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Grds 26 dec 2014 edited

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Grds 26 dec 2014 edited

  1. 1. Comparative analysis of cardiovascular development related genes in stem cells isolated from deciduous pulp and adipose tissue Loo Zhang Xin (Phd candidate) ICAET, 26-27 December 2014
  2. 2. Introduction AMI = acute myocardial infarction
  3. 3. Cell therapy as an alternative? • Left ventricular of the human heart contains ~5.8 x 109 cardiomyocytes. • Upon the onset of AMI, 25% of the cardiomyocytes will be lost. • Regeneration of the infarcted heart potentially requires: – a billion cardiomyocytes, – along with other supporting cells such as endothelial and smooth vascular cells for functional integration. • Possible few mechanisms for which these cells may influence the cardiovascular regeneration: – Differentiation of stem cells into cardiomyoctes, – Fusion of stem cells with endogenous cells, – Stem cells secretome signaling
  4. 4. List of suitable candidates Embryonic stem cells (ESC) Induced pluripotetent stem cells (IPS) Adult stem cells Pros • Totipotency • Laflamme et al. showsed that ESC were able to generate cardiomyocytes and had limited death rate in animal model. • Can generate a patient’s specific stem cells. • Ieda et al. used a combination of cardiac transcription factors to direct reprogram cardiac fibroblast into cardiomyocytes. • Cardiac stem cells (CSCs) offers better prospects. • Bolli et al. reported successful clinical trial using CSCs in human subjects with ischemic cardiomyopathy had been reported. Cons • Ethical issues  destruction of embryo • Complicated isolation methods • Tendency to form tumours • Risk of insertional mutagenesis is of particular concern. •Invasive procedures in isolating and culturing the cells • Escalating production cost due to autologous settings.
  5. 5. Upcoming candidates
  6. 6. Methodology • Characterized MSCs sources: human dental pulp (SHED) and human adipose stem cells (ASC)  Morphology, Tri-lineage differentiation and immunophenotyping  Human Stem Cell Transcription Factors Array for initial screening  Ingenuity Pathway Analysis • Cardiac differentiation using defined media  SHED and ASC were seeded onto 6-well plates at a density of 100,000 cells/cm2 .  Cardiac media: KO-DMEM containing 0.5% penicillin/streptomycin and 1% 1X Glutamax were supplemented with 100 ng/ml human recombinant Activin A for 24 hours followed by 10 ng/ml human recombinant BMP2 for 4 days.  The medium was then exchanged for KO-DMEM without supplementary cytokines, and cultures were refed every 2–3 days in a humidified incubator at 37°C and 5 % CO2 for up to 14 days.  Cell morphology was captured using an inverted microscope, RNA was extracted at day 7 and day 14, immunocytochemisty at day 14.
  8. 8. Results & Discussion
  9. 9. MSCs derived from SHED and ASC: Fibroblastic morphology Expressed MSCs markers  Capable of differentiating into osteoblasts, adipocytes and chondroblasts
  10. 10. WHY Human stem cell transcription factor array ? REASON: Plenty of experimental works emphasized on understanding stem cell characteristics such as proliferation pathways, quality of the cells under in vitro culture conditions & lack of information pertaining to stem cells at the basal level. Observation: Higher expression level for the HOX group of genes in the ASC [Development of obesity and body fat distribution] SHED expressed many markers related to pluripotency [More primitive
  11. 11. • Both types of cells are prone to angiogenesis as there are more interconnected molecules involved in formation of blood vessels and vasculogenesis. • This provides an explanation of how stem cells are able to improve or preserve cardiac function in an animal model through angiogenesis and not due to cardiomyocyte differentiation. • In hypoxic conditions, expression of HIF-α increases rapidly which in turn activates genes that encode for pro-angiogenic growth factors, such as VEGF, BGFG, Ang-1 and 2, PFG and PDGF-B. • These released cytokines encourage angiogenesis and the angiogenic effects of human multipotent stromal cell which explains the underlying improvement of cardiac function through any individual or a combination of mechanisms such as apoptosis inhibition, increase in survival, and angiogenesis stimulation by activation of the PI3K-Akt pathway.
  12. 12. ASC: Assumed more of a polygonal shape. SHED: Elongated and irregular There has been no reports yet of a successful protocol using dental stem cells to generate beating cardiomyocytes. Prior examples utilized dental pulp stem cells with a combination of VEGF, BFGF and IGF-1. In this case, spontaneous cellular beating was also not detected and the morphology similar to what we observed in our experiments. Only GATA4 and NKX 2.5 were detected in differentiated ASC
  13. 13. ASC expressed significantly more cardiomyocyte biomarkers compared to SHED at both day 7 and day 14 [Could be both adipose and heart development are mesoderm origin. This indicates that not all sources of MSCs are suitable for cardiac differentiation & selection of the MSC source for treating a disease highly depended on its origin. Spontaneous cellular beating, the hallmark indicator of successful cardiomyocyte differentiation was not detected in our experiment [Could be cTnI was suppressed in this condition whereby it can only be activated when the cells were cultured with neonatal rat cardiomyocytes, or when these cells were delivered in vivo to murine models of myocardial infarction]
  14. 14. Acknowledgement This work is part of a research collaboration between Hygieia Innovation and the Faculty of Dentistry, University of Malaya and is supported by the University of Malaya, High Impact Research Grant, Ministry of Higher Education, Malaysia. (Grant No UM.C/HIR/MOHE/DENT/02)

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