Intracellular Ca2+ mobilization via secondary messengers modulates multiple cell functions. Cyclic Adenosine 5’-Diphosphate-Ribose (cADPR) is one of the most well recognized endogenous Ca2+ mobilizing messengers. In mammalian, cADPR is mainly formed by CD38, a multi-functional enzyme, from nicotinamide adenine dinucleotide (NAD). It has previously been shown that the cADPR/CD38/Ca2+pathway mediates many cardiac functions, such as regulating the excitation-contraction coupling in cardiac myocytes and modulating the Ca2+ homeostasis during the ischemia injury of the heart. Thus it is reasonable to propose that the cADPR/CD38/Ca2+ pathway plays a role in cardiogenesis. The pluripotent mouse embryonic stem (mES) cells which can be induced to differentiate into all cell types provide an ideal model for studying cardiogenesis. The first part of this dissertation is to determine the role of CD38/cADPR/Ca2+pathwayin the cardiomyogenesis of mES cells. The data showed that CD38 expression was markedly up-regulated during the in vitro embryoid body (EB) differentiation of mouse ES cells, which indicated a regulatory role of CD38 in the differentiation process. Lentivirus mediated shRNA provides a convenient method to knockdown the expression of CD38 in mES cells. Surprisingly, beating clusters appeared earlier and more in CD38 knockdown EBs than that in control EBs. Likewise, the expressions of several cardiac markers were up regulated in CD38 knockdown EBs. In addition, more cardiomyocytes (CMs) existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs than those in control EBs. On the other hand, over-expression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, we showed that CMs derived from the CD38 knock down mES cells possessed the functional properties characteristic of CMs derived fromnormal ES cells. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2cascade during CM differentiation of mES cells, and transiently inhibition of Erk1/2 blocked the enhancive effects of CD38 knockdown on the differentiation of CM from mES cells. Taken together, our data indicate that the CD38/cADPR/Ca2+ signaling pathway suppresses the cardiac differentiation of mES cells. 　　One of the main goals of the researches on cardiac differentiation of ES cells is to enhance the production of CMs from ES cells, thereby providing sufficient amount of functional intact CMs for the treatment of severe heart disease. Nitric oxide (NO) has been found to be a powerful cardiogenesis inducer of mES cells, in that it can significantly increase the yield of ES-derived CM. The second objective of this dissertation is to explore the mechanism underlying the NO facilitated cardiomyogenesis of mES cells. We found that the NO did induce intracellular Ca2+ increases in mES cells, and this Ca2+ increase was due to internal Ca2+ release from ER through theIP3 pathway. Therefore, the expression of IP3 receptors (IP3Rs) in mES cells were knocked down by lentivirus-mediated shRNAs. Interestingly, only type 3 IP3R (IP3R3) knockdown significantly inhibited the NO induced Ca2+ release in mES cells. Moreover, NO facilitated cardiogensis of mES cells was abolished in IP3R3 knockdown EBs. In summary, our results indicate that the IP3R3-Ca2+ pathway is required for NO facilitated cardiomyogenesis of mES cells. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Diabetic cardiomyopathy (DCM), a disorder of the heart muscle, is one of the major and most rampant culprits claiming thousands and thousands of lives around the globe every year by interfering with the blood circulation and causing the development of heart failure eventually. The progression of the disease is asymptomatic and having a long latent period, and it is characterized functionally by ventricular dilation, diastolic dysfunction, interstitial fibrosis and cardiomyocytes hypertrophy. It was suggested the pathogenesis of the disease and the related complications are related to the effects of hyperglycemia on cardiomyocytes. So understanding the physiology of both the normal and pathological conditions, and the underlying mechanisms involved are of paramount importance to derive therapies to cope with this disease. However, it is difficult, if not impossible, to study the physiology in vivo using a live sample or to build a cellular model with adult cardiomyocytes due to the insufficient number of the cells harvested. This is not until the emergence of Embryonic Stem Cells (ESCs) that a cellular model with clinical sufficient number of cardiomyocytes could be built for investigation and drug screening. With a view to mimicking the situation of the Diabetic cardiomyopathy of the Type II Diabetes mellitus (DM) patients, mouse ESCs are used to differentiate into cardiomyocytes using the traditional hanging drop method to produce Embryoid body (EB). The cardiomyocytes were then enriched and plated so that different testing conditions could be applied. The effect of high glucose (HG), Insulin and the combination of high glucose and insulin were then analyzed. This was to show the significance of hyperglycemia, hyperinsulinemia due to insulin resistance and the role of insulin in hyperglycemia on cardiomyocytes respectively. The results agreed with previous findings that high glucose and insulin alone do induce cells apoptosis while the combination of insulin and glucose did decrease the number of apoptosis and while the co-culture of insulin with High dosage of glucose has shown to reduce the effect of hypertrophy. / published_or_final_version / Medicine / Master / Master of Medical Sciences

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

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

No description available.

No description available.

No description available.

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.