Upon aging, the intervertebral disc (IVD) inevitably undergoes degeneration characterized by biochemical and morphologic changes. IVD degeneration can lead to multiple clinical disorders such as back and neck pain, and myelopathy. Low back pain can disable up to 85% of the adult population and results in a significant restriction of social activities and inability to work. Such disorder incurs billions of dollars in medical expenditures each year. Despite advances in the detection and treatment of the degeneration, the regeneration of the IVD remains low because current therapies are limited by exogenous curing approaches. New strategies for the reversal of IVD degeneration, including gene, cytokine, and stem cell therapies that can influence the anabolic and catabolic pathways in disc cells have been under investigation. These therapies aim to rejuvenate or replace diminished nucleus pulposus cells in the degenerative IVD. Recent reports have put forth a proposal of “endogenous disc stem cells”, suggesting that cells derived from the degenerative IVD tissue possess stem cell properties. These putative stem cells are believed to regulate the development and homeostasis of the IVD tissue. In this study, we identified and characterized a stem cell population from the IVD of healthy Rhesus monkey, termed disc stem/progenitor cells (DSCs). We show that the DSCs possess clonogenicity, multipotency and self-renewal capacity. The DSCs are phenotypically similar to bone marrow mesenchymal stem cells (BMSCs) but they are not identical. The DSCs show a faster growth rate under hypoxia than normoxia. DSCs derived from nucleus pulposus (DSCNP) show a stable expression level of hypoxia inducible factor-1 alpha (Hif-1ａ) in response to hypoxia. DSCs derived from annulus fibrosus (DSCAF) are more resistant to apoptosis under hypoxia than DSCNP. More importantly, small leucine-rich proteoglycans (SLRPs) are identified as important DSC niche components. We show that biglycan (bgn) and decorin (dcn) reduce the susceptibility of DSCs to hypoxia-induced apoptosis by promoting the expression of hypoxia inducible factors (HIFs). Our findings suggest that DSCs rely on the unique niche components for survival. In summary, our findings propose the existence of endogenous stem cells in IVD. Further study of the DSCs may provide new insights into the biology of IVD and facilitate the design of new strategies to treat disc degeneration in future. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

A major challenge facing the tissue engineering discipline is cartilage tissue repair and engineering, because of the highly specialized structure and limited repair capacity that cartilage possesses. Dental pulp stem cells (DPSCs) were identified about a decade ago as a potential candidate for cell based therapy and tissue engineering applications. The present study aimed to utilize gene therapy with isolated DPSCs to induce chondrogenic transgene expression and chondrogenic lineage differentiation, with the ultimate goal of engineering cartilage tissue-like constructs. We isolated DPSCs from human teeth extracted for orthodontic treatment. We further enriched the isolated population using immunomagnetic bead selection, which increased stem cell markers: Stro-1 and CD146, compared to unselected population. The DPSCs showed the ability to differentiate into the chondrogenic lineage when induced with recombinant hTGFβ-3 and when transduced with hTGFβ-3 transgene. We successfully constructed the recombinant adeno-associated viral vector encoding the human TGFβ-3, and determined the best multiplicity of infection for DPSCs. The transduced DPSCs highly expressed hTGFβ-3 for up to 60 days. Expression of chondrogenic markers; Collagen IIa1, Sox9, and aggrecan was verified by immunohistochemistry and mRNA. We successfully fabricated an electrospun nano-fiber scaffold upon which morphology, proliferation and viability of the DPSCs were examined. DPSCs attached and proliferated on nano-fiber scaffolds demonstrating better viability compared to micro-fiber scaffolds. Transduced cells expressed hTGFβ-3 protein up to 48 days. Cells seeded on nanofiber scaffolds showed higher expression levels compared to micro-fiber scaffolds or culture plate. Scaffolds seeded with DPSCs were implanted in nude mice. Immunohistochemistry for TGFβ-3 DPSCs constructs (n=5/group) showed cartilage-like matrix formation with glucoseaminoglycans as shown by Alcian blue. Immunostaining showed positivity for Collagen IIa1, Sox9 and aggrecan. Semi-thin sections of the transduced DPSCs constructs examined by transmission electron microscopy (TEM) showed chondrocytic cellular and intra-cellular features, as well as extracellular matrix formation (n=2/group). In vivo constructs with the TGFβ-3 DPSCs showed higher collagen type II and Sox9 mRNA expression relative to non-transduced DPSCs constructs (n=5/group). Western blot analysis confirmed this expression pattern on the protein level (n=3/group). Engineered constructs mechanical properties were examined and compared to patellar bovine cartilage to assess functionality (n=5/group). TGFβ-3 transduced DPSCs constructs showed a higher equilibrium elastic modulus compared to nontransduced constructs. Micro-fiber scaffolds constructs showed a higher elastic modulus (0.11 MPa, 18% of bovine cartilage), compared to nano-fiber constructs modulus (0.032 MPa, 6% of bovine cartilage). Nano-fiber based constructs showed a similar Poisson‘s ration to bovine cartilage, while that of micro-fiber scaffolds was lower. As an alternative gene delivery method, electroporation parameters for DPSCs transfection were optimized, and compared to commonly used chemical transfection methods. TGFβ-3 transfected DPSCs showed a significantly higher relative TGFβ-3 mRNA and protein expression compared to non transfected control and to eGFP transfected DPSCs. Transfected DPSCs showed increased relative expression of chondrogenic markers; Collagen II, Sox9 and aggrecan, compared to non transfected DPSCs. Successful chondrogenic differentiation of DPSCs gene therapy with TGFβ-3 transgene, and seeding them on PLLA/PGA scaffolds makes it a potential candidate for cartilage tissue engineering and cell based therapy. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

When human mesenchymal stem cells (hMSCs) are cultured inside a 3D collagen meshwork, they become a potential tissue engineering bone graft alternative. However, the in vitro osteogenesis rate of hMSCs is slow, leading to a low mineral deposition. To enhance the osteogenic differentiation of hMSCs, low intensity pulsed ultrasound (LIPUS) was employed as an external stumulus. The present study demonstrated the feasibility of employing daily LIPUS exposure for enhancing osteogenesis in vitro. Exposure of seven consecutive days LIPUS, each of 30 minutes duration, did not affect the cell viability, and the organization of hMSCs within the collagen meshwork was not disturbed. The calcium deposition within the collagen meshwork was enhanced after seven days of exposure. The osteoinductivity was also upregulated at the early period of culture. In order to optimizing the enhancement effects of LIPUS, various ultrasound parameters, including intensity, exposure duration and exposure repetition were investigated. Results showed the LIPUS enhancement effects are dose dependent, LIPUS exposure should be longer than 10 minutes/day in order to elicit a significant effect. Calcium deposition was higher when LIPUS exposure was done twice per day instead of one. Although individual variation exists, optimal LIPUS intensity range was between 60-120 mW/cm2 ISATA (Spatial Average Temporal Average Intensity). The interaction mechanism between LIPUS and cells was also investigated. Microbubbles were added to the culture during LIPUS exposure to find out whether cavitation is involved in the interaction. Flow sensor primary cilium was also studied in order to verify that ultrasound is transduced through fluid flow. Results showed cavitation may not be a contributing factor to osteogenesis, and primary may be involved in the transduction of LIPUS stimulation. This study demonstrated that osteogenesis of hMSCs encapsulated in collagen constructs could be enhanced by LIPUS. The LIPUS parameters were also optimized. The LIPUS interaction pathways were also being better understood. This thesis study will be a paradigm for cellular mechanotransduction studies and put an important step forward for therapeutic ultrasound. / published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

Tumor heterogeneity has long been observed and recognized as a challenge to cancer therapy. The cancer stem cell (CSC) model is one of the hypotheses proposed to explain such a phenomenon. A specific cancer stem cell marker has not been determined for non-small cell lung cancers (NSCLC), preventing the definitive evaluation of whether the biology of NSCLC is governed by the CSC model. This study aimed to analyze the expression of candidate CSC markers and using the identified putative marker, to isolate CSC and determine the applicability of the CSC model in NSCLC. The expression or activities of four putative stem cell markers, CD24, CD44, CD133 and aldehyde dehydrogenase 1 (ALDH1) were measured by flow cytometry in eight NSCLC cell lines before and after chemotherapy for 24 hours. Markers with increased expression after treatment were considered potential CSC markers and used for isolating tumor cell subpopulations from the untreated cell lines by fluorescence-activated cell sorting (FACS). Confirmatory analyses using widely acceptable methodology were performed to test for CSC properties in the marker+ and marker- subpopulations. Isolated subpopulations were further characterized by functional and phenotypic studies. Flow cytometry showed amongst the 4 markers, only ALDH1 expression was significantly enhanced by chemotherapeutic treatment, suggesting ALDH1 could be a CSC marker. Untreated ALDH1+ cells formed significantly more and larger cell spheres in non-adherent, serum-free conditions than ALDH1- cells. Likewise, higher in vitro tumorigenic ability was observed in ALDH1+ subset using colony formation assay. Furthermore, a higher resistance to cytotoxic drugs was observed in ALDH1+ compared to ALDH1- cells. In vivo studies also showed ALDH1+ cells showed higher tumorigenicity than ALDH1- cells; as few as 2,500 ALDH1+ cells formed tumor in SCID mice which were serially transplantable to 2nd and 3rd recipients, while no tumor was formed from ALDH- cells with even ten times the number of cells. Also, expression analysis revealed higher expression of the pluripotency genes, OCT4, NANOG, BMI1 and SOX9, in ALDH1+ cells. In view of previous studies reporting CD44 as a CSC marker in lung cancer, double marker selection of putative CSC was performed to compare ALDH1+CD44+ and ALDH1-CD44+ subpopulations. Results of the spheroid body formation assay and cisplatin treatment experiments revealed the ALDH1+CD44+ subpopulation possessed higher self-renewal ability and chemo-resistance. Cell migration and invasion assays showed differences between the ALDH1+CD44+ and ALDH1- CD44+ subpopulations. The significance of these observations require further investigation. In conclusion, the result showed that ALDH1 could be a marker for NSCLC stem cells as evidenced by enhanced self-renewal and differentiation abilities in ALDH1+ subpopulation. Furthermore, this study observed the presence of at least two potential stem cell subpopulations in NSCLC cells with differential selfrenewal, chemotherapy resistance and cell mobility properties. Further investigations are required to validate these observations and to investigate the underlying mechanisms. Better understanding of these issues would help to solve the challenges brought by tumor heterogeneity in lung cancer therapy. / published_or_final_version / Pathology / Master / Master of Philosophy

　　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

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by persistent airway obstruction that is only partially reversible. It is the fourth leading cause of death and is predicted to be the third by 2030. The progression of the disease involves chronic inflammation, oxidative stress, excess protease activity, increased lung cell apoptosis and accelerated lung aging, but the exact pathogenesis is still unclear. The major cause of COPD is cigarette smoking(CS). Although COPD is associated with increasing social and economical burden, there have been few advances in pharmacological therapy of COPD. Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells which can be isolated from a broad range of sources including bone marrow (BM) and adipose tissue. Administration of BM-derivedMSCs (BM-MSC) or adipose tissue-derived MSCs was reported to attenuate CS-induced emphysema in murine models. Induced pluripotent stem cell-derived MSC (IPSC-MSC) are MSCs differentiated from induced pluripotent stem cells(IPSCs), which are pluripotent cells generated by somatic cell reprogramming in vitro. IPSC-MSCs have several advantages over BM-MSC, including more abundant sources and high capacity of doubling without loss of differentiation potency. A general exploration and comparison on the effects of human IPSC-MSC and BM-MSC treatments were carried out in a 56-day CS-exposed rat model. Compared to BM-MSC, IPSC-MSC showed a higher capacity to reside in lung tissue. The two treatments shared similar efficacy to attenuate CS-induced lung cell apoptosis, to restore CS-induced reduction of lungIL-10and to alleviate CS-induced elevation of systemic TGF-β1. In addition, IPSC-MSC was found to cause reduction in CS-induced elevation of systemic oxidative stress and reversal of CS-induced reduction of lung adiponectin. Furthermore, in order to understand the possible paracrine mechanism involved, human airway epithelial cells were treated with IPSC-MSC or BM-MSC-conditioned medium in a cell culture system in the presence of cigarette smoke medium (CSM). Potentiation rather than attenuation of CSM-induced release of pro-inflammatory cytokine IL-8, MCP-1 and IL-6 was observed with IPSC-MSC or BM-MSC conditioned medium. It is currently unknown whether cultured IPSC-MSCs or BM-MSCs will release pro-inflammatory mediators into the conditioned medium or not. In order to study CS-induced oxidative stress and inflammation in a short time frame, anacute (5-day) CS-exposed rat model was established in juvenile and adult groups. An age-dependent alteration of CS-induced oxidative and inflammatory responses was demonstrated in this model. In summary, our in vivo rat model provides a platform for elucidating the effects of stem cell treatment in CS-induced oxidative stress and inflammation, leading to lung damage. Our findings suggest that treatment of IPSC-MSC or BM-MSC might be able to slow down CS-induced disease progression, possibly through anti-oxidant, anti-inflammatory and anti-apoptotic properties. However, caution should be taken as our in vitro data revealed that conditioned medium from MSCs may provoke pro-inflammatory responses. Further studies on the regulation of the activity of MSCs in vivo will be needed before developing IPSC-MSC into cell therapies for COPD to halt the progression over time. / published_or_final_version / Medicine / Master / Master of Philosophy

Degenerative disc disease (DDD) is a medical condition whereby the intervertebral discs (IVD) of the human spine degenerates and may cause pain which significantly affects the quality of one掇 life. Its prevalence has sparked off much research in deciphering its causes and developing new treatments. Recently, attempts to treat this degenerative problem have turned to seeking answers from regenerative medicine. One approach is to deliver mesenchymal stem cells (MSCs) with or without carriers to the nucleus pulposus (NP) in degenerative disc to restore both its structural and functional properties. However, the optimal conditions and signals for inducing MSCs differentiation toward NP-like phenotype have not been identified. This work aimed to develop injectable microspheres with matrix microenvironment mimicking that of native NP tissue for MSCs delivery. Firstly, it was aimed to establish a collagen microencapsulation based 3D culture system for maintenance of the phenotype of nucleus pulposus cells (NPCs) and remodeling of the collagen matrix to one that was similar to the native NP. Secondly, it was aimed to optimize a decellularization protocol for complete removal of the encapsulated NPCs with minimal loss of remodeled extracellular matrix. Thirdly, it was aimed to investigate whether this acellular matrix produced by NPCs was inductive for MSCs discogenic differentiation. Finally, it was aimed to evaluate the efficacy of the MSC-seeded acellular matrix microspheres in a pilot rabbit disc degeneration model. It demonstrated that NPCs maintained their phenotype, survived within the collagen microspheres and produced NP-like ECM such as glycosaminoglycan (GAG) and collagen type II. GAG production of NPCs was found to positively correlate with the dosage of TGF-? within a short period, initial collagen concentration and cell seeding density. An optimized decellularization protocol with 50mM SB-10, 0.6mM SB-16 and 0.14% Triton X-200 was established to completely remove the encapsulated NPCs with partial retention of the GAG-rich matrix. The decellularized microspheres were able to be repopulated with human MSCs (hMSCs) or rabbit MSCs (rMSCs). Within the NPC-derived acellular matrix, the repopulated hMSCs were able to partially exhibit NPC phenotype with upregulated expression of a few NPC markers and NP-like ECM according to histological, biochemical, immunohistological and real-time PCR results. In the pilot in vivo evaluation study, preliminary results showed that intra-discal injection of MSCs reseeded acellular NPC-matrix microspheres maintained a better water content than the control MSC-microspheres without the NPC-derived acellular matrix. This work reconstituted in vitro a NP-like 3D matrix and provided preliminary evidence on discogenic differentiation of MSCs in such a matrix environment. This work laid down the foundation to future development of stem cell-based therapies for DDD. Further studies should focus on deciphering the soluble and insoluble composition of such a NP-like matrix environment and understanding the molecular mechanism of the cell-matrix interactions involved. / published_or_final_version / Mechanical Engineering / 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

Regeneration of the tissues to replace diseased, missing and traumatized dentin/pulp requires combining the recent progress in stem cell and tissue engineering research. Dental pulp stem cells (DPSCs) are considered as a promising population of cells in regenerative dentistry and shown to be able to produce dentin/pulp-like tissues following implantation in-vivo. Securing a good blood supply is critical in pulp regeneration, however, this is a challenging task due to the unique structure of the tooth, the anatomy of which permits only a microcirculatory system via a very small apical opening (<0.3-1mm). This limitation raises the need to develop novel methods to enhance angiogenesis during pulp regeneration. It was shown that DPSCs reside in the microvasculature region of the dental pulp and interact with perivascular cells. Therefore, endothelial cells could be a major source of modulators of pulp-dentin development and angiogenesis. If a pulp tissue substitute with pre-formed endothelial network could be engineered in-vitro, it would not only gain rapid anastomosis with host vasculature but also regulate DPSC function in pulp regeneration. In this study, for the first time, synergistic effects of DPSCs and human umbilical vein endothelial cells (HUVECs) on osteo/odontogenic differentiation and angiogenesis were investigated using two-dimensional and three-dimensional direct co-culture systems. Furthermore, the potential of three-dimensional DPSC constructs prevascularized with HUVECs in dental pulp regeneration in-vivo was exmined. HUVECs promoted odonto/osteogenic differentiation of DPSCs in direct two-dimensional co-cultures in-vitro. Further, addition of DPSCs stabilized the pre-existing vessel-like structures formed by HUVECs and increased the longevity of these structures on matrigel in-vitro. Using two different systems, scaffold-free self-assembling microtissue spheroids and peptide hydrogel scaffold, the interactions of DPSCs and HUVECs in three-dimensional cultures were investigated. The results demonstrated that DPSCs can self assemble into three-dimensional microtissue spheroids when cultured alone or with HUVECs. DPSCs promoted survival and vascular structure formation by HUVECs both in scaffold-free microtissue spheroids and peptide hydrogel scaffold. In contrast, HUVECs, when cultured alone, neither formed vascular structures nor survived in either of the 3D systems. The latter phenomenon was attributable to vascular endothelial growth factor secreted by DPSCs, a major factor responsible for endothelial function. Co-cultures also showed enhanced odonto/osteogenic differentiation in both three-dimensional microtissue spheroid and peptide hydrogel scaffold systems. Following implantation of tooth-root fragments filled with three-dimensional DPSC constructs into the subcutaneous space of immunodefficient mice, vascularised pulp-like tissue was regenerated within the root canals. Compared to DPSC-only group, DPSC/HUVEC co-culture groups showed higher vascularisation, extracellular matrix formation and mineralization in regenerated tissue. More importantly, HUVEC-lined vascular lumens were observed in regenerated tissues suggesting the successful integration of in-vitro formed pre-vascular structures to the host vasculature. In summary, the findings suggest that DPSCs and HUVECs display significant synergy during odonto/osteogenic differentiation and angiogenesis when co-cultured either in two-dimensional or three-dimensional culture systems. Unravelling these fundamental behavioural patterns of DPSCs provides novel insights into the process of pulp regeneration, leading to new avenues for more effective therapies in regenerative endodontics. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Despite major advances in diagnosis and prevention of coronary artery disease (CAD), the development of therapies to regenerate functional cardiomyocytes after myocardial infarction (MI) is very challenging. Studies have demonstrated that bone marrow derived mesenchymal stem cells (BM-MSCs) secrete a panel of growth factors and anti-inflammatory cytokines to activate resident cardiomyocytes and cardiac stem cells in myocardial repair after MI. However, the mechanisms of modulating BM-MSC secretions are not well understood. Recently, molecular candidates in regulating BM-MSCs paracrine secretion to improve cardiac protection have been explored. Amongst the molecular candidates, Nuclear casein kinase and cyclin-dependent kinase substrate 1 (Nucks1) is suggested as a regulatory protein in nuclear factor-kappa B (NF-κB) signaling pathway by interacting with TANK-binding kinase 1 (TBK1). TBK1 is a non-canonical I kappa B (IκB) kinase that can activate the NF-κB transcription factor and its transcriptional response. NF-κB signaling pathway controls many cellular responses such as cell survival, proliferation and cytokine productions. We hypothesizes Nucks1 may have potential roles in regulating mouse BM-MSCs secretion of growth factors and cytokine profiles in heart repairs after MI. To test our hypothesis, the cardiac protection efficacy of acute infarcted mouse myocardium was measured after the transplantation of WT versus Nucks1 KO BM-MSCs. To this end, we developed a mouse model of acute myocardial infarction (AMI) induced by ligation of left descendant coronary artery. Acute infarcted mouse myocardium receiving WT or Nuck1 KO BM-MSCs transplantation, demonstrated a significant improvement of left ventricular ejection fraction (LVEF), ESP, +dP/dt, ESPVR and vessel density, and reduced infarction size in comparison with PBS control group post-4 weeks of transplantation. Furthermore, acute infarcted mouse myocardium receiving Nucks1 KO BM-MSCs transplantation provided better cardioprotective effects than those receiving WT BM-MSCs transplantation. Immunostaining disclosed CD31 and smooth muscle actin (SMA) expression in acute infarcted mouse myocardium receiving Nucks1 KO BM-MSCs were relatively higher than those receiving WT BM-MSCs transplantation. Additionally, a distinct secretion profile of growth factors and cytokines between Nucks1 KO BM-MSCs versus WT BM-MSCs under in vitro ischemia was studied. Expression of vascular endothelial growth factor alpha (VEGFα) in Nucks1 KO BM-MSCs under hypoxia/ serum deprivation was significantly higher than that of WT BMMSCs. Taken together, our data suggested BM-MSCs provide cardiac protection in acute infarcted myocardium. Transplantation of Nucks1 KO BMMSCs may further enhance the cardiac repair of the acute infracted myocardium through an induction of VEGFα. / published_or_final_version / Medicine / Master / Master of Philosophy