Mesenchymal stem cells are more and more widely used in tissue engineering due to their pluripotency and no relative ethical problems. Traditional characterization techniques to detect mesenchymal stem cell states include flow cytometry, gene expressing profiling and immunohistochemistry. However, these methods can only provide transient and low level information from current RNA or protein levels about mesenchymal stem cells, which may cause problems when predicting the possible downstream lineages they will commit into. We have developed chromatin immunoprecipitation (ChIP)-based epigenetic technique to detect mesenchymal stem cell states. For the systems we tested, this epigenetic assessor successfully characterized cell state changes and gave similar results obtained from gene expression profiling or protein expression assay. This epigenetic technique can provide information about mesenchymal stem cells states from a more fundamental chromatin level, which is promising for predicting future lineages from current states.

Numerous intra and extra cellular factors take role in differentiation of cell towards a given lineage. These factors have crucial role in cell-cell and cell-environment interactions. In this study, the aim is to investigate the effect of mechanical forces on the adipogenic differentiation of preadipocytes and mesenchymal stem cells in an in vitro model. Human preadipocytes and mesenchymal stem cells were embedded in 2 % agarose discs. According to the stress-relaxation test results it was observed that initial mechanical properties of agarose-mesenchymal stem cell (MSC) discs did not change compared to acellular agarose whereas those of preadipocytes decreased significantly. The discs with cells were exposed to compression under different weights (1.4 &plusmn / 0.2 g, 7.5 &plusmn / 0.2 g, and 14.6 &plusmn / 0.3 g.) continuously in differentiation medium for 21 days. The control discs were treated with differentiation medium without any compressive weight on top of them. After 21 days, total ribonucleic acids (RNA) have been isolated. Adipogenic differentiation was investigated via reverse transcription coupled quantitative polymerase chain reaction (PCR). The expression of peroxisome proliferators-activated receptors (PPAR-gamma), CCAAT-enhancer binding protein (C/EBP-Beta), leptin, adiponectin, adipophilin and human stearoyl-CoA desaturase (hSCD) have been assessed as adipogenic markers. Differentiation to adipocytes has been further investigated by histochemical Sudan IV staining and immunochemistry and compared to control group. Decrease in the expression of adipogenic factors, size and number of lipid droplets were observed for both MSCs and preadipocytes subjected to compression in agarose discs. The decreases were correlated with the level of mechanical stress. The highest depletion of gene expression was observed in leptin and C/EBP&amp / #61538 / . From our results, it was shown for the first time that mechanical stress impaired the adipogenic differentiation of MSCs and preadipocytes in agarose discs. However, the differentiation pathways should be further investigated.

Due to the limited intrinsic healing ability of mature cartilage tissue, stem cell therapies offer the potential to restore cartilage lost due to trauma or arthritis. Mesenchymal stem cells (MSCs) are a promising cell source due to their ability to differentiate into various adult tissues under specific biochemical and physical cues. Current MSC chondrogenic differentiation strategies employ large pellets, however, we have previously developed a high-throughput technique to form small MSC aggregates (500-1,000 cells) that may reduce diffusion barriers while maintaining a multicellular structure that is analogous to cartilaginous condensations. The objective of this study was to examine the effects on chondrogenesis of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within these small MSC spheroids when cultured in the presence of transforming growth factor-β1 (TGF-β1) over 21 days. Spheroids +MP induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids -MP, although no large differences in immunostaining for these matrix molecules were observed by day 21. Collagen I and X was also detected in the ECM of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. Because chondrogenesis was not hindered by the presence of CSMA MPs, this study demonstrates the utility of this culture system to further examine the effects of matrix molecules on MSC phenotype, as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific target tissues.

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

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

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Discovery of the ability of mesenchymal stem cells (MSCs) to differentiate into cells of non-mesodermal tissues, particularly neuronal cells, have raised the possibility of utilising MSCs in regenerative/reparative therapies for neurological disorders. However, a number of hurdles remain to be resolved. This thesis aims to address some of these issues by investigating the characteristics of bone marrow-derived human MSCs (hMSCs) during long-term culture, the potential of hMSCs to differentiate in vitro toward the neuronal lineage under the influence of cytokines, and the effects of intracerebral transplantation in the hemiparkinsonian rat model. During expansion culture hMSCs were found to display the expected characteristics of MSC populations, and also constitutively expressed neural and pluripotency markers simultaneously with mesodermal markers. Analysis of hMSC long-term subcultivation revealed an optimal period for commencing neuronal differentiation (first 6-8 passages), and also showed the absence of spontaneous neural differentiation. Application of neural-inducing cytokines and culture conditions resulted in the generation of an immature neuronal-like phenotype by hMSCs. Through live cell microscopy it was demonstrated for the first time that cytokine-based hMSC neuronal differentiation occurs through active and dynamic cellular processes involving outgrowth and motility of cellular extensions. In addition, single- and multiple-stage cytokine-based strategies for inducing dopaminergic neuronal-like cells from hMSCs were investigated. These studies revealed that FGF-2 and EGF exerted the greatest benefits for hMSC neuronal differentiation. Undifferentiated and neuronal-primed hMSCs were transplanted intracerebrally into the striatum and substantia nigra of cyclosporine-treated hemiparkinsonian rats. Grafted hMSCs could be clearly identified at 1-day and 7-days post-transplantation; however, grafts were gradually lost over time, with complete absence by 21-days. Co-transplantation with olfactory ensheathing cells, neuronal-priming prior to grafting, and nigral as well as striatal grafting could not provide engraftment and differentiation advantages. Immunohistological analysis demonstrated the presence of innate inflammatory responses (microglia and astrocyte activation) at graft sites, fibronectin deposition by hMSCs, and lack of endogenous host neurogenesis. The results of my PhD work indicate that cytokine-based culture methods are capable of differentiating hMSCs to an immature neuronal-like phenotype, and host-mediated innate inflammatory responses may be a key contributing factor for the failure of in vivo hMSC engraftment.