonatal mice.

The primary sites of hematopoiesis change during murine ontogeny. The first blood cells emerge in two waves in the yolk sac; primitive erythroblasts, megakaryocytes, and macrophages emerge on embryonic d (E) 7.0, whereas definitive progenitor cells appear as clusters within the yolk sac vasculature on E8.25. Of interest, yolk sac cells isolated prior to d 10.5 fail to engraft in myeloablated adult recipient mice and do not reconstitute hematopoiesis. We describe a method of sublethally myeloablating newborn mice in which E9.0 yolk sac cells engraft and repopulate all lineages of the hematopoietic system for up to 12 mo in primary recipients and up to 6 mo in secondary recipients. The exact mechanisms that permit yolk sac engraftment in the conditioned newborn mice remain elusive, but this method has been used by a number of investigators to pursue transplantation studies using embryo- or fetal-derived donor cells.

Animals↗

PubMed2004

Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.

Endothelial progenitor cells (EPCs) can be isolated from adult peripheral and umbilical cord blood and expanded exponentially ex vivo. In contrast, human umbilical vein endothelial cells (HUVECs) or human aortic endothelial cells (HAECs) derived from vessel walls are widely considered to be differentiated, mature endothelial cells (ECs). However, similar to adult- and cord blood-derived EPCs, HUVECs and HAECs derived from vessel walls can be passaged for at least 40 population doublings in vitro. Based on this paradox, we tested whether EPCs reside in HUVECs or HAECs utilizing a novel single cell deposition assay that discriminates EPCs based on their proliferative and clonogenic potential. We demonstrate that a complete hierarchy of EPCs can be identified in HUVECs and HAECs derived from vessel walls and discriminated by their clonogenic and proliferative potential. This study provides evidence that a diversity of EPCs exists in human vessels and provides a conceptual framework for determining both the origin and function of EPCs in maintaining vessel integrity.

Age Factors↗