Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body while maintaining proliferative capabilities. The generation of human iPSC-derived hepatocytes (iPSC-Heps) has resulted in a new source for hepatic cells. The current available options for human hepatocytes are primary human hepatocytes (PHHs) and cell lines. PHHs isolated from healthy human donors are difficult to obtain, while cell lines exhibit reduced hepatotoxic sensitivity. iPSC-Heps are being investigated as an alternative option as they are derived from a continuous, stable source and are able to maintain their original donor genotype, which opens the door for patient-specific studies. iPSC-Heps show promise for utilization in tissue engineering, hepatotoxicity studies as well as screening for patient-specific therapeutics. Various reports have concluded that iPSC-Heps exhibit reduced hepatocyte function in comparison to PHHs. Prior reports on iPSC-Heps have focused on improving their adult phenotype functions through variations in differentiation protocols or by altering their in vitro culturing environment. This thesis focuses on incorporating hepatic non-parenchymal cells to more closely mimic the tissue and cell architecture found in the liver tissue. We designed and assembled a 3D iPSC-Hep model that integrates liver sinusoidal endothelial cells, with the goal of achieving functional maturity. Hepatotoxicants were administered to our models and various hepatic markers were measured to analyze the toxic response. This work demonstrates the need for the inclusion of hepatic non-parenchymal cells in iPSC-derived liver tissues, specifically for hepatotoxicity applications. / Master of Science / Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body while maintaining proliferative capabilities. The generation of human iPSC-derived hepatocytes (iPSC-Heps) has resulted in a new source for hepatic cells. The current available options for human hepatocytes are primary human hepatocytes (PHHs) and cell lines. PHHs originating from healthy human donors are difficult to obtain, while cell lines may exhibit reduced hepatotoxic sensitivity to chemicals. iPSC-Heps are being investigated as an alternative option since they are derived from a continuous source and are able to maintain their original donor genetic make-up, allowing for patient-specific studies. iPSC-Heps can be used in tissue engineering, hepatotoxicity studies as well as screening for patient-specific therapeutics. Various reports have concluded that iPSC-Heps exhibit reduced function in comparison to PHHs. Prior reports on iPSC-Heps have focused on improving their function through variations in differentiation procedures or by changing their culture environment. This thesis focuses on incorporating other hepatic cells to more closely mimic the tissue and cell architecture found in the liver tissue. We designed and assembled a 3D iPSC-Hep model that integrates liver sinusoidal endothelial cells, with the goal of improving hepatocyte function. Chemicals were administered to our models and various hepatic markers were measured to analyze the toxic response. This work demonstrates the need for the inclusion of additional hepatic cell types in iPSC-derived liver tissues, specifically for hepatotoxicity applications.

Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\' 2′-O-Methyltransferase enzymecreates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48 h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

"Recent data show that 7.6 million Americans have survived a myocardial infarction (MI), and 5.1 million Americans suffer from severe heart failure. Stem cell therapy has the potential to improve cardiac function after MI. Two promising cells for cardiovascular regeneration therapies include human mesenchymal stem cells (hMSCs) and pluripotent stem cell derived cardiomyocytes (hPS-CM) each with their own unique method for improving cardiac function post-infarct. However, a limiting factor to cell therapies is that the methods currently used to deliver cells to the myocardium, including intramyocardial injection (considered the gold standard), suffer from low retention rates. To promote localization of delivered cells to the infarct and increase retention rates, our lab has developed a fibrin biological suture that can deliver human mesenchymal stem cells (hMSCs) with an efficiency of 64% compared to just 11% with intramyocardial injection in the normal rat heart. In this dissertation we sought to examine the functionality of hMSC and hPS-CM seeded sutures and their impact on cardiovascular regeneration applications. We began by delivering hMSC seeded fibrin sutures to an infarcted rat heart and found that the sutures are an effective method to deliver cells to the infarcted myocardium and demonstrated a trend towards improved regional mechanical function in the infarct region over infarct alone. Next, we transitioned to using hPS-CM and developed methods to seed the sutures, as well as a method to measure hPS-CM contractility with high spatial and temporal resolution, while concurrently capturing calcium transients. This technique allowed us to examine the contractile behavior in terms of contractile strain and conduction velocity of hPS-CM seeded on fibrin microthreads over 21 days in culture. We found that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction and that extended culture promotes cell alignment along the length of the suture as well as improvements in contractile function in terms of increases in contractile strain and conduction velocity. Finally, we delivered the hPS-CM seeded microthreads to an uninjured rat heart and found a delivery efficiency of 67%. Overall, we further demonstrated the technology of the fibrin suture to deliver cells to an infarct as well as the ability to support the attachment, contraction and delivery of hPS-CM to cardiac tissue. "

Breast cancer is the most common cancer in Taiwanese women and the invasive ductal carcinoma (IDC) is the most common type. Increasing evidence shows that cancer stem cells (CSCs) have been implicated in tumorigenesis, tumor progression, and drug-resistance. In addition, four reprogramming factors (Octamer-binding Protein 4 (Oct4), Sex-determining Region Y (SRY)-related Box 2 (Sox2), Nanog and Lin28) employed to induce induced pluripotent stem (iPS) cells are associated with CSCs formation. The purpose of this study was to investigate the relationship of the protein expression levels of the reprogramming factors (Oct4, Sox2, Nanog and Lin28) with the tumorigenesis, clinicopathologic outcomes and prognosis of breast IDC patients. Immunohistochemistry (IHC) assay of tissue microarrays, made by 309 IDC and 20 breast fibrosis paraffin embedded samples, were used to examine the protein expression levels of Oct4, Sox2, Nanog and Lin28 in normal mammary ductal tissues, tumor adjacent normal mammary ductal tissues, ductal carcinoma in situ (DCIS), IDC and recurrence tissues. Our IHC results showed that Sox2 and Lin28 were expressed in half of breast IDC patients¡¦ tumor tissue (49.6% and 49.7%, respectively), but Oct4 and Nanog are less expressed (13.5% and 24.7%, respectively). The protein expression levels of the four proteins were positively correlated with each other. In addition, the expression levels of the four proteins were upregulated in tumor adjacent normal tissue as compared to breast fibrosis pateints¡¦ normal mammary ductal tissue. To compare the expression levels of the four proteins in different tissues; such as tumor adjacent normal, DCIS, IDC and recurrence tissues, the expression levels of the four protiens gradually decreased when tumor developed and progressed. However, their expression levels were comparable between IDC and recurrence tissues. Additionally, the high expression levels of four proteins were high in two good clinicopathological characteristics and a biomarker of breast cancer; such as nuclear Sox2 and Lin28 in those with pathology stage I; nucleus expression of the four proteins in those with well and moderate cell differentiation; and Sox2 in those with positive estrogen receptor. However, the four proteins¡¦ expression levels were not correlated with IDC patients¡¦ survival. In conclusion, the reprogramming factors: Oct4, Sox2, Nanog and Lin28 may play an important role in tumorigenesis of breast IDC, but their impacts on tumor progression were quite small.

Cellular fate, once established, is usually stable for the lifetime of the cell. However, the mechanisms that restrict the developmental potential of differentiated cells are in principle reversible, as demonstrated by the success of animal cloning from a somatic genome through somatic cell nuclear transfer (SCNT). An increased understanding of the molecular determinants of cell fate has also enabled the reprogramming of cell fate using defined transcription factors; recently, these efforts have culminated in the discovery of four genes that convert somatic cells into induced pluripotent stem cells (iPSCs), which resemble embryonic stem cells (ESCs) and can give rise to all the cell types in the body. As a first step toward generating clinically useful iPSCs, we identified a small molecule, RepSox, that potently and simultaneously replaces two of the four exogenous reprogramming factors, Sox2 and cMyc. This activity was mediated by the inhibition of the Transforming Growth Factor-\(\beta\) \((Tgf-\beta)\) signaling pathway in incompletely reprogrammed intermediate cells. By isolating these stable intermediates, we showed that RepSox acts on them to rapidly upregulate the endogenous pluripotency factor, Nanog, allowing full reprogramming to pluripotency in the absence of Sox2. We also explored lineage conversion as an alternative approach for producing a target cell type in a patient-specific manner, without first generating iPSCs. A combination of pro-neural as well as motor neuron-selective factors could convert fibroblasts directly into spinal motor neurons, the cells that control all voluntary movement. The induced motor neurons (iMNs) displayed molecular and functional characteristics of bona fide motor neurons, actuating muscle contraction in vitro and even engrafting in the developing chick spinal cord when transplanted. Importantly, functional iMNs could be produced from fibroblasts of adult patients with the fatal motor neuron disease, amyotrophic lateral sclerosis (ALS). Given the therapeutic value of generating patient-specific cell types on demand, defined-factor reprogramming is likely to serve as an important tool in regenerative medicine. It is hoped that the different approaches presented here can complement existing technologies to facilitate the study and treatment of intractable human disorders.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by over expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Using serial live cell immunofluorescence imaging of human fibroblasts undergoing reprogramming, we traced the emergence of nascent iPS cell colonies among heterogeneous cell populations and defined the kinetics of marker expression. We identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters, and differentiation into teratomas, we determined that only one colony type represented bona fide iPS cells, whereas the others represented reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B, and REX1 distinguished the fully reprogrammed state, whereas Alkaline Phosphatase, SSEA-4, GDF3, hTERT and NANOG proved insufficient as markers. Reprogramming in chemically defined medium favored formation of bona fide iPS cell colonies relative to partially reprogrammed colonies. These data highlight the need for rigorous characterization and standardization of putative iPS cells.

In addition to the well-characterized Induced Pluripotent Stem cells (iPSCs) that closely resemble Embryonic Stem cells (ESCs), a recent study has proven the existence of a stable state, resembling partially reprogrammed cells, termed F-class iPSCs. To study these distinct iPSC states, a reprogramming dataset has been generated, featuring the parallel analysis of multiple molecular platforms. MicroRNAs (miRNAs) are small RNA regulators of gene expression whose critical role in reprogramming is now being realized. In the present study, small RNA deep sequencing data from this novel reprogramming dataset was used to identify miRNAs that are likely to enhance reprogramming by detecting significantly up-regulated miRNAs in ESC-like iPSCs versus F-class iPSCs. These candidate miRNAs were cloned and overexpressed in reprogramming mouse embryonic fibroblasts and their effect on reprogramming efficiency was measured. miR-214 was discovered to increase iPSC generation efficiency, marking the first reprogramming-related role for this microRNA.

In addition to the well-characterized Induced Pluripotent Stem cells (iPSCs) that closely resemble Embryonic Stem cells (ESCs), a recent study has proven the existence of a stable state, resembling partially reprogrammed cells, termed F-class iPSCs. To study these distinct iPSC states, a reprogramming dataset has been generated, featuring the parallel analysis of multiple molecular platforms. MicroRNAs (miRNAs) are small RNA regulators of gene expression whose critical role in reprogramming is now being realized. In the present study, small RNA deep sequencing data from this novel reprogramming dataset was used to identify miRNAs that are likely to enhance reprogramming by detecting significantly up-regulated miRNAs in ESC-like iPSCs versus F-class iPSCs. These candidate miRNAs were cloned and overexpressed in reprogramming mouse embryonic fibroblasts and their effect on reprogramming efficiency was measured. miR-214 was discovered to increase iPSC generation efficiency, marking the first reprogramming-related role for this microRNA.

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<p><p> Articular cartilage is a smooth connective tissue that covers the ends of bones and protects joints from wear. Cartilage has a poor healing capacity, and the lack of treatment options motivates the development of tissue engineering strategies. The widespread cartilage degeneration associated with osteoarthritis (OA) is dramatically accelerated by joint injury, but the defined initiating event presents a therapeutic window for preventive treatments. In vitro model systems allow investigation of OA risk factors and screening of potential therapeutics. This dissertation develops stem-cell based strategies to 1) treat cartilage injury and OA using tissue-engineered cartilage, 2) prevent the development of OA by delivering stem cells to the joint after injury, and 3) study cartilage by establishing systems to model genetic and environmental contributors to OA.</p><p> Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are promising human adult cell sources for cartilage tissue engineering, but require distinct chondrogenic conditions. As compared to ASCs, MSCs demonstrated enhanced chondrogenesis in both alginate beads and cartilage-derived matrix scaffolds. </p><p> We hypothesized that MSC therapy would prevent post-traumatic arthritis (PTA) by altering the balance of inflammation and regeneration. Highly purified MSCs (CD45-TER119-PDGFR&#945;+Sca-1+) rapidly expanded under hypoxic conditions. Unexpectedly, MSCs from control C57BL/6 (B6) mice proliferated and differentiated more than MSCs from MRL/MpJ (MRL) "superhealer" mice. We injected B6 or MRL MSCs into mouse knees immediately after fracture, and MSCs of either strain were sufficient to prevent PTA. </p><p> Genetically reprogramming adult cells into induced pluripotent stem cells (iPSCs) generates large numbers of patient-matched cells with chondrogenic potential for therapy and cartilage modeling. We produced murine iPSC-derived cartilage constructs with a multi-phase approach involving micromass culture with bone morphogenetic protein-4, flow cytometry cell sorting of chondrocyte-like cells, monolayer expansion, and pellet culture with transforming growth factor-beta 3. Successful differentiation was confirmed by increased chondrogenic gene expression, robust synthesis of glycosaminoglycans and type II collagen, and the repair of an in vitro cartilage defect. </p><p> The diverse applications pursued in this research illustrate the power of stem cells to deepen the understanding of cartilage and guide the development of therapies to prevent and treat cartilage injury and OA.</p> / Dissertation