As células tronco espermatogoniais (SSCs) são caracterizadas pela capacidade de autorrenovação, proliferação e transmissão das informações genéticas. Em caninos a primeira tentativa de xenotransplante não obteve o sucesso da produção de espermatozoides, no entanto, há evidências de que as células testiculares xenogênicas podem ser transplantadas no testículo do animal hospedeiro, e gerar espermatozoides viáveis do doador. Portanto, este estudo tem como objetivo realizar o xenotransplante das células germinativas caninas em camundongos imunosuprimidos, e com isto promover à produção de espermatozoides caninos viáveis, geneticamente modificados. E por meio desta técnica, analisar a eficiência da espermatogênese pós-transplante. Células germinativas testiculares foram caracterizadas, isoladas e cultivadas de cães pré-púberes, por meio de sistemas de cultura de enriquecimento e fatores de crescimento. As células foram transduzidas com um gene repórter GFP e LacZ, e por um vetor lentiviral para indentificar as SSCs nos testículos receptores. As SSCs transduzidas foram transplantadas nos testículos de camundongos (C57BL/6) tratados com Busulfan, após diferentes períodos os animais receptores foram eutanasiados e analisados. Aos 10 dias de cultivo as células germinativas adultas foram positivas para CD49f, CD117, e com 5 dias uma expressão semelhante de GFRA1 e DAZL, demonstrando a presença de SSCs e algumas células em meiose. Transplantamos 105 células e 20-43% das células transplantadas foram identificadas na membrana basal dos túbulos seminíferos do animal receptor. Portanto, o transplante das células germinativas caninas, mostrou que a purificação e o cultivo realizados são possíveis para obter SSCs caninas, as quais colonizaram os túbulos seminíferos dos camundongos imunodeficientes e mantiveram-se vivas na membrana basal por 90 dias após transplante, mesmo que estes animais tenham distância filogenética / The spermatogonial stem cells (SSCs) are characterized by the capacity for self-renewal, proliferation and transmission of genetic information. In canines the first attempt to xenotransplantation did not achieve the success of sperm production. However, there is evidence that testicular xenogeneic cells can be transplanted into the testis of the host animal, and generate viable sperm donor. Therefore, this study aims conduct xenotransplantation of the canine germ cells in immunosuppressed mice, and thereby promote the production of viable sperm canines, genetically modified. And by this technique, analyze the efficiency of post-transplant spermatogenesis. Testicular germ cells were identified, isolated and cultured prepuberes dogs through enrichment culture systems and growth factors. Cells were modificated with a reporter gene GFP and LacZ. The SSCs canine was transplanted in mice (C57Bl/6), after different period and then the recipient animals were euthanized and analyzed. After 10 days in culture the germ cells were positive for CD49f, CD117, and 5 days a similar expression of GFRA1 and DAZL was observed, demonstrating the presence of SSCs and some cells in meiosis. 105 cells were transplanted and 20-43% of the transplanted cells were identified in the basement membrane of the seminiferous tubules after 90 days after transplantation. Therefore, the transplantation of canine germ cells showed that the cultivation and purification are performed possible for canine SSCs, it can colonize the seminiferous tubules the mice infertility remained alive in the basement membrane for 90 days after transplanting, even though these animals have phylogenetic distance

Objetivos: Avaliar o potencial terapêutico e a segurança do uso intravítreo de (FMMO) contendo células CD34+ em pacientes portadores de degeneração macular relacionada com a idade na forma atrófica. Casuística e Métodos: Foram avaliados 10 pacientes com degeneração macular relacionada à idade (DMRI) seca e acuidade visual no pior olho <=20/100. Foi obtido aspirado da medula óssea de todos os pacientes, e após o processamento do material no hemocentro, foi injetado 0,1 ml da suspensão de FMMO intravítreo no olho com pior acuidade. Os pacientes foram avaliados no baseline, 1, 3, 6, 9 e 12 meses após a injeção. Todos realizaram medida da melhor acuidade visual corrigida (MAVC), microperimetria, eletrorretinografia multifocal (ERGmf), autofluorescência, Tomografia de coerência óptica (OCT) e responderam o questionário VFQ-25 em todos seguimentos. Também foi realizada angiografia fluoresceínica antes da injeção, seis e doze meses após. Resultados: Todos os pacientes completaram o seguimento de seis meses, e seis finalizaram os doze meses. Antes da injeção, a média da MAVC foi de 1,18 logMAR (20/320-1); variando de 20/125 a 20/640-2. Aos doze meses, a média foi de 1,0 logMAR (20/200), com melhora significativa em todos os meses de seguimento. A média do limiar de sensibilidade na microperimetria mostrou melhora significativa a partir do sexto mês (p=0,009). Ao se dividirem os pacientes com área de atrofia maior e menor, por meio da medida da hipoautofluorescência, observou-se que a melhora foi significativa apenas no grupo de menor atrofia. Não houve diferença significativa na ERGmf. A angiofluoresceinografia não apresentou crescimento de neovasos ou tumores. O questionário de qualidade de vida mostrou diferença significativa na saúde mental (p=0,003) e na visão de cores (p=0,005) e forte tendência à significância na análise que abordou a visão geral (p=0,05) e dependência (p=0,067). Houve declínio em relação à saúde geral (p=0,77). Conclusões: Os resultados indicam que o uso de FMMO intravítreo em pacientes com DMRI é seguro e está associado à melhora da acuidade visual e microperimetria. Pacientes com área menor de atrofia têm melhor resposta. Não é possível afirmar, mas acredita-se no resgate funcional das células em sofrimento, que ainda não se degeneraram. / Objectives: To assess the therapeutic potential and safety of the intraviteral use of a bone marrow mononuclear fraction (BMMF) containing CD 34+ cells in patients with the atrophic form of age-related macular degeneration (AMD). Casuistic and Methods: The study was conducted on 10 patients with the atrophic form of ARMD with worse eye visual acuity <=20/100. Bone marrow was aspirated from each patient under local anesthesia. After processing and separation of mononuclear cells, 0.1 ml of the suspension was injected intravitreally into the eye of lower acuity. The patients were evaluated at baseline and 1, 3, 6, 9 and 12 months after the injection. All were submitted to measurement of best corrected visual acuity (BCVA), microperimetry, multifocal electroretinography (ERGmf), autofluorescence, and optical coherence tomography (OCT) and all responded to the VFQ-25 questionnaire at all follow-up visits. Fluorescein angiography was performed before the injection and six and 12 months later. Results: All patients completed the six month follow-up and six completed the 12 month follow-up after the injection. Before the injection, mean BCVA was 1.18 logMAR (20/320-1), ranging from 20/125 to 20/640-2 and at 12 months it was 1.0 logMAR (20/200), with a significant improvement over all followup months. Mean perimetric threshold sensitivity improved significantly starting at the sixth month (p=0.009). When the patients with a larger area of atrophy measured by hypoautofluorescence were considered separately, a significant improvement was observed only in the group with lower atrophy. There was no significant difference in electroretinography. Angiofluoresceinography did not reveal neovessel or tumor growth. The quality of life questionnaire showed a significant difference in mental health (p=0.003) and color vision (p=0.005), and a strong tendency in the analysis of general vision (p=0.05) and dependence (p=0.067). There was a decline in general health (p=0.77). Conclusions: The results indicate that the use of intravitreal BMMF in patients with AMD is safe and is associated with improved visual acuity and microperimetry. Patients with a smaller atrophy area obtained a better response. The paracrine effect of these cells may explain the functional improvement observed in the present study; however, a larger series should be study to confirm these clinical findings.

Avaliou-se a proliferação das células tronco da polpa de dentes decíduos esfoliados humanos (SHED) após aplicação única do laser de baixa potência. Foi realizada a análise da viabilidade das SHED cultivadas sob déficit nutricional e em condições ideais após irradiação com o laser de baixa potência vermelho de Indio Gálio Alumínio e Fósforo - InGaAlP (660nm, 40mW e 10J/cm2) e infravermelho (780nm, 40mW e 10J/cm2) durante 4 e 8 segundos, nos períodos de 24, 48 e 72 horas através dos ensaios de redução do MTT e do ensaio colorimétrico de Busatti e Gomes. Para análise estatística utilizou-se o teste ANOVA complementado pelo teste de Tukey com nível de significância de 5% (p< 0,05). Observou-se tanto com o MTT quanto com o ensaio colorimétrico de Busatti e Gomes uma tendência de aumento da proliferação celular diretamente relacionada à dose do LBP, estatisticamente significante nos períodos de 24, 48 e 72 horas. Ao analisar os resultados e considerando os parâmetros utilizados e o protocolo de LBP, pode-se concluir que o LBP promoveu a proliferação das SHED tanto a 660 nm quanto a 780nm, pode influenciar a viabilidade e a proliferação das SHED nas doses e comprimentos de onda utilizados e os ensaios do MTT e colorimétrico de Busati e Gomes demonstraram dentro de suas limitações ser eficientes para determinar a viabilidade e proliferação das SHED. / It was evaluated the proliferation of stem cells from human exfoliated deciduous teeth (SHED) after a single application of low power laser. The viability of SHED grown under ideal conditions and under nutritional deficit after irradiation with red laser (660/780nm, 10J/cm2 and 40mW) during periods of 4 and 8 seconds was analyzed through the MTT reduction assays and rapid colorimetric assay of Busatti and Gomes. Statistical analysis was performed using the ANOVA and Tukey´s multiple comparisons test with a significance level of 5% (p < 0.05). It was observed with the MTT assay and Busatti and Gomes assay a trend of cell proliferation increase directly releated to the irradiation dose, statistically significant. After 24, 48 and 72 hours, all the groups showed higher cell proliferation when compared to control. Analyzing the results and considering the used parameters and LBP protocol, it can be concluded that LBP promoted the proliferation of SHED both 660nm and 780nm according to the dosage and wavelengths used, and MTT assay and colorimetric Busatti and Gomes demonstrated within their limitations to be effective in determining the viability and proliferation of SHED.

O camundongo mdx é um modelo animal muito utilizado para estudar a distrofia muscular degenerativa de Duchenne (DMD) e apesar de o modelo apresentar uma degeneração mais branda, inúmeras pesquisas com este animal são realizadas devido à alta reprodutibilidade de fenótipos, obtenção comercial de várias linhagens e baixo custo de manutenção. A degeneração muscular progressiva ocorre devido a uma mutação genética que culmina na ausência ou diminuição da produção da proteína distrofina. Esta alteração gera uma cascata de eventos que pioram a degeneração muscular. Atualmente, a terapia preconizada é o uso de corticóide, que visa modular a inflamação muscular e possui uma série de efeitos colaterais, por isso uma série de estudos propondo outras formas de tratamento vem sendo desenvolvidos. Um tratamento promissor parece ser a terapia celular com células-tronco mesenquimais, a qual pode interferir no processo degenerativo por imunomodulação celular. Como a acupuntura pode controlar o processo inflamatório de doenças degenerativas, tal qual na doença de Parkinson, acredita-se que o uso consorciado das duas terapias possa diminuir o efeito degenerativo da DMD. Nesta pesquisa foram avaliados 22 camundongos mdx, machos, 4 a 6 semanas de idade. As células-tronco utilizadas para a aplicação foram de polpa dentária humana carregando a proteína verde fluorescente (EGFP) e a quantidade aplicada em cada acuponto foi de 1x104 células. Os acupontos selecionados foram B47 (Hunmen), B49 (Yishe) e B52 (Zhishi). Os animais foram distribuídos aleatoriamente em quatro tratamentos (A) células-tronco em acupontos falsos, (B) solução fisiológica em acupontos verdadeiros, (C) células-tronco em acupontos verdadeiros e (D) controle, sem nenhum tratamento. No total três injeções foram realizados em um período de 56 dias de estudo. As avaliações do tratamento foram feitas através das análises da força de tração dos músculos, da creatinofosfoquinase sérica (CPK), histológica através da estruturação das miofibrilas e morfometria do colágeno e imuno-histoquímica pela expressão da proteína distrofina. Ao décimo dia após a terceira aplicação de células-tronco observa-se uma diferença somente no grupo controle, a qual manifestou um aumento da CPK em relação aos outros tratamentos e sem diferença estatística para força de tração pelos membros torácicos. Na histologia dos músculos tibial cranial e diafragma foram observou-se infiltrados inflamatórios, regeneração tecidual e diminuição do colágeno nos grupos tratados p<0,05. Na imuno-histoquímica verificou-se uma melhora da quantidade de distrofina nos animais tratados principalmente no tratamento células-tronco em acupontos verdadeiros p< 0,05 e, pela análise do rastreamento das células-tronco com EGFP não se pode determinar o seu caminho uma vez que não observamos imunorreatividade nas análises utilizando o anti-GFP. Conclui-se que os tratamentos com terapia celular nos acupontos verdadeiros, acupontos falsos e solução fisiológica nos acupontos podem melhorar a doença degenerativa muscular no mdx pelo aumento da expressão da proteína distrofina. / Muscular dystrophy in mdx mouse is an animal model used to study Duchenne muscular dystrophy (DMD) in humans. Although this model presents a milder degeneration, there are several studies with this animal due to the high reproducibility of phenotypes, obtaining various lines available and low maintenance cost. It is known that progressive muscle degeneration occurs due to a genetic mutation that culminates in the absence or decreased production of dystrophin protein. This change generates a cascade of events which aggravate the muscle degeneration. Currently, the recommended therapy is the use of corticosteroids to modulate muscle inflammation; however, there are a number of collateral effects, so a series of studies suggesting other forms of treatment have been developed. A promising treatment seems to be a therapy with mesenchymal stem cells, which can interfere with the degenerative process by cellular immunomodulation. As acupuncture can control the inflammatory process of degenerative diseases such as Parkinson\'s disease, the use of both syndication therapies may reduce the DMD degenerative effect. In this study 22 mdx mice, males, 4-6 weeks of age were evaluated. Stem cells were used for the application of human dental pulp carrying the green fluorescent protein (EGFP) and the quantity applied to each acupoint was 1x104 cells. The acupoints selected were B47 (Hunmen), B49 (Yishe) and B52 (Zhishi). The animals were randomly assigned to four treatments groups: (A) stem cells in false acupoints, (B) saline in true acupoints, (C) stem cells in true acupoints and (D) control without any treatment. In total, three injections were performed over a period of 56 days of study. Evaluations of treatment were made by analyzing the tensile strength of muscles, measuring the serum creatine phosphokinase (CPK), structuring of myofibrils by histology and morphometry of collagen immunohistochemistry and by the amount of dystrophin protein. At day ten after the third application of stem cells there is a difference only in the control group, which manifests an increase in CPK compared to other treatments and no statistical difference in tensile strength. In the histology of cranial tibial muscles and diaphragm, inflammatory infiltrates were observed, as well as tissue regeneration and decreased collagen in the treated groups (p <0.05). On immunohistochemistry there was an improvement in the amount of dystrophin in animals treated mainly in acupoints true (p <0.05) and through analysis and tracking of stem cells with EGFP it was not possible to determine its path once immunoreactivity was not observed in analyzes using anti-GFP. We conclude that treatment with stem cell therapy on true and false acupoints and with saline solution on acupoints can improve muscular degenerative disease in mdx by increased expression of dystrophin protein.

MicroRNAs play an important role in biological regulation. Recently miR-34a has been reported to regulate tumour cell cycle progression and apoptosis. However, the functional role of miR-34a in smooth muscle cell (SMC) differentiation from stem cells is yet unclear. Main objectives of this PhD project are to determine the functional role of miR-34a and its target genes in SMC differentiation and underlying mechanisms. Mouse embryonic stem (ES) cells were seeded on collagen coated flasks in differentiation medium to allow SMC differentiation. Upon analysis, miR-34a was significantly up-regulated during SMC differentiation. Results demonstrated that overexpression of miR-34a significantly promoted SMC-specific gene expression, while knockdown of miR-34a inhibited expression of SMC specific gene. Enforced expression and knockdown of miR-34a in differentiating ES cells up-regulated and down-regulated, respectively, several SMC transcription factors in a similar manner. It was also found that miR-34a overexpression in stem cells promoted SMC differentiation in vivo. Furthermore, deacetylase sirtuin 1 (Sirt1) was identified as one of the top targets of miR-34a. Surprisingly, Sirt1 was demonstrated to be positively regulated by miR-34a during SMC differentiation in a cellular context and RNA sequence dependent manner. VIII Mechanistically, the data suggested that miR-34a promoted differentiating stem cells arrest at G0/G1 phase, and a significant decreased incorporation of miR-34a and SirT1 RNA into Ago2-RISC complex was observed upon SMC differentiation. The results demonstrated that Sirt1 acted as a transcriptional activator in the regulation of SMC gene during ES cell differentiation. Finally, H3K9 tri-methylation around the promoter regions of the SMαA and SM22α genes was also found to be significantly inhibited by SirT1 overexpression. These findings suggest that miR-34a plays an important role in SMC differentiation from ES cells. Meanwhile, Sirt1 can be regulated by miR-34a through an unexpected pathway and it was identified as a functional modulating target in miR-34a mediated SMC differentiation.

The mammary gland is a branched epithelial organ comprised of myoepithelial, ductal and alveolar cells that are derived from resident stem and progenitor cells. The progression from mammary gland stem cell(s) to the differentiated mammary gland cell types is poorly understood. Here, I describe the identification and characterization of two luminal progenitor cell populations in the mouse mammary gland, and investigate the role of the transcription factor C/EBPβ in their development. In Chapter 2, I describe the isolation of two luminal progenitor cell populations (Sca1+ and Sca1- luminal cells) and show that they are differentially primed in their gene expression towards ductal and alveolar cell fates, respectively. Furthermore, I show that in vivo genetic priming affects the in vitro differentiation potential of Sca1+ and Sca1- luminal cells. In Chapter 3, I show that C/EBPβ is required for the appropriate specification of ductal and alveolar lineages, and in its absence, alveolar lineage priming is lost, and ductal lineage priming is up-regulated in both Sca1+ and Sca1- cells. Preliminary data also shows that in addition to severe proliferation defects, the changes in in vivo lineage priming in Cebpb-/- mice also affect the in vitro differentiation potential of Cebpb-/- Sca1+ and Sca1- luminal progenitors. Lastly, in Chapter 4, I describe the genome-wide binding characteristics of C/EBPβ in Sca1+, Sca1- and P16.5 alveolar cells. These experiments reveal that genome-wide C/EBPβ occupancy is correlated with alveolar cells fate, and that C/EBPβ target genes perform distinct cellular functions in alveolar cells (Sca1- cells and P16.5). Furthermore, I show that Elf5 is directly regulated by C/EBPβ, and posit that direct regulation of Elf5 by C/EBPβ may be one mechanism through which C/EBPβ exerts its alveolar cell fate programming.

Tissue engineering offers a potential alternative therapy to overcome the limitations of organ transplantation, by employing biomaterials as scaffold for cell growth. For example, poly-lactic-co-glycolic acid (PLGA) is a synthetic biomaterial widely used in tissue engineering. However, the hydrophobicity of PLGA results in scaffolds that are poorly wettable, and which, therefore, possess poor mass transfer properties for the delivery of nutrients and the removal of waste. The present work aimed to develop more hydrophilic PLGA scaffolds, specifically hollow fibre membranes, within a bioreactor system, which enables co-culture of cells in order to direct stem cell differentiation. Large quantities of costly growth factors are required over long periods for stem cell differentiation. Therefore, this project also aimed to use a cell line as a “factory” for the inexpensive, in situ growth factor production. Hollow fibres were fabricated by wet spinning and a hydrophilic polymer, polyvinyl alcohol (PVA), was added to the PLGA solution at three different concentrations (1.25, 2.5, 5% w/w) in order to obtain a more hydrophilic membrane. Results indicated that 5% PVA-PLGA hollow fibres were the only membranes which allowed permeation of water, BSA and cell-secreted hepatocyte growth factor (HGF), thus indicating that they are the most suitable membranes for use in bioreactor devices. However, these membranes failed to improve cell-attachment. Cell secreted HGF was shown to be more stable in a dynamic culture environment than commercial HGF, thus suggesting its suitability for applications in bioreactor devices. However, using both commercial and cell-secreted HGF, mesenchymal stem cell differentiation was unsuccessful. In conclusion, this work has developed a hollow fibre membrane which is more permeable to water and proteins for a higher mass transfer of nutrients, and has realised a model system for the inexpensive production of growth factors for use in bioreactor devices and the differentiation of stem cells.

Understanding the establishment of polarity and the cell fate specification of epithelial cells is important for developmental biology, regenerative medicine and the study of cancer. In this thesis, models of pre-implantation epithelial development are used to investigate the relationship between these two processes. The trophoblast is an extraembryonic epithelial tissue which contributes to the placenta. Addition of BMP4 to mouse and human embryonic stem (mES) cells grown in culture has been suggested to induce differentiation of cells to the trophoblast lineage. The use of this differentiation method was investigated as a possible model of trophoblast polarisation and cell fate specification. Unfortunately, with the protocol and reagents available this model did not appear to physiologically recapitulate trophoblast development and was not reliable. The primitive endoderm is an epithelium which arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. When mES cells are grown in suspension they form aggregates of differentiating cells known as embryoid bodies. The outermost cell layer of an embryoid body is an epithelial cell type comparable to the primitive endoderm. Embryoid bodies were used here to study the polarisation and cell fate specification of the primitive endoderm. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. The acquisition of epithelial polarity occurred prior to the maximal expression of cell fate markers. Fgfr/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier which normally blocks free diffusion across the epithelial cell layer occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erkmediated polarisation. This data shows that the formation of a polarised primitive endoderm layer in embryoid bodies requires the Fgfr/Erk signalling pathway.

Current treatment options are limited for patients with myocardial infarction or heart failure. Cellular cardiomyoplasty is a promising therapeutic strategy being investigated as a potential treatment, which aims to deliver exogenous cells to the infarcted heart, for the purpose of restoring healthy myocardial mass and mechanical cardiac function. While several cell types have been studied for this application, only bone marrow cells and human mesenchymal stem cells (hMSCs) have been shown to be safe and effective for improving cardiac function in clinical trials. In both human and animal studies, the delivery of hMSCs to infarcted myocardium decreased inflammatory response, promoted cardiomyocyte survival, and improved cardiac functional indices. While the benefits of using hMSCs as a cell therapy for cardiac repair are encouraging, the desired expectation of cardiomyoplasty is to increase cardiomyocyte content that will contribute to active cardiac mechanical function. Delivered cells may increase myocyte content by several different mechanisms such as differentiating to a cardiomyocyte lineage, secreting paracrine factors that increase native stem cell differentiation, or secreting factors that increase native myocyte proliferation. Considerable work suggests that hMSCs can differentiate towards a cardiomyocyte lineage based on measured milestones such as cardiac-specific marker expression, sarcomere formation, ion current propagation, and gap junction formation. However, current methods for cardiac differentiation of hMSCs have significant limitations. Current differentiation techniques are complicated and tedious, signaling pathways and mechanisms are largely unknown, and only a small percentage of hMSCs appear to exhibit cardiogenic traits. In this body of work, we developed a simple strategy to initiate cardiac differentiation of hMSCs in vitro. Incorporating environmental cues typically found in a myocardial infarct (e.g. decreased oxygen tension and increased concentrations of cell-signaling factors), our novel in vitro conditioning regimen combines reduced-O2 culture and hepatocyte growth factor (HGF) treatment. Reduced-O2 culturing of hMSCs has shown to enhance differentiation, tissue formation, and the release of cardioprotective signaling factors. HGF is a pleiotropic cytokine involved in several biological processes including developmental cardiomyogenesis, through its interaction with the tyrosine kinase receptor c-Met. We hypothesize that applying a combined conditioning treatment of reduced-O2 and HGF to hMSCs in vitro will enhance cardiac-specific gene and protein expression. Additionally, the transplantation of conditioned hMSCs into an in vivo infarct model will result in differentiation of delivered hMSCs and improved cardiac mechanical function. In testing our hypothesis, we show that reduced-O2 culturing can enhance hMSC growth kinetics and total c-Met expression. Combining reduced-O2 culturing with HGF treatment, hMSCs can be conditioned to express cardiac-specific genes and proteins in vitro. Using small-molecule inhibitors to target specific effector proteins in a proposed HGF/c-Met signaling pathway, treated reduced-O2/HGF hMSCs show a decrease in cardiac gene expression. When implanted into rat infarcts in vivo, reduced-O2/HGF conditioned hMSCs increase regional cardiac mechanics within the infarct region at 1 week and 1 month. Further analysis from the in vivo study showed a significant increase in the retention of reduced-O2/HGF conditioned hMSCs. Immunohistochemistry showed that some of the reduced-O2/HGF conditioned hMSCs express cardiac-specific proteins in vivo. These results suggest that a combined regimen of reduced-O2 and HGF conditioning increases cardiac-specific marker expression in hMSCs in vitro. In addition, the implantation of reduced-O2/HGF conditioned hMSCs into an infarct significantly improves cardiac function, with contributing factors of improved cell retention and possible increases in myocyte content. Overall, we developed a simple in vitro conditioning regimen to improve cardiac differentiation capabilities in hMSCs, in order to enhance the outcomes of using hMSCs as a cell therapy for the diseased heart.

Cell therapy, including human mesenchymal stem cell (hMSC) therapy, has the potential to treat different pathologies, including myocardial infarctions (heart attacks). Biological sutures composed of fibrin have been shown to effectively deliver hMSCs to infarcted hearts. However, hMSCs rapidly degrade fibrin making cell seeding and delivery time sensitive. To delay the degradation process, we propose using aprotinin, a proteolytic enzyme inhibitor that has been shown to slow fibrinolysis. This project investigated the effects of aprotinin on hMSCs and suture integrity. Viability of hMSCs incubated with aprotinin, examined using a LIVE/DEAD stain, was similar to controls. No differences in proliferation, as determined by Ki-67 presence, and were observed. hMSCs incubated in aprotinin differentiated into adipocytes, osteocytes, and chondrocytes, confirming multipotency. CyQuant assays were used to determine the number of cells adhered to fibrin sutures. The number of adhered cells was increased through aprotinin supplementation at Days 2, 3, and 5 time points. To examine the effect of aprotinin on suture integrity, sutures were loaded to failure to determine ultimate tensile strength (UTS) and modulus (E). Sutures exposed to aprotinin had higher UTS and E when compared to sutures exposed to standard growth media. Degradation of fibrin was quantified using an ELISA to quantify fibrin degradation products (FDP) and by measuring suture diameter. Fibrin sutures incubated in aprotinin had larger diameters and less FDP compared to the controls, confirming decreased fibrinolysis. These data suggest that aprotinin can reduce degradation of biological sutures, providing a novel method for extending the implantation window and increasing the number of cells delivered for hMSC seeded biological sutures.