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Modular tissue engineering involves assembling tissue constructs with integral vasculature from units containing adipose-derived mesenchymal stromal cells (adMSCs) and endothelial cells. Here, the effects of implant volume and adMSC density on the vascularization of modular tissues were explored. Both parameters affected the contributions of host- and graft-derived vessels, without affecting total vessel density. Increasing implant volume from 0.01 to 0.10 mL increased HIF1α expression and graft-derived vessel density, suggesting a role of hypoxia in graft-derived vessel formation. However, increasing adMSC density within small-volume implants did not increase HIF1α expression. Vascularization of small-volume implants of high (4.3•10^6 cells/mL) and low (1.0•10^6 cells/mL) adMSC densities was dominated by host vessel ingrowth at day 7. By increasing adMSC density, a high proportion of host-derived vessels was maintained to day 14, presumably via paracrine effects. Further dissection of the role of hypoxia in modular tissue engineering remains a promising avenue to pursue.

Modular tissue engineering involves assembling tissue constructs with integral vasculature from units containing adipose-derived mesenchymal stromal cells (adMSCs) and endothelial cells. Here, the effects of implant volume and adMSC density on the vascularization of modular tissues were explored. Both parameters affected the contributions of host- and graft-derived vessels, without affecting total vessel density. Increasing implant volume from 0.01 to 0.10 mL increased HIF1α expression and graft-derived vessel density, suggesting a role of hypoxia in graft-derived vessel formation. However, increasing adMSC density within small-volume implants did not increase HIF1α expression. Vascularization of small-volume implants of high (4.3•10^6 cells/mL) and low (1.0•10^6 cells/mL) adMSC densities was dominated by host vessel ingrowth at day 7. By increasing adMSC density, a high proportion of host-derived vessels was maintained to day 14, presumably via paracrine effects. Further dissection of the role of hypoxia in modular tissue engineering remains a promising avenue to pursue.

Acute myeloid leukemia (AML) often remains refractory to current chemotherapy and transplantation approaches despite many advances in our understanding of mechanisms in leukemogenesis. The bone marrow “niche” or microenvironment, however, may be permissive to leukemia development and studying interactions between the microenvironment and leukemia cells may provide new insight for therapeutic advances. Mesenchymal stem cells (MSCs) are central to the development and maintenance of the bone marrow niche and have been shown to have important functional alterations derived from patients with different hematological disorders. The extent to which MSCs derived from AML patients are altered remains unclear. The aim of this study was to detect changes occurring in MSCs obtained from human bone marrow in patients with AML by comparing their function and gene expression pattern with normal age-matched controls. MSCs expanded from patients diagnosed with acute leukemia were observed to have heterogeneous morphological characteristics compared to the healthy controls. Immunohistochemistry and flow data confirmed the typical cell surface immunophenotype of CD90+ CD105+ CD73+ CD34- CD45-, although MSCs from two patients with AML revealed reduced surface expression of CD105 and CD90 antigens respectively. Differentiation assays demonstrated the potential of MSCs from AML patients and healthy donors to differentiate into bone, fat and cartilage. However, the ability of MSCs from AML samples to support hematopoietic function of CD34+ progenitors was found to be impaired while the key hematopoietic genes were found to be differentially expressed on AML-MSCs compared to nMSCs. These studies indicate that there exist differences in the biologic profile of MSCs from AML patients compared to MSCs derived from healthy donors. The results described in the thesis provide a formulation for additional studies that may allow us to identify new targets for improved treatment of AML.

Severe influenza, recognized as a clinical syndrome characterized by hyper-induction of pro-inflammatory cytokine production, results in approximately 250–500 thousand deaths annually worldwide. Current influenza research is focused on therapeutics to target the influenza virus or modulate influenza virus-induced inflammation as potential treatment options to improve clinical outcome in experimental influenza A (H1N1) virus infection. The goals of this work were: (1) to evaluate the utility of inhaled nitric oxide (iNO) for decreasing influenza virus production in the lungs, and (2) investigate the use of mesenchymal stromal (stem) cells (MSCs) for mitigating deleterious host responses to influenza infection. Here, we report that MSCs and iNO, administered alone either prophylactically or post-influenza virus infection, fail to modulate host inflammation, fail to improve acute lung injury, fail to dampen lung viral load, and fail to improve survival of infected mice.

Severe influenza, recognized as a clinical syndrome characterized by hyper-induction of pro-inflammatory cytokine production, results in approximately 250–500 thousand deaths annually worldwide. Current influenza research is focused on therapeutics to target the influenza virus or modulate influenza virus-induced inflammation as potential treatment options to improve clinical outcome in experimental influenza A (H1N1) virus infection. The goals of this work were: (1) to evaluate the utility of inhaled nitric oxide (iNO) for decreasing influenza virus production in the lungs, and (2) investigate the use of mesenchymal stromal (stem) cells (MSCs) for mitigating deleterious host responses to influenza infection. Here, we report that MSCs and iNO, administered alone either prophylactically or post-influenza virus infection, fail to modulate host inflammation, fail to improve acute lung injury, fail to dampen lung viral load, and fail to improve survival of infected mice.

Discovery of the ability of mesenchymal stem cells (MSCs) to differentiate into cells of non-mesodermal tissues, particularly neuronal cells, have raised the possibility of utilising MSCs in regenerative/reparative therapies for neurological disorders. However, a number of hurdles remain to be resolved. This thesis aims to address some of these issues by investigating the characteristics of bone marrow-derived human MSCs (hMSCs) during long-term culture, the potential of hMSCs to differentiate in vitro toward the neuronal lineage under the influence of cytokines, and the effects of intracerebral transplantation in the hemiparkinsonian rat model. During expansion culture hMSCs were found to display the expected characteristics of MSC populations, and also constitutively expressed neural and pluripotency markers simultaneously with mesodermal markers. Analysis of hMSC long-term subcultivation revealed an optimal period for commencing neuronal differentiation (first 6-8 passages), and also showed the absence of spontaneous neural differentiation. Application of neural-inducing cytokines and culture conditions resulted in the generation of an immature neuronal-like phenotype by hMSCs. Through live cell microscopy it was demonstrated for the first time that cytokine-based hMSC neuronal differentiation occurs through active and dynamic cellular processes involving outgrowth and motility of cellular extensions. In addition, single- and multiple-stage cytokine-based strategies for inducing dopaminergic neuronal-like cells from hMSCs were investigated. These studies revealed that FGF-2 and EGF exerted the greatest benefits for hMSC neuronal differentiation. Undifferentiated and neuronal-primed hMSCs were transplanted intracerebrally into the striatum and substantia nigra of cyclosporine-treated hemiparkinsonian rats. Grafted hMSCs could be clearly identified at 1-day and 7-days post-transplantation; however, grafts were gradually lost over time, with complete absence by 21-days. Co-transplantation with olfactory ensheathing cells, neuronal-priming prior to grafting, and nigral as well as striatal grafting could not provide engraftment and differentiation advantages. Immunohistological analysis demonstrated the presence of innate inflammatory responses (microglia and astrocyte activation) at graft sites, fibronectin deposition by hMSCs, and lack of endogenous host neurogenesis. The results of my PhD work indicate that cytokine-based culture methods are capable of differentiating hMSCs to an immature neuronal-like phenotype, and host-mediated innate inflammatory responses may be a key contributing factor for the failure of in vivo hMSC engraftment.

Discovery of the ability of mesenchymal stem cells (MSCs) to differentiate into cells of non-mesodermal tissues, particularly neuronal cells, have raised the possibility of utilising MSCs in regenerative/reparative therapies for neurological disorders. However, a number of hurdles remain to be resolved. This thesis aims to address some of these issues by investigating the characteristics of bone marrow-derived human MSCs (hMSCs) during long-term culture, the potential of hMSCs to differentiate in vitro toward the neuronal lineage under the influence of cytokines, and the effects of intracerebral transplantation in the hemiparkinsonian rat model. During expansion culture hMSCs were found to display the expected characteristics of MSC populations, and also constitutively expressed neural and pluripotency markers simultaneously with mesodermal markers. Analysis of hMSC long-term subcultivation revealed an optimal period for commencing neuronal differentiation (first 6-8 passages), and also showed the absence of spontaneous neural differentiation. Application of neural-inducing cytokines and culture conditions resulted in the generation of an immature neuronal-like phenotype by hMSCs. Through live cell microscopy it was demonstrated for the first time that cytokine-based hMSC neuronal differentiation occurs through active and dynamic cellular processes involving outgrowth and motility of cellular extensions. In addition, single- and multiple-stage cytokine-based strategies for inducing dopaminergic neuronal-like cells from hMSCs were investigated. These studies revealed that FGF-2 and EGF exerted the greatest benefits for hMSC neuronal differentiation. Undifferentiated and neuronal-primed hMSCs were transplanted intracerebrally into the striatum and substantia nigra of cyclosporine-treated hemiparkinsonian rats. Grafted hMSCs could be clearly identified at 1-day and 7-days post-transplantation; however, grafts were gradually lost over time, with complete absence by 21-days. Co-transplantation with olfactory ensheathing cells, neuronal-priming prior to grafting, and nigral as well as striatal grafting could not provide engraftment and differentiation advantages. Immunohistological analysis demonstrated the presence of innate inflammatory responses (microglia and astrocyte activation) at graft sites, fibronectin deposition by hMSCs, and lack of endogenous host neurogenesis. The results of my PhD work indicate that cytokine-based culture methods are capable of differentiating hMSCs to an immature neuronal-like phenotype, and host-mediated innate inflammatory responses may be a key contributing factor for the failure of in vivo hMSC engraftment.

Thesis (M.Sc.). / Written for the Division of Experimental Medicine. Title from title page of PDF (viewed 2009/06/18). Includes bibliographical references.

Made available in DSpace on 2015-03-03T11:52:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-03-07Bitstream added on 2015-03-03T12:06:10Z : No. of bitstreams: 1 000799295.pdf: 1434919 bytes, checksum: 36275b6266ae49abeed7a7bba084d604 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Células-tronco mesenquimais (CTMs) obtidas a partir da medula óssea são capazes de se diferenciarem, sobretudo, em condrócitos, adipócitos e osteoblastos. Durante a osteogênese in vitro, alguns parâmetros são utilizados para caracterizar este processo, tais como atividade da fosfatase alcalina (FAL), mineralização e expressão de proteínas associadas à osteoblastos. Ratos espontaneamente hipertensos (SHR) são um modelo animal de hipertensão essencial humana e desenvolvem hipertensão após 4 semanas de idade. Esta linhagem apresenta alterações significativas no metabolismo ósseo. O objetivo do presente estudo foi investigar se, o genótipo hipertensivo poderia interferir na diferenciação osteoblástica das CTMs de ratos SHR e qual mecanismo está alterado quando comparadas com a linhagem progenitora, ratos Wistar. Para isso, nós obtivemos CTMs da medula óssea de ratos Wistar e SHR com 4 semanas de idade, sem a hipertensão estabelecida, afim de avaliar somente o possível efeito do genótipo hipertensivo na diferenciação osteogênica in vitro. Nós induzimos, ou não, a diferenciação osteogênica in vitro por meio da utilização dos indutores osteogênicos: ácido ascórbico, ?-glicerofosfato e dexametasona. Os resultados demonstraram que, CTMs indiferenciadas de SHR (SHRC) demonstraram taxa de proliferação aumentada em comparação a CTMs, na mesma condição, de Wistar (WC), e após a indução da osteogênica, a taxa de proliferação apresentou uma diminuição acentuada no grupo SHR (SHRMO) do que no grupo Wistar na mesma condição (WMO). Embora não fora observada diferença significativa na atividade da FAL entre SHRMO e WOM no 7° dia, a mineralização e a diferenciação osteoblástica foram menores no grupo SHRMO no mesmo período experimental. Os fatores de transcrição Osterix e ?-catenina parecem estar envolvidos na diferenciação reduzida no grupo SHRMO... / Mesenchymal stem cells (MSCs) from bone marrow are able to differentiate mainly into chondrocytes, adipocytes and osteoblasts. During in vitro osteogenesis, some parameters are used to characterize this process, such as the activity of alkaline phosphatase (ALP), mineralization and osteoblast-associated proteins expression. Spontaneously hypertensive rats (SHR) is an animal model of human essential hypertension. This animals developing hypertension after 4 weeks of age. This strain shows significant changes in bone metabolism. The aim of this study was to investigate whether the hypertensive genotype could influence the osteoblastic differentiation of MSCs from SHR and which mechanism are altered when compared to the parental strain, Wistar rats. For that, we have obtained bone marrow MSCs from Wistar and SHR rats at 4 weeks of age, without hypertension established in order to evaluate only the possible effect of hypertensive genotype on osteogenic differentiation in vitro. We induced or non-osteogenic differentiation in vitro using osteogenic inducers: ascorbic acid, dexamethasone and ?-glycerophosphate. The results demonstrate that undifferentiated MSCs SHR (SHRC) showed increased proliferation rate compared to MSCs, in the same condition Wistar (WC) and after osteogenic induction, proliferation rate showed a marked decrease in SHR (SHRMO) than in Wistar group in the same condition (WMO). Although it was not observed significant difference in ALP activity between WMO and SHRMO on day 7, mineralization and osteoblast differentiation were lower on group SHRMO in the same experimental period. The transcription factors Osterix and ?-catenin appear to be involved in reduced differentiation in SHRMO group because they showed lower expression in this experimental group. Furthermore, the decreased... / FAPESP: 12/01924-9 / FAPESP: 11/06070-5 / FAPESP: 11/19458-1