Heart failure (HF) following myocardial infarction (MI) is the leading cause of mortality and morbidity worldwide. Existing medical and interventional therapies can only reduce the cardiomyocytes (CMs) lost during MI. They are unable to replenish the permanent loss of CMs and this contributes to progressive pathological left ventricular (LV) remodeling and HF. Cell-based therapies using adult stem cells or embryonic stem cells (ESCs) and their cardiac derivatives have frequently been explored as a potential therapeutic approach to restore cardiac function in HF. The objectives of this thesis are to evaluate the efficacy and safety of different approaches of stem cell based therapy to improve cardiac function using small and large animal MI models. In Chapter 3, we studied the functional consequences of direct intramyocardial transplantation of ESCs and ESC-derived cardiomyocytes (ESC-CMs) in a murine model of acute MI. LV ejection fraction (LVEF) and maximal positive or negative pressure derivative (dP/dt) improved 4 weeks after transplantation of either ESCs or ESC-CMs. Nevertheless there was a higher incidence of inducible ventricular tachyarrhythmia (VT) and higher mortality in animals transplanted with ESC-CMs than those with ESCs. At a single cell level, ESC-CMs exhibited immature electrophysiological properties such as depolarized resting membrane potential (RMP), longer action potential duration (APD) and automaticity. In Chapter 4, we tested the hypothesis that genetic modification of these immature electrophysiological properties of ESC-CMs by overexpression of Kir2.1 gene encoding the ion channels for IK1, may alleviate the pro-arrhythmic risk. In this study, Kir2.1 channels expression could be controlled with the administration of doxycycline (DOX). The DOX-treated ESC-CMs were more mature with hyperpolarized RMP and shorter APD than their counterparts without DOX treatment. A similar improvement in LV systolic function was observed 4 weeks after both DOX treated and untreated ESCCMs transplantation, although those animals transplanted with DOX-treated ESC-CMs had a significantly lower incidence of spontaneous and inducible VT. Histological analysis in both studies suggested that the major mechanisms of improvement in cardiac function were related to angiogenesis and low apoptosis rate of native cardiomyocytes mediated via paracrine effects. Importantly, very limited retention of ESC-CMs was observed 4 weeks after transplantation. Cell-based patches that use different bioengineering techniques have been proposed to improve cell retention and survival following transplantation. In Chapter 5, the efficacy of a passive epicardial patch was tested in a chronic large animal MI model with HF created with catheter-based coronary embolization. The implantation of an epicardical patch over the infarcted LV region was performed 8 weeks after MI in pigs with impaired LVEF. At week 20, pigs implanted with epicardical patches had significantly thicker LV wall thickness at the infarction sites, smaller LV dilation and better LV systolic function compared with control animals. The expression of MMP-9 was significant lower in the epicardical patch group at the peri-infarct zones. These findings suggested that a passive epicardial patch can improve LV function in HF and provides important proof-of-principle data to support its use as a platform for delivery of cell-based therapies after MI. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Dendritic Cells (DCs) are professional antigen presenting cells that play a crucial role in the induction of immune tolerance. Although DCs have been a potential target for immunotherapy, the amount of DCs in blood source is limited and ex vivo expansion has been inefficient. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide a great source in cell-based therapy because of their self-renewal ability and pluripotency. My project focuses on generating tolerogenic DCs (tDCs) from human pluripotent stem cells (i.e. hESCs and iPSCs) and their characterization. Specifically, hESCs and hiPSCs were first differentiated to hematopoietic progenitor cells (HPCs) using three different methods (i.e. bone-marrow stromal cell co-culture and two previously reported defined medium methods). The hESC/iPSC-differentiated hematopoietic progenitor cells (HPCs) were characterized by their surface phenotype using flow cytometry. Then the hESC/iPSC-differentiated immature DCs were further expanded and differentiated from the hESC/iPSCdifferentiated CD34+ HPCs with the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin 4 (IL-4). Tolerogenic properties were introduced by treating hESC-differentiated DCs with rapamycin. The treated DCs were characterized for their tolerogenicity by examining their expression of PDL1, PDL2, ICOS and CD40 etc., and their ability to promote regulatory T cells (Treg) differentiation. All these were compared with monocyte-derived tDCs. In summary, this study has examined the potential of using pluripotent stem cells-derived DCs as a cell source for immune tolerance induction therapy. / published_or_final_version / Anatomy / Master / Master of Philosophy

Although tremendous efforts have been made to search for other novel growth factors in promoting marrow recovery after irradiation or chemotherapy, there have not been any efficient and safe agents discovered so far. Danggui Buxue Tang (當歸補血湯) as a traditional Chinese herbal decoction, is commonly used for replenishing blood loss in menstruating women, or enhancing erythropoiesis and immune responses in various settings. Our previous study confirmed that Danggui Buxue Tang promotes haematopoiesis and thrombopoiesis both in vitro & in vivo. Recent studies also showed that parenteral Astragalus regulates haematopoiesis in myelosuppressed mice and has protection effect on UV irradiated human dermal fibroblasts. However, astragaloside IV, as the major component of Astragalus, the "Monarch" (君葯) in Danggui Buxue Tang, the bioactivity and its possible mechanism on haematopoiesis remains unclear. My studies showed that astragaloside IV had promoting effect on different lineages of haematopoietic CFUs forming including erythrocytes, granulocytes, monocytes and megakaryocytes both in normal and irradiated mice. In the K562 and CHRF apoptotic model, astragaloside IV exerted proliferation effect and induced K562 into megakaryocytic differentiation. Astragaloside IV up-regulated phosphorylation of ERK and it was abolished by PD98059. Meanwhile, astragaloside IV increased phosphorylated ERK migration into nuclei which enhanced cell survival and differentiation. EGFR inhibitor also attenuated the enhancing effect of astragaloside IV on ERK phosphorylation. It suggested that astragaloside IV is likely to function through EGFR with subsequent activation of ERK1/2 pathway. Furthermore, astragaloside IV also increased Bcl-2/Bax ratio by up-regulating Bcl-2 alone. Bone marrow derived mesenchymal stromal cells are the major supporting cells involved in the haematopoietic microenvironment. My studies demonstrated that astragaloside IV also indirectly enhanced haematopoiesis by stimulating cytokine release from MSCs, especially IL-6, IL-8, MCP-1 and GRO1. I also found that matured and activated population of neutrophils was increased after cultured with mesenchymal stromal cells conditional medium stimulated by astragaloside IV. This finding further supported why there was a significant increment of CFU-GM in vitro culture with murine bone marrow collected from mouse model after astragaloside IV treatment, where MSCs serve as the feeder layer in such system in mice. In conclusion, my studies explored the directly and indirectly dynamic and multiple targeted function of astragaloside IV on haematopoiesis. In addition to activating haematopoietic cells, astragaloside IV also stimulated mesenchymal stromal cells to secret cytokines that could modulate haematopoiesis and up-regulated neutrophil production and maturation. It provided a holistic view on how astragaloside IV induced synergistic effect on haematopoietic cells and mesenchymal stromal cells in the marrow microenvironment. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

Human mesenchymal stromal cells (hMSCs) are part of bone marrow micro-environment that supports hematopoiesis. However, hMSCs also enhance tumor progression and survival when they become part of the cancer micro-environment. I aimed to investigate the interaction between hMSCs and cancer cells during chemotherapy. Firstly, I studied the interaction between hMSCs and T-lineage acute lymphoblastic leukemia (T-ALL) cells under pegylated arginase I (BCT-100) treatment. Three T-ALL cell lines were sensitive to BCT-100 but not hMSCs. Conversely, hMSCs could partly protect all T-ALL cell lines from BCT-100 induced cell death under transwell co-culture condition. Concerning the possible mechanism, the intermediate metabolite L-ornithine could not rescue most T-ALL cells from BCT-100 treatment. But the downstream L-arginine precursor, L-citrulline could partly rescue all T-ALL cells from BCT-100 treatment. Ornithine transcarbamylase (OTC) converts L-ornithine into L-citrulline. OTC expression level in hMSCs remained relatively high during BCT-100 treatment but OTC expressions in T-ALL cell lines declined drastically. It suggested that hMSCs may protect T-ALL cells against BCT-100 treatment by having sustained OTC expression. Suppression of hMSCs by vincristine (VCR) disrupted the protective effect of hMSCs to most T-ALL cells during BCT-100 treatment. This suggests that by transiently suppressing hMSCs, we may abolish the protective effect of hMSCs to T-ALL cells during BCT-100 treatment. Then I studied the interaction between hMSCs and neuroblastoma under cisplatin treatment. Two neuroblastoma cell lines were used for both of them are cisplatin sensitive while hMSCs are cisplatin resistant. hMSCs could partly protect neuroblastoma cells from cisplatin induced cytotoxicity. On the other hand, exogenous IL-6 but not IL-8 could also partly rescue them from cisplatin induced cytotoxicity. IL-6 activated STAT3 phosphorylation dose-dependently and enhanced expression of detoxifying enzyme (glutathione S-transferase π, GST-π) in neuroblastoma. Such effect could be counteracted by anti-IL-6R neutralizing antibody tocilizumab (TCZ). However, TCZ failed to suppress hMSCs’ protection to neuroblastoma during cisplatin treatment. This suggests involvement of multiple factors. Up-regulation of serum GST-πin some hTertMSCs/neuroblastoma co-engrafted SCID mice compared to neuroblastoma engrafted mice provided a clue that GST-π might be a possible stromal-protection factor. Caffeic acid phenethyl ester (CAPE) is a known GST inhibitor after tyrosinase activation. Neuroblastoma cells expressed tyrosinase and CAPE enhanced cisplatin cytotoxicity on them, with or without hMSCs. Paradoxically, CAPE enhanced GST-πexpression with or without cisplatin treatment in neuroblastoma suggesting possible negative feedback to GST-π inhibition. However, such additive effect of CAPE to cisplatin cytotoxicity was not observed in vivo. Further delineation of the in vivo study design may help to verify the additive effect of CAPE to cisplatin cytotoxicity in vivo. Finally, I studied the effect of apoptotic cancer cells (AC) on the immune function of hMSCs. hMSCs could phagocytose apoptotic neuroblastoma cells with respective up-regulation of many immune-mediators including two highly-expressed cytokines IL-6 and IL-8. Up-regulation of these immune-mediators may enhance immune cells chemotaxis. Further detailed investigation on the effect of AC-engulfed hMSCs to other immune cells will help us to understand the dynamic interaction between cancer cells and stromal cells during chemotherapy. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Proteinuria has been recognized as a common feature in many forms of chronic kidney disease (CKD). As traditional medications for proteinuric nephropathy, such as blockade of the renin-angiotensin system (RAS), has only achieved limited clinical success, more effective renoprotective strategies need to be explored. Bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) have recently shown promise as a therapeutic tool in acute kidney injury (AKI) models. The therapeutic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) in proteinuric nephropathy models is unknown. Using a co-culture model of human proximal tubular epithelial cells (PTECs) and BM-MSCs, I first examined the potential effect of BM-MSCs in albumin-induced pro-inflammatory response and epithelial-to-mesenchymal transition (EMT) in PTECs. The unstimulated BM-MSCs exerted moderate suppressive effect on tubular inflammation as only albumin-induced CCL-2 and CCL-5 expression was attenuated in PTECs. When concomitantly stimulated by albumin excess, however, BM-MSCs remarkably suppressed albumin-induced tubular IL-6, IL-8, TNF-α, CCL-2, and CCL-5 expression, suggesting albumin overloaded milieu to be a prerequisite for them to fully exhibit their anti-inflammatory effects. This effect was mediated via deactivation of tubular NF-κB signaling as BM-MSCs prevented the overexpression of p-IκB and nuclear translocation of NF-κB. In addition, albumin-induced tubular EMT, as shown by the loss of E-cadherin and induction of α-SMA, FN-1 and collagen IV in PTECs, was also prevented by BM-MSC co-culture. To dissect the mechanism of action, I next explored the paracrine factors secreted by BM-MSCs under an albumin-overloaded condition and studied their contribution to the protective effect on tubular inflammation and EMT. Albumin-overloaded BM-MSCs per se overexpressed 34 paracrine factors, of which hepatocyte growth factor (HGF) and TNFα-stimulating gene (TSG)-6 were regulated by P38 and NF-κB signaling. These paracrine factors suppressed both the proinflammatory and profibrotic phenotypes in albumin-induced PTECs. Neutralizing HGF and TSG-6 abolished the anti-inflammatory and anti-EMT effects of BM-MSC co-culture in albumin-induced PTECs, respectively. Finally, in albumin-overloaded mice, a well established murine model reminiscent of human CKD, treatment with mouse BM-MSCs markedly reduced BUN, tubular CCL-2 and CCL-5 expression, interstitial macrophage, α-SMA and collagen IV accumulation independent of changes in proteinuria, together with upregulated renal cortical expression of HGF. Exogenous BM-MSCs were detected in their kidneys by PKH-26 staining. Collectively, these in vitro and in vivo data suggest a modulatory effect of BM-MSCs on albumin-induced tubular inflammation and fibrosis and underscore a therapeutic potential of BM-MSCs for CKD in the future. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs) for disease modeling, cardiotoxicity screening, drug discovery and cell‐based therapies. However, as shown in previous studies conducted by our lab (Poon, Kong et al. 2011), human embryonic stem cells (hESCs)‐derived CMs display immature〖Ca〗^(2+)–handing properties with smaller transient amplitudes, slower rise and decay kinetics than those of adult CMs. Although the cytosolic 〖Ca〗^(2+) signaling of hESC‐CMs has only recently been understood, there is no investigation on the nuclear 〖Ca〗^(2+) signal in hESC‐CMs, despite its importance. In this dissertation, delayed kinetics of nuclear 〖Ca〗^(2+), as compared to that of cytosol during 〖Ca〗^(2+)waves or 〖Ca〗^(2+) transients, was found in hESC‐derived ventricular (V) CMs, indicating that nuclear 〖Ca〗^(2+) was initiated by 〖Ca〗^(2+) diffusion from cytosol. Besides global 〖Ca〗^(2+) signals, local nuclear 〖Ca〗^(2+) signals were observed and identified as Ca2+ release from ryanodine receptors (RyRs), and nucleoplasmic reticulum (NR) served as their structural basis. In addition, targeted expression of 〖Ca〗^(2+) buffering protein parvalbumin (PV) in cytosol or nucleus altered 〖Ca〗^(2+) transient and stimuli‐induced apoptosis of hESC‐VCMs. For cytosolic 〖Ca〗^(2+) signaling in hESC‐VCMs, the mechanistic basis of excitation‐contraction coupling of hESC‐VCMs was studied by using 〖Ca〗^(2+) sparks, which are the unitary 〖Ca〗^(2+) ‐events. The results indicated that RyRs could be sensitized by 〖Ca〗^(2+) in permeabilized hESC‐VCMs. Increasing external 〖Ca〗^(2+) dramatically escalated the basal 〖Ca〗^(2+) and spark frequency. Furthermore, RyR‐mediated Ca2+ release sensitized nearby RyRs, leading to compound 〖Ca〗^(2+) sparks, whereas inhibition of mitochondrial 〖Ca〗^(2+) + uptake promoted Ca2+ waves. The aforementioned immature 〖Ca〗^(2+)–handing properties of hESC‐CMs can be attributed to their differential expression of crucial Ca2+–handling proteins. During diastole, SERCA and NCX sequester and extrude 〖Ca〗^(2+) ions, respectively, to return cytosolic 〖Ca〗^(2+) to the resting level. As previously published in our lab, NCX, robustly expressed in hESC‐CMs but much less so in the adult counterparts, is a functional determinant of immature 〖Ca〗^(2+) homeostasis. Unlike NCX, SERCA is expressed less in hESC‐CMs than in adult‐CMs. The present study first demonstrated the effects of lentivirus‐based genetic manipulation of SERCA2a and NCX1 in hESC‐VCMs, and the results indicated that SERCA2a overexpression shortened the decay phase of low‐frequency (0.5 Hz) electrical stimulation‐elicited Ca2+ transient. Increasing pacing frequency from 0.5 Hz to 2 Hz led to a decrease of relative transient amplitude, showing that hESC‐VCMs harbored a negative‐frequency response. At a high‐stimulation frequency of 2 Hz, it was revealed that SERCA overexpression, but not NCX1 suppression, increased the amplitude of 〖Ca〗^(2+) transient by accelerating 〖Ca〗^(2+) sequestration to sarcoplasmic reticulum (SR), indicating partial rescue of the negative‐frequency response. Taken collectively, the findings provide 1) novel information on nuclear 〖Ca〗^(2+) signaling in hESC‐VCMs, 2) the first lines of direct evidence that hESC‐VCMs have functional 〖Ca〗^(2+)‐induced‐〖Ca〗^(2+)+‐release (CICR), and 3) evidence of driving hESC‐VCMs maturation by SERCA2a overexpression, which may facilitate clinical and other applications of hESC‐VCMs. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

abstract / Biochemistry / Master / Master of Philosophy

Cancer stem cells are a subpopulation of cells needed for cancer initiation and progression. Previous works have revealed CD26-expressing colorectal cancer (CRC) stem cells are not only endowed with tumor-initiating properties, but also capable of conferring metastasis. However, whether the CD26 molecule plays role in metastasis and the underlying mechanism by which CD26 may mediate metastasis remain unclear. This study aims to reveal the biology and the molecular characteristics of the CD26-expressing CRC stem cells. Here, by the gene manipulation experiment, we showed that CD26 molecule is a functional marker that confers metastasis as transient and stable knock-down of the CD26 molecule in the CRC stem cells resulted in reduced wound healing, migration and invasion abilities in vitro and the capability to generate metastatic liver nodules in vivo, respectively. With the use of genome-wide expression array and immuno-blotting analysis, Smad-dependent TGF-β signaling, orchestrated by the SMAD2, SMAD3 and SMAD4 molecules, was up-regulated and activated in the CD26 expressing colorectal CSCs. In addition, expressions of the SMAD2 and SMAD3 molecules were found to be positively correlated with the CD26 molecule in clinical samples by qPCR and immunohistochemistry studies. Furthermore, no metastasis through EMT could be achieved once the Smad-dependent TGF-β signaling was down-regulated in the CD26 expressing CRC stem cells, which suggested that Smad-dependent TGF-β signaling was necessary for CD26-expressing CRC stem cells to induce metastasis. Finally, our result showed that the Smad-dependent TGF-β signaling was regulated by the CD26 molecule possibly through the down-regulation of CAV1 protein. To conclude, our findings have not only revealed the functional role of CD26 molecule, but have also unveiled a linkage between the CD26 molecule and Smad-dependent TGF-β signaling. Further study of this connection may introduce a novel mechanism, through which CRC metastasis can be induced by this functional CD26 marker of CRC stem cells. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

The vascular endothelium is a dynamic cellular interface that displays a unique phenotypic plasticity. This plasticity is critical for vascular function and when dysregulated is pathogenic in several diseases. The development of new human endothelial genotype-phenotype studies, personalized vascular medicine efforts and cell based regenerative therapies are limited by the unavailability of patient-specific endothelial cells. Induced pluripotent stem cells (iPSC) offer great promise as a new personalized source of endothelium; however, the reproducibility, fidelity and functionality of iPSC-derived endothelial cells remains poorly understood. / Engineering and Applied Sciences

Human neurodegenerative disorders are among the most difficult to study. In particular, the inability to readily obtain the faulty cell types most relevant to these diseases has impeded progress for decades. Amyotrophic lateral sclerosis is a late onset neurodegenerative disease in which the upper and lower motor neurons degenerate, leading to paralysis and eventually death. Recent advances in pluripotent stem cell technology now grant access to significant quantities of disease pertinent neurons both with and without predisposing mutations. The two studies described in this thesis demonstrate the feasibility of using MNs, generated from pluripotent stem cell lines harboring known ALS mutations, to establish in-vitro models of the disease. Specifically, we first used gene targeting to establish genetically controlled systems, able to identify causal relationships between a familial ALS mutation and in vitro phenotypes. Next, using transcriptional profiling, we identified novel pathways altered by the mutation and demonstrated functional consequences of these pathways' misregulation. Furthermore, by monitoring the physiology of the pluripotent stem cell derived MNs, we discovered an increased firing rate in the mutant MNs, and identified an FDA-approved drug, retigabine, capable of rescuing this defect. Lastly, to aid in the discovery of additional therapeutic compounds, we combined gene targeting, transcriptional profiling, and a fluorescent reporter human embryonic stem cell line to establish a well-controlled in vitro system capable of identifying genetic modifiers of the phenotypes described herein.