Embryonic stem cells (ESCs) are being considered as a cell source for cardiac regeneration because of their potency and availability. We studied the electrophysiological implications using co-cultures of ESCs and neonatal rat ventricular myocytes (NRVM) grown on a multi-electrode array (MEA). To mimic expected engraftment rates 5% mouse ESCs were co-cultured with NRVMs. Comparing cultures without and with 5% ESCs at 4 days, the mean bipolar field potential duration (FPD) of NRVMs increased from 26.3 ± 2.2 ms (n=10) to 44.3 ± 6.2 ms (n=9; p < 0.05), the interspike interval (ISI) increased from 358.3 ± 62.8 ms (n=10) to 947.8 ± 214.6 ms (n=7; p < 0.01), and conduction velocity (CV) decreased from 14.2 ± 1.3 cm/s (n=8) to 4.6 ± 1.2 cm/s (n=5; p < 0.01). To evaluate whether ESC were having direct or paracrine effects on NRVMs, media conditioned by 3x106 ESCs for 24 hr was diluted 1:1 with fresh media and then introduced to NRVM cultures on the day of plating. Conditioned media was changed daily and altered mean FPD, ISI, and CV to 46.1 ± 7.8 ms, ISI to 682.0 ± 128.5 ms, and 4.2 ± 0.4 cm/s (n=8; p < 0.01 for each measure), respectively at 4 days. However, changes were not seen in media that was incubated for 24hrs and diluted 1:1 with fresh media and introduced to NRVM cultures in a similar fashion (n=7; p > 0.05). Slowed CV is associated with increased arrhythmic risk and reports demonstrate an inverse relationship between CV and nonphosphorylated Cx43(NP-Cx43). Western blots for total Cx43 expression revealed a decrease in ratio of P-Cx43/NP-Cx43 in the 5% mouse ESCs and ESC conditioned media cultures as compared to controls (n=8; p < 0.01 for each). There was not significant increase in the total Cx43 expression (n=6; p > 0.05). Culturing ESCs with NRVMs resulted in a decreased ISI, prolonged FPD, and slowed CV of the co-cultures as compared to controls leading to pro-arrhythmic conditions. Similar effects on NRVMs were observed when applying media conditioned by ESCs, suggesting that the electrophysiological changes were mediated by soluble factors. The increase in NP-Cx43 leads to gap junction uncoupling being a potential mechanism for these arrhythmogenic substrates. Further research into preventing NP-Cx43 in cultures is currently underway.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Dissertation (PhD)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 154-187).. Also available online.

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Biomedical Engineering." Includes bibliographical references (p. 45-48).

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references.

Mammalian cell types and tissues have diverse functional roles within an organism but can be derived by the differentiation of the embryonic stem cells (ESCs). ESCs are pluripotent cells with self-renewal properties. During development subsets of genes in ESCs are activated or silenced for manifestation of the cell type specific function. Gene expression changes occur transiently in early developmental stages, through signals received and executed by a variety of transcription factors (TFs), regulatory elements (promoters, enhancers) and epigenetic modifications of chromatin. Post-translational modifications of the histone tails are regulated by chromatin modifiers and transform the chromatin architecture. Polycomb (PcG) and Trithorax (TrxG) group proteins are the most commonly studied histone modifiers. They were first discovered as repressors (H3K27me3) and activators (H3K4me3) respectively of Homeobox (Hox) genes in Drosophila and they are conserved in mammals. Bivalent chromatin is defined as the simultaneous presence of silencing (H3K27me3) and activating (H3K4me3) histone marks and was first discovered as a feature of many developmental gene promoters of ESCs. Bivalent promoters are thought to be in a ‘poised’ state for later activation or repression during differentiation due to the presence of the two counter-acting histone modifications and a pausing variant of RNA polymerase II (RNAPII) accompanied with intermediate-low levels of expression. By integrative analysis of publicly available ChIP sequencing (ChIP-seq) datasets in murine and human ESCs, we predicted 3,659 and 4,979 high–confidence (HC) bivalent promoters in mouse and human ESCs respectively. Using a peak-based method, we acquire a set of bivalent promoters with high enrichment for developmental regulators. Over 85% of Polycomb targets were bivalent and their expression was particularly sensitive to TF perturbation. Moreover, murine HC bivalent promoters were occupied by both Polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions. HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters and a ‘TCCCC’ sequence motif was specifically enriched in bivalent promoters. Using the recent technology of single cell RNA sequencing (scRNA-seq) we focused on gene expression heterogeneity and how it may affect the output of differentiation. We collected single cell gene expression profiles for 32 human and 39 murine ESCs and studied the correlation between diverse characteristics such as network connectivity and coefficient of variation (CV) across single cells. We further characterized properties unique to genes with high CV. Highly expressed genes tended to have a low CV and were enriched for cell cycle genes. In contrast, High CV genes were co-expressed with other High CV genes, were enriched for bivalent promoters and showed enrichment for response to DNA damage and DNA repair. Bivalent promoters in ESCs grouped in four distinct classes of variable biological functions according to Polycomb occupancy and three RNAPII variants. To study the dynamics of epigenetic and transcription control at promoters during development, we collected ChIPseq data for two chromatin modifications (H3K4me3 and H3K27me3) and RNAPII (8WG16 antibody) as well as expression data (RNA-seq) across 8 cell types (ESCs and seven committed cell types) in mouse. Hierarchical clustering of 22,179 unique gene promoters across cell types, showed that H3K4me3 peaks are in agreement with the expression data while H3K27me3 and RNAPII peaks were not highly consistent with the hierarchical tree of gene expression. Unsupervised clustering of ChIP-seq and RNA-seq profiles has resulted in 31 distinct profiles, which were subsequently narrowed down to nine major profile groups across cell types. TF enrichment at individual clusters using ChIP sequencing data did not fully agree with the classification of 8 major profile groups. Considering all the above results, three major epigenetic profiles (active, bivalent and latent) seem to be conserved across the species and cell types in our study. These states could recapitulate only a fraction of the transcriptional information - adding other chromatin marks could enrich it - since they are seemingly unaffected by their respective expression profiles. H3K27me3 only state has low CpG density and shows stronger signatures at differentiated cell types. Transcriptional control is tighter in active than bivalent promoters and the different occupancy levels of PcG subunits and RNAPII can be reflected at the expression variance of bivalent genes, where a fraction of them are involved in developmental functions while others are more tissue-specific. Last, there is a striking similarity in the pausing patterns of RNAPII in the progenitor cell types, which suggests that RNAPII pausing is correlated with the developmental potential of the cell type. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during development.

Immunotherapy traditionally made use of biological agents such as cytokines and monoclonal antibodies. Such first generation therapies lack antigen specificity and fail to induce immunological memory, suggesting that cell therapies may provide the next generation of treatments that are more discerning in their mode of action. Nevertheless, difficulties in obtaining sufficient immunologically-relevant cell types from patients has limited their success. Given that induced pluripotent stem cells (iPSC) may be generated from patients, we have investigated the feasibility of deriving two cell types whose availability is restricted in vivo: regulatory T cells (T_regs) and CD141⁺ cross-presenting dendritic cells (DCs). We describe the optimization of protocols for differentiation and purification of CD141⁺ DCs, focussing on their utility as a therapeutic vaccine for HIV-1. We investigate their phenotype, chemotactic capacity, phagocytic ability and propensity to harbour infectious virus. We also assess their immunostimulatory capacity and ability to cross-present exogenous antigen to MHC class I-restricted T cells. Our findings led us to speculate that iPSC-derived DCs (iPDCs) possess fetal phenotype, which is characterised by excessive secretion of IL-10 and failure to secrete IL-12, under all but the most stringent conditions. We hypothesised that constitutive secretion of IL-10 may be responsible for maintaining the fetal phenotype, a hypothesis we tested by developing an appropriate mouse model. iPSCs were derived from WT and IL-10^-/- mice and were shown to differentiate into iPDCs which recapitulate the fetal phenotype observed among human cells. However, loss of the endogenous Il-10 gene failed to restore full immunogenicity and IL-12 secretion. Finally, we developed protocols for differentiation of FoxP3+ T_regs from iPSCs, a feat that has not previously been achieved. These findings pave the way for the differentiation of T_regs from iPSCs reprogrammed from antigen-specific pathogenic T cells, thereby creating a source of T_regs with matched specificity for therapeutic intervention.

abstract: Several debilitating neurological disorders, such as Alzheimer's disease, stroke, and spinal cord injury, are characterized by the damage or loss of neuronal cell types in the central nervous system (CNS). Human neural progenitor cells (hNPCs) derived from human pluripotent stem cells (hPSCs) can proliferate extensively and differentiate into the various neuronal subtypes and supporting cells that comprise the CNS. As such, hNPCs have tremendous potential for disease modeling, drug screening, and regenerative medicine applications. However, the use hNPCs for the study and treatment of neurological diseases requires the development of defined, robust, and scalable methods for their expansion and neuronal differentiation. To that end a rational design process was used to develop a vitronectin-derived peptide (VDP)-based substrate to support the growth and neuronal differentiation of hNPCs in conventional two-dimensional (2-D) culture and large-scale microcarrier (MC)-based suspension culture. Compared to hNPCs cultured on ECMP-based substrates, hNPCs grown on VDP-coated surfaces displayed similar morphologies, growth rates, and high expression levels of hNPC multipotency markers. Furthermore, VDP surfaces supported the directed differentiation of hNPCs to neurons at similar levels to cells differentiated on ECMP substrates. Here it has been demonstrated that VDP is a robust growth and differentiation matrix, as demonstrated by its ability to support the expansions and neuronal differentiation of hNPCs derived from three hESC (H9, HUES9, and HSF4) and one hiPSC (RiPSC) cell lines. Finally, it has been shown that VDP allows for the expansion or neuronal differentiation of hNPCs to quantities (>1010) necessary for drug screening or regenerative medicine purposes. In the future, the use of VDP as a defined culture substrate will significantly advance the clinical application of hNPCs and their derivatives as it will enable the large-scale expansion and neuronal differentiation of hNPCs in quantities necessary for disease modeling, drug screening, and regenerative medicine applications. / Dissertation/Thesis / Masters Thesis Bioengineering 2016

Estudos com células tronco (CT) têm despertado grande interesse devido ao seu promissor potencial terapêutico. Neste contexto, as CT mesenquimais (CTm) representam uma alternativa para o tratamento de diversas patologias em diferentes órgãos, inclusive o rim e as glomerulopatias que o acometem. As doenças glomerulares constituem uma freqüente causa de doença renal crônica e se caracterizam por apresentar proteinúria. Neste processo, os podócitos são células que apresentam um papel crucial, sendo que as podocitopatias se associam com o aparecimento de proteinúria e desenvolvimento de esclerose glomerular. A obtenção de um modelo de podocitopatia através da administração de aminonucleosídeo de puromicina (PAN), permite a melhor compreensão dessas células altamente diferenciadas que não possuem potencial de proliferação ou regeneração. O presente projeto teve como objetivo estabelecer o modelo experimental de nefropatia crônica induzida por PAN associado à nefrectomia unilateral (UniNx) para induzir lesões glomerulares mais exuberantes e, neste modelo experimental, avaliar o efeito da infusão de CTm derivadas da medula óssea. Ratos Wistar (n=52) foram divididos em três grupos: Controle (UniNx), PAN (PAN+UniNx) e PAN+CTm (PAN+UniNx+CTm). As CTm foram inoculadas na região subcapsular renal no dia 0 e os animais foram sacrificados após 30 e 60 dias. O efeito da infusão das CTm no tecido renal foi avaliado através de parâmetros clínicos e laboratoriais, além de análise histológica, imunohistoquímica, microscopia eletrônica e PCR em tempo real. Paralelamente, o projeto analisou a diferenciação in vitro de CTm em podócitos através do estímulo com colágeno tipo IV e através de cocultura de glomérulos isolados de ratos com CTm. A diferenciação celular das CTm foi analisada por citometria de fluxo, imunocitoquímica e PCR em tempo real para genes de proteínas podocitárias. No modelo in vivo foi possível observar a presença de CTm até 15 dias após a inoculação na região subcapsular renal. As CTm foram capazes de diminuir significativamente a proteinúria e a albuminúria com 30 e 60 dias, assim como a pressão arterial aos 60 dias. Não houve diferença nos valores de creatinina, uréia sérica, glomeruloesclerose e fibrose intersticial entre o grupo PAN e o grupo PAN+CTm. As CTm foram responsáveis pela diminuição significativa da fusão dos pedicelos à microscopia eletrônica, com melhora da expressão relativa de WT1 aos 60 dias e melhora parcial da expressão gênica de nefrina, podocina e sinaptopodina. A expressão proteica de WT1 também foi significativamente maior no grupo PAN+CTm em comparação ao grupo PAN. Além disso, houve melhora significante da expressão relativa de IL-4 e IL-10, e diminuição de IL-1? e TNF-? no grupo tratado. Ainda, as CTm promoveram aumento significativo da expressão gênica de VEGF aos 60 dias. Nos resultados in vitro não houve diferenciação das CTm em podócitos quando cultivadas com colágeno IV, assim como a cocultura com glomérulos não proporcionou alteração na expressão de marcadores de superfície das CTm. Concluímos que a terapia celular com CTm foi capaz de induzir proteção renal caracterizada por diminuição da proteinúria, da albuminúria e da pressão arterial, associado a menor fusão dos pedicelos, maior expressão gênica de proteínas podocitárias e de expressão celular de WT1. As citocinas inflamatórias IL-1?, TNF-?, IL-4 e IL-10, em conjunto com o VEGF, foram os possíveis mediadores responsáveis por estes resultados / Stem cells (SC) have emerged as a potential therapeutic approach for several diseases. In this context, the mesenchymal SC (mSC) are considered an alternative for the treatment of kidney diseases such as glomerulopathies. Glomerular diseases are an important cause of chronic kidney disease (CKD) and are characterized by proteinuria. In this process, the podocytes are cells that have a critical role, and the podocytopathies are associated with the onset of proteinuria and glomerular sclerosis. The achievement of a podocytopathy model through administration of puromycin (PAN) allows a better understanding of these highly differentiated cells which do not have the potential for proliferation or regeneration. The aim of the present study was to establish an experimental model of chronic nephropathy induced by PAN associated with unilateral nephrectomy (UniNx), to induce early and marked glomerular lesions and, in this experimental model, to evaluate the effect of bone marrow mSC infusion. Wistar rats (n=52) were randomly divided into three groups: Control (UniNx), PAN (PAN+UniNx) and PAN+mSC (PAN+UniNx+mSC). mSC were inoculated into the subcapsular renal region on day 0, and the animals were sacrificed after 30 and 60 days. The mSC infusion effects in renal tissue were evaluated by clinical and laboratory parameters, histology, immunohistochemistry, electron microscopy and real-time PCR. In parallel, we analyzed whether mSC could differentiate in vitro into podocytes through stimulation with collagen type IV or by means of co-culture of isolated rat glomeruli with mSC. The cell differentiation was analyzed by flow cytometry, immunocytochemistry and real-time PCR. In the in vivo model, mSC were detected until 15 days after inoculation in the renal subcapsular region. mSC were able to significantly reduce the proteinuria and albuminuria with 30 and 60 days, as well as blood pressure at 60 days. There was no difference in the values of creatinine, BUN, glomerulosclerosis and interstitial fibrosis between the groups PAN and PAN+mSC. The treated group showed lower effacement of foot process by electron microscopy, with significant improvement in the relative expression of WT1 in 60 days and partial improvement of nephrin, podocyn and synaptopodin. The WT1 protein expression was also significantly higher in the PAN+mSC group compared to the PAN group. In addition, mSC treatment significantly reduced gene expression of IL-1? and TNF-?, as well as increased the expression of IL-4 and IL-10. At 60 days mSC promoted significant increase of VEGF relative expression. In vitro results, mSC cultived with collagen type IV did not show differentiation to podocytes and the co-culture with glomeruli provided no change in expression of mSC surface markers. In conclusion, mSC therapy in the PAN model was able to induce renal protection characterized by the reduction of albuminuria, proteinuria and blood pressure, associated with a lower effacement of foot process, increased gene expression of podocytes proteins and cellular expression of WT1. Inflammatory cytokines IL-1?, TNF-?, IL-4 and IL-10 associated with VEGF were the probable mediators of these results, promoting podocyte protection