Vasculogenic cell-based therapy combined with tissue engineering is a promising revascularization strategy for patients with hibernating myocardium, a common clinical condition. We used a clinically relevant swine model of hibernating myocardium to examine the benefits of biopolymer-supported delivery of circulating angiogenic cells (CACs) in this context. Twenty-five swine underwent placement of an ameroid constrictor on the left circumflex artery (LCx). After 2 weeks, positron emission tomography measures of myocardial blood flow (MBF) and myocardial flow reserve (MFR) were reduced in the affected region (both p<0.001). Hibernation (mismatch) was specific to the LCx territory. Swine were randomized to receive intramyocardial injections of PBS control (n=10), CACs (n=8), or CACs + a collagen-based matrix (n=7). At follow-up, stress MBF and MFR were increased only in the cells+matrix group (p<0.01), and mismatch was lower in the cells+matrix treated animals (p=0.02) compared to controls. Similar results were found using microsphere-measured MBF. Wall motion abnormalities and ejection fraction improved only in the cells+matrix group. This preclinical swine model demonstrated ischemia and hibernation, which was improved by the combined delivery of CACs and a collagen-based matrix. To our knowledge, this is the first demonstration of the mechanisms and effects of combining progenitor cells and biopolymers in the setting of myocardial hibernation, a common clinical condition in patients with advanced coronary artery disease.

Fetal growth in utero depends on nutrient and oxygen reaching the fetus through the uterine and placental microcirculations, both undergoing massive expansion during pregnancy. Aberrations of the placental vasculature are associated with Intrauterine Growth Restriction (IUGR), a common pathological outcome of pregnancy; however, the cellular components responsible for vessel formation in the placenta and the uterus remain unknown. Endothelial Progenitor Cells (EPC) are a group of morphologically and functionally varied bone marrow derived vasculogenic cell types, divided into two major subsets: (i) Circulating Angiogenic Cells (CACs), which promote vessel formation by interfering with the extracellular matrix and (ii) Endothelial Colony Forming Cells (ECFCs), which provide the source for new endothelium. This role has been demonstrated in pathophysiological studies, but not in normal physiological events in vivo. Fetal ECFCs are more proficient than their adult counterparts, but it is unclear in what specific fetal or maternal physiological situations fetal ECFCs are involved. Based upon these considerations, it was hypothesised that: (i) fetal-derived ECFCs play a role in placental vasculogenesis, (ii) these cells transmigrate the placenta and home to loci of vessel formation in the pregnant uterus, and that (iii) intrinsic alterations in their capabilities are associated with fetal growth restriction during intrauterine life. To support these hypotheses the following experiments were performed;(i) EPCs in blood from pairs of human umbilical arteries and veins were counted by flow cytometry. Numbers of EPCs in these samples showed an arterio-venous gradient suggesting their placental sequestration. Furthermore, ECFCs were isolated from human umbilical blood using established culture techniques. Labelled human fetal ECFCs were transplanted into the circulation of murine fetuses using an ultrasound-guided intra-cardiac injection. Using a fluorescent imager and microscopy these cells were shown to home to the murine placenta and participate in vasculogenesis.(ii) Male mice ubiquitously expressing eGFP were crossbred with native females, and fetal (eGFP-positive) endothelial-like cells integrated into the uterine microvasculature. Human fetal ECFCs injected into murine fetuses were shown to migrate to the maternal uterus and became functionally involved with the microvasculature. In humans, microvessels were isolated from uterine biopsies of mothers with male offspring. Copies of the male specific SRY gene (quantified by RT-QPCR) indicated that cells of fetal origin constituted 12% of the endothelium in these vessels. In cross-sections, hybridisation of the Y-chromosome demonstrated the presence of fetal cells in the maternal endothelium of the human uterus. (iii) Using flow cytometry, fewer EPCs were defined within the peripheral circulation of growth-restricted babies. Functional assays showed that ECFCs derived from these growth-restricted cases had intrinsically impaired proliferation, migration, matrix-metalloproteinase (MMP-2) production, and generated fewer blood vessels in a murine vasculogenic bioassay. These results demonstrated the vasculogenic capacity of human fetal ECFCs in vivo and established them as key players in human placental vasculogenesis and uterine vessel expansion. Notably, these results also showed a link between impaired function of fetal ECFCs and IUGR, which is associated with increased cardiovascular risk of both the fetus as an adult, and mother in later life. From these findings it could be speculated, that intrinsic changes in ECFC-biology may be the causative link between IUGR and fetal and maternal cardiovascular susceptibility. Insight into these processes may contribute to early diagnosis, prevention and treatment of IUGR and associated conditions.

Vasculogenic cell-based therapy combined with tissue engineering is a promising revascularization strategy for patients with hibernating myocardium, a common clinical condition. We used a clinically relevant swine model of hibernating myocardium to examine the benefits of biopolymer-supported delivery of circulating angiogenic cells (CACs) in this context. Twenty-five swine underwent placement of an ameroid constrictor on the left circumflex artery (LCx). After 2 weeks, positron emission tomography measures of myocardial blood flow (MBF) and myocardial flow reserve (MFR) were reduced in the affected region (both p<0.001). Hibernation (mismatch) was specific to the LCx territory. Swine were randomized to receive intramyocardial injections of PBS control (n=10), CACs (n=8), or CACs + a collagen-based matrix (n=7). At follow-up, stress MBF and MFR were increased only in the cells+matrix group (p<0.01), and mismatch was lower in the cells+matrix treated animals (p=0.02) compared to controls. Similar results were found using microsphere-measured MBF. Wall motion abnormalities and ejection fraction improved only in the cells+matrix group. This preclinical swine model demonstrated ischemia and hibernation, which was improved by the combined delivery of CACs and a collagen-based matrix. To our knowledge, this is the first demonstration of the mechanisms and effects of combining progenitor cells and biopolymers in the setting of myocardial hibernation, a common clinical condition in patients with advanced coronary artery disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Cigarette smoking (CS) continues to be a significant modifiable factor contributing to a variety of diseases including cardiovascular, pulmonary and renal pathologies. It was suggested that smoking have detrimental effect of the body’s progenitor cells of bone marrow and peripheral organs. Since the concept of cell therapy that utilizes adipose stem/stromal cells (ASC) is gaining momentum it becomes critical to assess the therapeutic activities of the progenitors isolated from smokers. This study has revealed that CS negatively impacts the vasculogenic potential of ASC, in vitro, as well as weakening their therapeutic activity in vivo when tested in mouse model of hindlimb ischemia. We hypothesized that the decrease in vasculogenic activity of ASC is attributed to a higher level of expression of an angiostatic factor Activin A by ASC from CS donors. These findings clearly suggest that smokers should be evaluated for potential exclusion from early clinical trials of autologous cell therapies, or assessed as a separate cohort. The donor’s health status should be considered when choosing between autologous vs allogeneic cell therapies. We then examined the effect of CS on development of kidney pathology in mice. CS exposure led to decrease in kidney weights, capillary rarefaction, and cortical blood perfusion, and in parallel led to increase in kidney fibrosis and iron deposition. Interestingly, infusion of healthy ASC to the mice following CSexposure reversed CS-induced damages. This strongly support the notion that ASC-based therapy may provide rejuvenation effect. In the other subset of studies, we hypothesized that CS-induced lung emphysematous changes are preceded by suppression of bone marrow (BM) hematopoietic progenitor cells (HPC). We have revealed that intermittent BM mobilization with AMD3100 may mitigate the CS-induced myelo-suppression and deterioration of lung function and morphology. We observed that treatment of mice with AMD3100, while exposed to CS, preserves HPC at the levels of healthy control mice. Furthermore, AMD3100 treatment preserved lung parenchyma from pathological changes. These data suggest that while CS has a myelo-suppressive effect, administration of AMD3100 preserved BM-HPC and ameliorated lung damage.

No description available.

Development and regeneration of the vertebrate eye are the result of complex interactions of regulatory networks and spatiotemporally controlled gene expression events. During embryonic retinal development, the coordination of cell signaling and transcriptional regulation allows for a relatively homogenous sheet of neuroepithelial cells to proliferate and differentiate in-to a multilayered, light sensitive retinal tissue. Following injury, the retinas of many cold-blooded vertebrates, such as the zebrafish, undergo a proliferative response that results not only in new retinal cells of the correct type in the correct location, but also functional integration of these cells and restoration of vision. In order for embryonic retinal neurogenesis to proceed correctly, systems must be in place that restrict subsets of progenitor cells from differentiation. Pools of actively proliferating retinal progenitor cells are maintained to fill the needs of developmental processes and normal growth of the retina. In addition, subsets of radial glia in the retina retain the ability to de-differentiate into proliferating progenitor cells to meet the demands of the regenerating retina. All of these processes rely on the tight coordination of extrinsic and intrinsic cues, as well as regulation of gene expression by transcription factors. Although a considerable amount of work has been conducted to identify key regulators of retinal development and regeneration, many gene regulatory networks which include both master signaling pathways as well as individual transcription factors remain poorly characterized. Some of these factors implicated in retinal development and regeneration are members of the Hairy/Enhancer of Split (Hes) superfamily of genes, including the Hairy-related (Her) factors Her4 and Her9. Her transcription factors are basic-helix-loop-helix-orange (bHLH-O) transcription factors that bind to palindromic E- and N-box canonical sequences in the promoters of target genes. Her factors have been previously shown to play roles in a diverse array of developmental and neurogenic processes, including neural tube closure, floor plate development, somitogenesis, and development of various components of the central nervous system as well as the cranial sensory placodes. The roles of her4 and her9 in retinogenesis, however, remain undefined. To determine the possible roles of her4 and her9 factors in the retina, I characterized the expression patterns of these factors during developmental retinal neurogenesis and/or regeneration, examined loss of function phenotypes, and identified signaling pathways that modulate expression of these factors. Chapter 1 of this dissertation provides an overview of vertebrate retina and retinal development, the known functions of her4 in other tissues, and the Notch-Delta signaling pathway. Chapter 2 provides evidence that her4 is a primary effector of the Notch pathway during retinal development, and examines the role of her4 expressing cells during regeneration of the mature zebrafish retina within the context of both chronic and acute photoreceptor damage paradigms. In addition, generation and validation of the transgenic her4:Kaede zebrafish which was used to identify the lineage of her4-expressing cells is described. Characterization of her9 during retinal development, identification of the retinoic acid signaling pathway as a regulator of her9 expression in the retina, and the role her9 plays during retinal vasculogenesis are discussed in Chapter 3. Chapter 4 discusses the generation of her9 knock-out zebrafish lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterization of mutant phenotypes in mosaic her9 mutant F0 fish. In addition, in Chapter 4 I also discuss the screening processes used to identify and characterize genetic lesions in the her9 allele and establish various lines that stably transmit deleterious her9 alleles in the germline, and provide preliminary data of the her9 mutant phenotype. Finally, in Chapter 5 I discuss conclusions from the data generated from this dissertation, additional studies that would expand upon this work, and the implications of these results on the broader understanding of retinal development and regeneration. My dissertation incorporates reverse genetic analysis in zebrafish, biochemical analysis, transgenesis, and various molecular approaches to help better understand the roles of her4 and her9 during retinal neurogenesis. Moreover, these studies may also contribute to a better understanding of retinal development, and disease pathogenesis. It is my hope that this work could also ultimately contribute, even if in some small way, to the goal of enabling human patients who have suffered from vision loss a means by which a damaged retina could be regenerated and functional vision restored.

A odontologia moderna, mesmo usando as suas técnicas mais primorosas, na prática, ainda recupera a perda dental com implantes metálicos recobertos por coroas protéticas. Há um empenho coletivo dos cientistas em criar técnicas de desenvolvimento dental in vitro na busca por maneiras de recuperar, de maneira biológica, a ausência dental. Já é possível criar estruturas similares a dentes a partir de células-tronco de origem dental (polpa de dentes permanentes e decíduos) e não dental (células-tronco embrionárias, células-tronco da medula óssea e da crista neural) por meio de recombinação dos tecidos epiteliais e mesenquimais de germes dentais. As técnicas de reconstrução tecidual nunca estiveram tão perto do desenvolvimento da terceira dentição mas a ciência ainda tem muito a aprender no que concerne o estudo da biologia dental e engenharia de tecidos. Não basta saber como um dente se desenvolve; há de se entender como ele interage com o organismo do qual faz ou fará parte. É com esta preocupação que nos propomos a estudar se pode haver uma correlação entre o desenvolvimento do sistema nervoso e vascular de um germe dental transplantado com a posição que ele é implantado e/ou com a presença de tecido ósseo que no leito receptor. Ademais, buscamos saber se o estágio de desenvolvimento do germe dental a ser transplantado pode influenciar a formação de tecido nervoso e vascular na polpa dental ou não. Nossos resultados revelaram que o local do sítio do implante influencia diretamente o desenvolvimento dental e que isto é tempo dependente. A vascularização e a reinervação da polpa dental nos espécimes implantados nas tíbias é mais semelhante ao grupo controle que os implantados nos rins e isto independe da posição de implantação dental. Entretanto, a polpa dental dos germes implantados nos rins parece estar comumente mais sadia, conter mais odontoblastos viáveis e ser capaz de produzir tecidos mineralizados como a osteodentina. / Contemporary dentistry, even using modern techniques, still deal with missing teeth using metal implants coated by prosthetic crowns. However, there is a worldwide effort to develop a biotooth using in vitro techniques. In this way it is already possible to generate structures similar to teeth using recombination of odontogenic and non odontogenic cells in tissue engineering experiments. The transplant of the recombined cells into a host is a necessary and major step to obtain the biotooth. In this context, at the same time that the development of an appropriate sensorial and vascular system in the biotooh is required, there are many unclear questions about it. Therefore, herein we intend to analyze (I) whether may exist a correlation between the stage of development and vascular and nervous re-growth in the dental pulp after tooth transplantation; (II) if the absence or presence of bone could influence this processes or (III) if the position of implantation could change the vascular and/or nervous development in the transplanted tooth. Our results showed that the site of implantation directly alter tooth development modifying morphogenesis and expression of different vascular, perivascular and neural markers in a time dependant way. The re-growth of the vascular and neural tissue on samples transplanted to the tibia is more similar to the control group than the kidney ones and it is non dependant of the position of implantation. However, the pulp tissue of the samples transplanted under the kidney capsule seemed to be healthier as they were capable of producing mineralized tissue such as osteodentin and still had live odontoblasts.

A odontologia moderna, mesmo usando as suas técnicas mais primorosas, na prática, ainda recupera a perda dental com implantes metálicos recobertos por coroas protéticas. Há um empenho coletivo dos cientistas em criar técnicas de desenvolvimento dental in vitro na busca por maneiras de recuperar, de maneira biológica, a ausência dental. Já é possível criar estruturas similares a dentes a partir de células-tronco de origem dental (polpa de dentes permanentes e decíduos) e não dental (células-tronco embrionárias, células-tronco da medula óssea e da crista neural) por meio de recombinação dos tecidos epiteliais e mesenquimais de germes dentais. As técnicas de reconstrução tecidual nunca estiveram tão perto do desenvolvimento da terceira dentição mas a ciência ainda tem muito a aprender no que concerne o estudo da biologia dental e engenharia de tecidos. Não basta saber como um dente se desenvolve; há de se entender como ele interage com o organismo do qual faz ou fará parte. É com esta preocupação que nos propomos a estudar se pode haver uma correlação entre o desenvolvimento do sistema nervoso e vascular de um germe dental transplantado com a posição que ele é implantado e/ou com a presença de tecido ósseo que no leito receptor. Ademais, buscamos saber se o estágio de desenvolvimento do germe dental a ser transplantado pode influenciar a formação de tecido nervoso e vascular na polpa dental ou não. Nossos resultados revelaram que o local do sítio do implante influencia diretamente o desenvolvimento dental e que isto é tempo dependente. A vascularização e a reinervação da polpa dental nos espécimes implantados nas tíbias é mais semelhante ao grupo controle que os implantados nos rins e isto independe da posição de implantação dental. Entretanto, a polpa dental dos germes implantados nos rins parece estar comumente mais sadia, conter mais odontoblastos viáveis e ser capaz de produzir tecidos mineralizados como a osteodentina. / Contemporary dentistry, even using modern techniques, still deal with missing teeth using metal implants coated by prosthetic crowns. However, there is a worldwide effort to develop a biotooth using in vitro techniques. In this way it is already possible to generate structures similar to teeth using recombination of odontogenic and non odontogenic cells in tissue engineering experiments. The transplant of the recombined cells into a host is a necessary and major step to obtain the biotooth. In this context, at the same time that the development of an appropriate sensorial and vascular system in the biotooh is required, there are many unclear questions about it. Therefore, herein we intend to analyze (I) whether may exist a correlation between the stage of development and vascular and nervous re-growth in the dental pulp after tooth transplantation; (II) if the absence or presence of bone could influence this processes or (III) if the position of implantation could change the vascular and/or nervous development in the transplanted tooth. Our results showed that the site of implantation directly alter tooth development modifying morphogenesis and expression of different vascular, perivascular and neural markers in a time dependant way. The re-growth of the vascular and neural tissue on samples transplanted to the tibia is more similar to the control group than the kidney ones and it is non dependant of the position of implantation. However, the pulp tissue of the samples transplanted under the kidney capsule seemed to be healthier as they were capable of producing mineralized tissue such as osteodentin and still had live odontoblasts.

<p>The present study was conducted in order to assess a role of the SH2 domain-containing adapter protein SHB in development and cell differentiation.</p><p>Embryonic stem (ES) cells overexpressing SHB and SHB with an inactive SH2 domain (R522K-SHB) were obtained. Microarray analysis in the SHB clone revealed altered expression of genes connected with neural cell function. The R522K-SHB clone exhibited altered expression of several transcription factors related to development. ES cells were differentiated by forming aggregates named embryoid bodies (EBs). The morphology of EBs was altered in the R522K-SHB clones, which showed fewer cavities. Expression of endodermal markers was decreased in the R522K-SHB EBs. </p><p>To further investigate the role of SHB in differentiation, murine ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-) were generated. SHB deficient clones increased the expression of mesendodermal and endodermal markers and decreased expression of two receptors, VEGFR2 and FGFR1, connected with blood vessel differentiation. Similarly, blood vessels showed an altered morphology in SHB+/- and SHB-/- EBs after VEGF stimulation. SHB-/- ES cells also formed fewer blood colonies than control ES cells.</p><p>Finally, the role of the SHB adapter protein in vivo was analyzed by generating a SHB deficient mouse (SHB-/-). SHB-/- animals are viable, fertile, but suffer from leukopenia and anemia. SHB-/- animals demonstrate an abnormal morphology of blood vessels in the liver and kidney. Breeding of SHB+/- animals revealed an abnormal segregation of the mutant allele with an increased number of SHB+/- animals and a decreased number of SHB-/- and SHB+/+animals. Backcross analysis of SHB+/- females with SHB+/+ males displayed an increased number of SHB+/- offspring already at the blastocyst level. Simultaneously, embryos from SHB+/- mothers show an increased malformation rate in comparison to embryos from SHB+/+ mothers.</p><p>In summary, the study suggests a role of SHB in reproduction and development and in mesodermal and endodermal specification. </p>

The present study was conducted in order to assess a role of the SH2 domain-containing adapter protein SHB in development and cell differentiation. Embryonic stem (ES) cells overexpressing SHB and SHB with an inactive SH2 domain (R522K-SHB) were obtained. Microarray analysis in the SHB clone revealed altered expression of genes connected with neural cell function. The R522K-SHB clone exhibited altered expression of several transcription factors related to development. ES cells were differentiated by forming aggregates named embryoid bodies (EBs). The morphology of EBs was altered in the R522K-SHB clones, which showed fewer cavities. Expression of endodermal markers was decreased in the R522K-SHB EBs. To further investigate the role of SHB in differentiation, murine ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-) were generated. SHB deficient clones increased the expression of mesendodermal and endodermal markers and decreased expression of two receptors, VEGFR2 and FGFR1, connected with blood vessel differentiation. Similarly, blood vessels showed an altered morphology in SHB+/- and SHB-/- EBs after VEGF stimulation. SHB-/- ES cells also formed fewer blood colonies than control ES cells. Finally, the role of the SHB adapter protein in vivo was analyzed by generating a SHB deficient mouse (SHB-/-). SHB-/- animals are viable, fertile, but suffer from leukopenia and anemia. SHB-/- animals demonstrate an abnormal morphology of blood vessels in the liver and kidney. Breeding of SHB+/- animals revealed an abnormal segregation of the mutant allele with an increased number of SHB+/- animals and a decreased number of SHB-/- and SHB+/+animals. Backcross analysis of SHB+/- females with SHB+/+ males displayed an increased number of SHB+/- offspring already at the blastocyst level. Simultaneously, embryos from SHB+/- mothers show an increased malformation rate in comparison to embryos from SHB+/+ mothers. In summary, the study suggests a role of SHB in reproduction and development and in mesodermal and endodermal specification.