Day 1 :
Keynote Forum
Mariusz Grudniak
Polish Stem Cell Bank, Warsaw, Poland FamiCord Group
Keynote: New insights into Alzheimer disease and potential mesenchymal stromal cell therapy
Time : 9:30-10:15
Biography:
Mariusz Grudniak has completed his PhD at the age of 37 years from Medical University of Warsaw. He is an expert of Research and Development Department of Polish Stem Cell Bank. He has published 6 papers in reputed journals.
Abstract:
Alzheimer’s Disease is a non treatable condition affecting more and more people every year. There are many clinical trials concerning treatment of this disease, but none has finished with positive results so far. The aim of this study was to evaluate possible correlations between Alzheimer’s Disease and the alterations in cytokine/chemokine level in the serum of 20 patients in the various stage of the disease and 10 healthy volunteers as a control. The stem cell therapy is a very fast developing branch of medicine. Very interesting from the scientific point of view seem to be the co-culture of stem cells and limfocytes obtaining from patients and healthy donors. Cord tissue was the source of mesenchymal stromal cells (MSC). Investigated interactions between MSC and limfocytes was measured by obtaining supernatant after 1, 3, and 7 days of co-culture. The conditions allowed or preserve of direct cell-to-cell interaction from paracrinne effect due to physical barriers. The preliminary findings of this investigation showed interesting relations between cells resulting in cytokine levels. One of the proposed explanation of beta-amyloid plaques creation is the dysregulation of immunological system. Th2 lymphocytes cannot compensate increased activity of Th1 lymphocytes and their overproduction of proinflamatory cytokines. This situation has the negative outcomes. It is the inability to control the production of beta-amyloid that in particular is not eliminated and leads to neuronal death. Additional is microglia incorrect activity which is able in physiological conditions to establish protein homeostasis in central nervous system.
Keynote Forum
Raghvendra V Tey
Associate Professor, Pathophysiology, Saint Georges University, Grenada, W.
Keynote: Regenerative Medicine in MD course: Current Scenario, Challenges & Future
Time : 10:15-11:00
Biography:
Dr Tey has been persuing his goal of promoting integrative approach in Medical education and Medical practice since his Medical graduation days. He strongly believes that molecular level changes are responsible for disease manifestations. A few years back there were many gaps in our knowledge, which are rapidly filling with evolution and strong emphasis on evidence based medicine. We are now more than ever, ready to explain many diseases starting from the molecular changes. He therefore strongly vouches for taking the clinician back to the basics and start treating at the molecular or cellular level using cell therapy. This realization came after a personal struggle & success in doing so by clubbing his MD postgraduate training in Clinical Biochemistry with Internal Medicine and few other specialties, after battling for a curriculum change. He believes that this approach would decentralize medicine, multidisciplinary cooperation would increase and bring in many noble innovations in future.
Abstract:
Current Scenario: As an insider, a simple approach like cell therapy involving, administering a few millions of highly flexible cells which can transform the functional and structural behaviour of that site, turned out to be a revolutionary idea. Though the idea looks quite simple and feasible, it has met lot of resistance. The age old approach where a synthetic or refined form of a natural chemical can cure or manage the disease is been quiet steadily replaced by Cell therapy, in effect to the realization of the side effects, cost & effectiveness of the prior approach.
Challenges: The idea that cells can be used as therapies and can be procured and processed in an ordinary clinic is very empowering to the medical fraternity and poses an existential challenge to the big pharmaceuticals with huge investments. It’s very disruptive to the manner in which medicine is practiced today. It breaks all the inter-disciplinary barriers and that is the reason, its intimidating to many established disciplines and acceptance is slow but steady.
Future: The Idea of Integrated curriculum approach being adopted in many medical universities (Mayo Clinic, USA) across the globe. In here, doctors in every year of their training are helped in understanding the problem in every organ system using their basic science knowledge. How a disturbance at a cellular/molecular level manifests as a disease with clinical presentation can all be explained in detail. The story of Stem cells changing the course of a disease process fits naturally & nearly perfectly into this background understanding with integrated curriculum approach.
Conclusion: The future looks bright for cell based therapies in medical curriculum & practice as they are in line with today’s medical curriculum. This can bridge the shortage of physicians globally. Looking beyond profits, cell therapy would reinstate people’s belief in their own hidden powers, unknown to mankind until recently.
Keynote Forum
Suman Chandra Nath
University of Calgary Calgary, Alberta, Canada
Keynote: Bioprocess Development for High-Density Culture of Human Induced Pluripotent Stem Cells in Bioreactor
Time : 11:00-11:45
Biography:
Suman Chandra Nath is working as a postdoctoral fellow in the Rancourt lab, Cumming School of Medicine, University of Calgary, Canada after completing his PhD from Kino-oka lab, Osaka University, Japan. His research is focused on applying human stem cells for curing degenerative diseases. Currently, he is developing bioprocesses for reprogramming human skin cells to induced pluripotent stem cells (iPSCs) in bioreactor and differentiating them to specific cell types for treating degenerative diseases. Before starting his works in the University of Calgary, his research performed in Kino-oka lab was remarkable in the field of stem cell engineering especially for establishing simple method for passaging hiPSCs aggregates and developing bioprocesses for reducing culture cost in the stirred suspension bioreactor.
Abstract:
Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank for clinical applications. Although suspension culture yields large cell numbers, there are still unresolved challenges for the expansion of hiPSCs because large size aggregates show low growth rate during long-term culture. In this study, we have investigated the size- and time-dependent growth properties of hiPSCs aggregates, and set up a boundary condition to maintain high growth rate in suspension culture. We have also established a simple method for hiPSC aggregate break–up into small sizes by using botulinum hemagglutinin (HA). hiPSCs showed size–dependent growth heterogeneity, and aggregates ranging from 100-200 µm showed high growth rate during early-stage of culture (24–72 h). hiPSCs proliferation was also dependent on culture time because the growth rate decreased significantly during late-stage of culture (72–120 h) at which point collagen type I accumulated on the periphery of aggregate. Therefore, to maintain high growth rate, controlling aggregate size, and shortening culture time is important. To obtain high cell density in suspension culture, hiPSC aggregates were exposed to HA, and broken into small sizes by pipetting. A maximum cell density of (4.5 ± 0.2) × 10^6 cells/mL was obtained by aggregate break–up into small ones, which was 3 times higher than that by the conventional culture without aggregate break–up. This method allowed high-density culture of hiPSCs by breaking-up aggregates into small sizes without the need for enzymatic treatment or centrifugation in suspension culture. Therefore, considering the boundary conditions for aggregate size and culture time is important to obtain high growth rate, as well as high cell density in stirred suspension bioreactor culture.
- Stem cells | Genetics and stem cell biology | Cancer stem cells | Tissue engineering | Stem cell therapy | Stem cell transplantation | Stem cell niche | Regenerative Medicine | Adult stem cells | Embryonic stem cells
Session Introduction
Xianmin Zeng
Buck Institute, Novato, CA, USA
Title: Making multiple therapeutic cell products from a cGMP-compliant iPSC lin
Time : 1:30-2:00
Biography:
Dr. Xianmin Zeng received her PhD in Molecular Biology from the Technical University of Denmark in 2000 and had her postdoctoral training at the NIH. She joined the faculty of the Buck Institute Research on Aging in 2005 where she builds the Institute’s Stem Cell Program, and is current Professor at the Buck Institute. Dr. Zeng is a recipient of several major funding including a translational grant to develop clinically grade dopaminergic neurons from pluripotent stem cells for Parkinson’s disease from California Institute for Regenerative Medicine (https://www.cirm.ca.gov/our-progress/people/xianmin-zeng), and an iPSC-based toxicity screen grant from the NIH. She is also the Founder and Chief Executive Officer of XCell Science Inc, a biotech company dedicated to providing reagents and services in neural space, and NxCell Inc, an iPSC-based therapy company.
Abstract:
We have developed several current Good Manufacture Practice (cGMP)-compliant induced pluripotent stem cell (iPSC) lines for clinical applications. Here we show that multiple cellular products currently being considered for therapy can be generated from a single master cell bank of a clinically compliant iPSC line. Here we tested differentiation into therapeutic relevant cell types of the three germ layers by standardized protocols using a stock prepared from the cGMP-compliant working cell bank. Cells we generated include 1) neural stem cells, dopaminergic neurons and astrocytes, 2) retinal cells (RPE and photoreceptors) and 3) hepatocyte, endothelial and mesenchymal cells. To confirm that these protocols can also be used for other iPSC lines we tested the reproducibility of our methodology with a second clinically compliant line. Our results confirmed that the same protocols could be used with minimal modifications with multiple qualified lines. In addition, we introduced a constitutively expressed GFP cassette in Chr13 safe harbor site and observed no significant difference in growth and differentiation between the engineered line and the control line. We believe that our demonstration that multiple products can be made from the same WCB, and that the same protocols can be used with multiple lines offers a path to a cost effective strategy for developing cellular products from iPSC lines.
Chandravanshi Bhawna
School of Regenerative medicine, GKVK Post, Bellary Road, Yelahanka, Bangalore
Title: Stem cell therapy facilitates tolerance in renal transplantation
Time : 2:00-2:30
Biography:
Bhawna Chandravanshi during the tenure of her project mastered several basic as well as specialized cell culture techniques such as maintenance of normal and cancer cell lines, isolation of islets from murine pancreas, isolation and cultivation of stem cells from Human umbilical cord, amnion, placenta etc. She explored the possibility of in vitro generation of islets from the mesenchymal stem cells (MSCs) derived from discarded tissues of human origin. She succeeded in obtaining large number of functional islets from these MSCs and could demonstrate restoration of normoglycemia upon transplantation of these islets in STZ diabetic mice. To her credit she has 5 first author publications, 5 second author publications with 2 book chapters. She has already visited Japan twice for oral and poster presentation and has bagged best oral presentation award.
Abstract:
The auto-immune destruction of pancreatic β cells leads to occurrence of Type 1 diabetes. Two main therapeutic approaches for the treatment of T1D are islet transplantation and exogenous insulin injection. However both the approaches have not attained significant success. One of the main reasons for the decline in the success of islet transplantation is the shortage of donor pancreas and their poor survival upon transplantation. Hence in the present study we generated ample number of islet like cell aggregates ( ICAs) from Wharton’s jelly mesenchymal stem cells (WJ-MSC) as published earlier and stored them at ultra low temperature (-196ËšC) employing a cocktail of small molecules (Eicosapentaenoic acid+Docosahexaenoicacid+Metformin)) in addition to 5% DMSO. The ICAs were revived after 30days, cultured for 24h and then were assessed for their viability, oxidative status and functionality. The viability was determined by MTT and FDA/PI assay and it was found that the combinations of small molecules maintained the viability by 4fold when compared to controls Further the small molecules guarded the islets against oxidative stress and hence significantly reduced total ROS (p<0.05) (55% reduction), superoxide ions (p<0.05) (65% reduction) and nitric oxide (p<0.01) (70%) free radicals. The expression of HIF1α was also enhanced in the presence of small molecules. There was significant down-regulation of the stress markers like CHOP, NOS2a and IL1β, apoptotic markers like p53 and Caspase 3.Further this was confirmed by western blot analysis where we found down-regulation of Caspase 3 and phosphor p38 and up-regulation of anti-apoptotic marker, BCl2. Finally the ICAs also responded to glucose and showed significant enhancement in insulin secretion in presence of high glucose (5 fold). Hence our data demonstrates for the first time that a cocktail of small molecules with only 5% DMSO protect islets against cryo injury thus opening new dimension for islet banking prior to transplantation.
Sholeh Etehad Asnaf
Azad University,North Tehran Branch Department of biology,Iran
Title: Evaluation of gene expression of NLRP3 and NLRC4 as Inflammasome receptors in peripheral blood of SM injured patient by Real-Time
Time : 2:30-3:00
Biography:
She worked in laboratory and Baghiyatollah hospital on chemical warfare victimsand and successfully defend her thesis In the title of Evaluation of gene expression of NLRP3 and NLRC4 as Inflammasome receptors in peripheral blood of SM injured patient by Real-Time She is highly experienced in different techniques including PCR, Electrophoresis, cell culture and western blot.
Abstract:
Mustard gas is one of the most widely used chemical warfare agents used during the Iraqi wars against Iranian warriors. Some mechanisms of chronic pulmonary damage caused by mustard gas such as oxidative stress, protease-antiprotease imbalance and inflammation have been studied. In the context of inflammation, the role of inflammacom and the genes involved in it, especially the receptors of this inflammatory complex, is important. Therefore, the present study evaluated the expression of NLRP3 and NLRC4 genes as inflammatory receptors in peripheral blood of veterans of pulmonary chemo using Real- Time PCR. The present study was conducted as a cross-sectional analytical study on 15 chemical warfare victims exposed to mustard gas, 15 COPD patients and 15 healthy individuals as controls that were referred to the lung clinic of Baqiyatallah Hospital (AS). After extracting RNA from the blood sample and synthesizing cDNA, expression of the two NLRP3 and NLRC4 genes was evaluated using Real Time PCR. Finally, the data were analyzed by SPSS version 20 The two NLRP3 and NLRC4 genes did not change significantly in veterans and people with COPD compared to healthy subjects. Although the expression of these two genes was higher in COPD patients than veterans (0.17 to 0.05 and 0.15 to 0.04, respectively), there was no statistically significant relationship. According to the results of the present study, it has been found that two NLRP3 and NLRC4 genes have the potential to be involved in inflammation and chronic obstructive pulmonary embolism. As a result, it is hoped that with the full knowledge of the process of chronic damage to the lungs caused by mustard gas, one can use it to create a pattern of diagnosis and prevent its further complications
Umang G. Thakkar
Department of Stem Cell Therapy and Regenerative Medicine
Title: Co‑infusion of Autologous Adipose Tissue Derived Neuronal Differentiated Mesenchymal Stem Cells and Bone Marrow Derived Hematopoietic Stem Cells, a Viable Therapy for Post‑traumatic Brachial Plexus Injury: A Case Report
Time : 3:30-4:00
Biography:
Abstract:
Stem cell therapy is emerging as a viable approach in regenerative medicine. A 31-year-old male with brachial plexus injury had complete sensory–motor loss since 16 years with right pseudo‑meningocele at C5–D1 levels and extra‑spinal extension up to C7–D1, with avulsion on magnetic resonance imaging and irreversible damage. We generated adipose tissue derived neuronal differentiated mesenchymal stem cells (N-AD-MSC) and bone marrow derived hematopoietic stem cells (HSC-BM). Neuronal stem cells expressed β-3 tubulin and glial fibrillary acid protein which was confirmed on immunofluorescence. On day 14, 2.8 ml stem cell inoculum was infused under local anesthesia in right brachial plexus sheath by brachial block technique under ultrasonography guidance with a 1.5-inch-long 23 gauge needle. Nucleated cell count was 2 × 104/µl, CD34+ was 0.06%, and CD45-/90+ and CD45-/73+ were 41.63% and 20.36%, respectively. No untoward effects were noted. He has sustained recovery with re-innervation over a follow-up of 4 years documented on electromyography-nerve conduction velocity study. (Biomed J 2014;37:237-240)
Dudhal Swati
PhD scholar at Université Paris Diderot, France
Title: Selenoprotein N, mutated in SEPN1-related myopathy, restrains muscle stem cell differentiation’
Time : 4:00-4:30
Biography:
I am an aspiring researcher (Indian), currently working as a PhD scholar at Université Paris Diderot. I have completed MSc in Molecular Biology and Human Genetics from Manipal University and an undergraduate degree in Biomedical Science (BSc Hons) from University of Central Lancashire, UK. During my studies and mainly internships, I got interested in subjects dealing with the stem cells therapeutic applications. This was the genesis of my decision to enrol for PhD and training in translational research. During my clinical training, industrial training, research institutes and public university experience; I worked on various types of stem cells eg. Human Umbilical Cord Mesenchymal Stem Cells (HUCMSC’s), differentiation studies of murine fetal mesenchymal stem cells (FMSC’s) into DA neurons, murine Muscle Stem Cells (MuSC's) and several cell lines. I also learned Stereotactic surgery and behavioural tests for mice which was useful for treatment of neurodegenerative disorders like Parkinson’s disease. (Kumar A, et al, 2016)
Abstract:
Mutations of selenoprotein N (SEPN1) cause a congenital myopathy, SEPN1-related myopathy (SEPN1-RM), characterized by severe weakness and wasting of neck and trunk muscles, scoliosis and lethal respiratory failure. SEPN1-RM has been associated with oxidative stress, reduced satellite cell population and defective muscle regeneration. To investigate the underlying mechanisms, particularly a potential role of SEPN1 in regulating the balance between self-renewal and differentiation of the satellite cell pool, we used Sepn1 KO mouse primary satellite cells and C2C12 cells knocked down for Sepn1, at different stages of differentiation (quiescent cells, myoblasts and myotubes).
Using a suspension system to generate synchronized quiescence on C2C12, we found that SEPN1 absence does not prevent cell cycle exiting and re-entering but prevents normal downregulation of myogenic factors in G0 cells and leads to higher cyclin D1 levels in quiescence conditions. Microarray, qRT-PCR and protein studies showed that SEPN1 depletion leads to significant increase of MYOG and MYOD1 expression in proliferative C2C12. Moreover, primary muscle cells from the Sepn1 KO mice showed increased myoblast fusion at early myogenic differentiation.
We are currently exploring the mechanistic pathways leading to this cell phenotype. While we found no abnormalities of the AMPK-mediated pathway, our data suggest that HDAC5 could be involved in the accelerated differentiation phenotype. Other mechanistic studies are in progress.
In conclusion, lack of SEPN1 leads to incomplete quiescence and accelerated myogenic differentiation. Thus, we identify SEPN1 as a novel regulator of the muscle progenitor’s cell fate decision process, and SEPN1 depletion favors differentiation over self-renewal. These results potentially explain the depletion of the satellite cell population and the regeneration defect in SEPN1-RM models, and identify novel biomarkers useful to assess potential therapeutic interventions.