Programme. Time Title Speaker. Session 1 (Chairperson: Prof Kong Bung Choo)

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2 Programme Time Title Speaker am Registration Session 1 (Chairperson: Prof Kong Bung Choo) am Opening remarks Prof. Soon Keng Cheong (FMHS, UTAR) am Mesenchymal stem cells: bench to clinic Prof. Soon Keng Cheong am Feeder-independent induction of pluripotent stem cells Dr. Shigeki Sugii (A*Star, Singapore) am Tea break Session 2 (Chairperson: Prof Soon Keng Cheong) am Non-viral delivery of erythropoietin gene into a mouse model using human mesenchymal stem cells Pooi Ling Mok (PPUKM-MAKNA) am GDF-5 as a regulator of tenogenic differentiation in mesenchymal stem cell: the effect, the process and the outcome Sik Loo Tan (Faculty of Medicine, UM) am MicroRNA profiling in induced pluripotent stem cells: a first glimpse Prof Kong Bung Choo (FMHS/CSCR, UTAR) pm Adipose stem cell system as potential therapeutic platforms for metabolic diseases Dr. Shigeki Sugii pm Disclosing the secrets of your samples with live cell imaging Rageshwary Ramupillai (Carl Zeiss) pm Lunch (sponsored by Carl Zeiss & Next Gene Scientific) 2 P a g e

3 MESENCHYMAL STEM CELLS: FROM BENCH TO CLINIC Soon Keng Cheong Faculty of Medicine & Health Sciences, Universiti Tunku Abdul Rahman, Bandar Sungai Long, Selangor. Stem cells can be divided into three broad categories: pluripotent, multipotent and unipotent stem cells. Embryonic stem cells and induced pluripotent stem cells are examples of pluripotent stem cells. Adult stem cells such as haemopoietic stem cells (HPSC) and mesenchymal stem cells (MSC) are examples of multipotent stem cells. These two types of adult stem cells can be isolated from the bone marrow, though MSC are have also been isolated from other tissues. MSC are currently the hottest adult stem cells in the radar. They were discovered way back in the seventies. In the last 15 years or so, their many appealing characteristics have generated much interest in the laboratories and the clinics. After initial Phase I clinical trials in the late nineties, MSC have been cultured and expanded to treat osteogenesis imperfecta, graft versus-host disease post HPSC transplantation, myocardial infarction, intractable heart failure, ischemic stroke, and arthritic diseases. In the last decade, MSC have shown promising results in Phase II trials for graftversus host disease. In Malaysia, MSC have been successfully isolated and expanded in the laboratories and scaled up for treatment of intractable heart failure with very promising outcome. Applications in other disorders are in progress. 3 P a g e

4 FEEDER-INDEPENDENT INDUCTION OF PLURIPOTENT STEM CELLS Shigeki Sugii Singapore Bioimaging Consortium, A*Star, Singapore. Discovery of induced pluripotency has tremendous implication in regenerative medicine. However, a number of issues remain, which include a low efficiency and use of animal sources (in culture medium and feeder layers) in generating induced pluripotent stem (ips) cells. Using adult human and mouse multipotent mesenchymal stem cells isolated from fat tissue, we found that these cells can be reprogrammed to ips cells, with substantially higher efficiencies than those reported for other cell types. Unexpectedly, both human and mouse ips cells can be produced in feeder-free conditions. We also developed human ips cells in the xeno (animal sources)-free manner, which is a key step toward production of clinical grade cells. These results demonstrate a great potential for mesenchymal stem cell types in regenerative therapeutics. 4 P a g e

5 NON-VIRAL DELIVERY OF ERYTHROPOIETIN GENE INTO A MOUSE MODEL USING HUMAN MESENCHYMAL STEM CELLS Pooi Ling Mok, a,c Soon Keng Cheong, a,b Chooi Fun Leong, c Kien Hui Chua, d & Othman Ainoon c,e a PPUKM-MAKNA Cancer Centre, Universiti Kebangsaan Malaysia, Kuala Lumpur. b Faculty of Medicine & Health Sciences, Universiti Tunku Abdul Rahman, Kajang, Selangor. c Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur. d Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur. e Faculty of Medicine and Health Sciences, Universiti Sains Islam Malaysia, Kuala Lumpur. Erythropoietin (EPO) stimulates red blood-cell production and is produced in the fetal liver and adult kidney. Since the first successful cloning of the human EPO gene in 1985, recombinant EPO (rhuepo) has become a therapeutic option for renal anemia in chronic renal failure (CRF) patients and several forms of non-renal anemia. In light of predicted rise in CRF in aging populations globally, the need to develop a cost-effective alternative is apparent. Transplanting EPO-producing cells of human origin is an ideal therapeutic alternative for CRF patients because it would be more cost effective and less likely to result in the serious complications of pure red-cell aplasia due to auto-regulation. The success of producing a biologically functional EPO depends on glycosylation of the EPO protein. The N-glycans, for example, play a major role in secretion, molecular stability, solubility, receptor binding affinity and in vivo elimination of EPO. The choice of host cells, the culturing conditions and the purification procedures determine the composition of the glycan isoforms of a rhuepo preparation. To date, human EPO gene has been transfected in myoblast and skin cells and has successfully corrected anemia in renal failure in a murine model. Mesenchymal stromal cells (MSC) are an attractive cell-targeting vehicle for gene delivery. The objective of our study was to test the ability of the human MSC (hmsc) to deliver the erythropoietin (EPO) gene in a nude mice model following nucleofection using a MIDGE construct. MIDGE (an acryonym for Minimalistic, Immunologically Defined Gene Expression) construct is relatively safer than the viral or plasmid expression system as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. hmsc nucleofected with MIDGE encoding the EPO gene was injected subcutaneously in Matrigel at the dorsal flank of nude mice. Subcutaneous implantation of nucleofected hmsc resulted 5 P a g e

6 in increased hemoglobin level with presence of human EPO in the peripheral blood of the injected nude mice in the first two weeks post-implantation compared with the control groups. The basal layer of the hair shaft in the dermal layer was found to be significantly positive for immunohistochemical staining of a human EPO antibody. However, only a few basal layers of the hair shaft were found to be positively stained for CD105. In conclusion, hmsc harboring MIDGE-EPO could deliver and transiently express the EPO gene in the nude mice model. These cells could be localized to the hair follicle and secreted EPO protein might have possible role in hair regeneration. 6 P a g e

7 GDF-5 AS A REGULATOR OF TENOGENIC DIFFERENTIATION IN MESENCHYMAL STEM CELLS: THE EFFECTS, THE PROCESSES AND THE OUTCOMES Sik Loo Tan & Tunku Kamarul National Orthopaedics Centre of Excellence in Research & Learning, Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. Growth Differentiation Factor 5 (GDF-5), a member of the human BMP family, has been identified as a key biological molecule that can accelerate tendon healing. However there are limited studies investigating the isolated effect of GDF-5 on hmsc proliferation and differentiation in vitro. Our current work investigated the optimum concentration of GDF-5 that can induce maximal phenotypic expression of the tenogenic hmsc in vitro. A gradient of GDF-5 concentration was tested, considering that progenitor cells can differentiate into various types of mature cells, eg. osteocytes, chondrocytes and tenocytes, in response to the amount of signal molecules received. In our studies, no significant changes were observed in the cell proliferation rate in hmscs treated at different concentration of GDf-5. This growth factor appeared to induce tenogenic differentiation at 100ng/ml, as reflected by a significant increase in total collagen expression, similar to that of native human tenocytes culture. Furthermore, a significant up regulation in candidate tenogenic marker genes expression, i.e. scleraxis, tenascin-c and type-i collagen were observed in hmscs culture in presence of GDF-5. A significant down regulation of the non-tenogenic marker genes RUNX2 and SOX9 further confirmed that hmsc does not differentiated into non-tenogenic lineages upon GDF-5 induction. From our global gene expression analysis results, we postulated that the GDF-5 acts as a molecular cue that regulates tenocyte-specific transcription factor, particularly scleraxis, through the Smad/BMP signaling cascade, which in turn regulates the expression of type-i collagen in tendon tissues. In our in vivo studies, MSCs differentiated with GDF-5, showed more efficacious in animal rotator cuff tendon healing than undifferentiated MSCs. This work provides new insights into the control of tenogenesis that may ultimately lead to a stem cell based therapy for tendonitis. 7 P a g e

8 MICRORNA PROFILING IN INDUCED PLURIPOTENT STEM CELLS: A FIRST GLIMPSE Chiu-Jung Huang 1, Shigeki Sugii 2 & Kong Bung Choo 3,4 1 Department of Animal Sciences & Institute of Biotechnology, Chinese Culture University, Taipei, Taiwan 2 Singapore Bioimaging Consortium, A*Star, Singapore 3 Faculty of Medicine and Health Sciences & Centre for Stem Cell Research, University Tunku Abdul Rahman, Selangor, Malaysia Three types of stem cells have been characterised: (i) embryonic stem cells (ESC) derived from the totipotent inner cell mass of blastocysts; (ii) mesenchymal stem cells (MSC) derived from various sources of adult tissues; (iii) induced pluripotent stem cells (ipscs) which are products of reprogramming from MSCs and other somatic cells and are described as ESClike for their ability to differentiate into all three germ layers. In this work, we aimed to investigate if unique molecular signatures exist to distinguish each of the three stem cell types. If so, what do the signatures mean in terms of the molecular mechanism(s) that govern the establishment of each cell type. mirna profiles of each stem cell types were established using the nanolitre-scale real-time PCR microarray platform of the WaferGen BioSystems which included 1,036 mature mirnas. The data obtained were first subjected to in silico validation in which mirnas known or predicted to reprogramme MSC into ipsc were used to verify the microarray data. In cases of inconsistency, real-time PCR was performed to support our microarray data. In this first-glimpse analysis under stringent analysis criteria, only 32 mirnas are found to be differentially expressed in ipsc relative to ESC, consistent with ESC-like nature of ipsc. On the other hand, 261 mirnas are found to be differentially expressed in ipsc relative to the MSC from which the ipscs were derived, echoing the distinct differences in differentiation potencies of the two stem cell types. While the two ipscs originated from two adipose-derived MSCs (AD-MSCs) are similar in their mirna profiles, significant differences in the mirna profiles are evident when the AD-MSC-iPSCs are compared with an ipsc derived from preadipocytes, indicating that ipscs derived from different cell sources are dissimilar. In a preliminary hierarchical clustering analysis of a small number of mirnas, we are able to identify mirnas that are consistently and specifically expressed in each stem cell type. Further analysis of validated or predicted target genes of these unique sets of mirnas would lead to an understanding of the regulatory mechanisms that govern reprogramming and differentiation potency of ESC, MSC and ipsc. 4 Supported by UTARRF grant 6200/C38 (CKB) and in part by NSC (Taiwan) grant B (HCJ). 8 P a g e

9 ADIPOSE STEM CELL SYSTEM AS POTENTIAL THERAPEUTIC PLATFORMS FOR METABOLIC DISEASES Shigeki Sugii Singapore Bioimaging Consortium, A*Star, Singapore. Adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells. It also possesses anti-diabetic potentials; we found that specific activation of PPARγ in adipocytes leads to improved adipokine profiles, lipid metabolism, insulin signaling and inflammation in vivo. As presented in the first talk, fat tissue is a great source for induced pluripotency. Adipose-derived stem cell s ability to generate ips cells with high efficiency and in the absence of feeder cells makes it ideal for various applications including regenerative therapeutics, development of drug screening platforms and modelling of human diseases. In order to promote use of fat-derived stem cells, however, it is necessary to understand and define their origin and cellular characteristics more in detail. Potential use of these cells for treating metabolic diseases such as diabetes, and possible roles of metabolic regulators, nuclear receptor superfamily, in cellular reprogramming will be discussed. 9 P a g e

10 DISCLOSING THE SECRETS OF YOUR SAMPLES WITH LIVE CELL FLUORESCENCE IMAGING Rageshwary Ramupillai Carl Zeiss, Malaysia The technique of fluorescence microscopy has become widely established as a research instrument for investigating fixed and live biological specimens. Nevertheless, all biological specimens from the thinnest cell monolayer to large whole organism inherently have some level of thickness. Historically, the only way to get clear microscopic images of a thicker sample was to physically cut very thin sections of the sample which was time-consuming and not conducive to imaging of living samples. During the last few decades a large number of techniques have been developed that allow faster and more reliable way to get clear Live Cell Fluorescence Images. What is Live Cell- Fluorescence Imaging? This brief talk will provide an overview of Live Cell Fluorescence Imaging with Introduction of Basic Fluorescence Microscopy, Brief explanation of various Fluorescence Labeling Techniques along with control parameters for Live Cell Imaging. And finally, an Overview of the Right Microscope solution helps scientists to handle the most comprehensive applications in research. 10 P a g e

11 Organising Committee Advisor: Prof Emeritus Dr Cheong Soon Keng Chairperson: Prof Choo Kong Bung Committee members: Teoh Hoon Koon (Secretary; Symposium lead) Amanda Teoh Siak Mun (Workshop lead; Reception of Singapore team) Soon Yuen Loon (Treasurer) Malcolm Tan (Transportation) Erica Choong Pei Feng Wong Chee Yin Lee Han Chung Teh Hui Xin Nalini Devi a/p Verusingam 11 P a g e