12^th International Conference & Exhibition on Tissue Preservation, Life care and Biobanking (B2B & Networking) November 9-10, 2018 Atlanta, Georgia, USA

Kejin Hu received his PhD degree in animal molecular biology in 2003 from Hong Kong University. He received postdoctoral training in human pluripotent stem cell biology at University of Wisconsin – Madison. He has more than 12 years of experience in pluripotent stem cells and cellular reprogramming. He has established his own laboratory at the Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham (UAB). He is currently an Assistant Professor in stem cell biology and cellular reprogramming at UAB. He has been involved in the improvement of iPSC technology. His method of iPSC reprogramming is widely used, and human iPSC lines he generated are also widely used. He has authored more than 20 scientifi c articles. His lab is deciphering the molecular regulation of pluripotency, and dissecting the molecular mechanisms of cellular reprogramming. The works from his laboratory enhance our understanding of iPSC reprogramming process. His research is supported by Alabama Institute of Medicine, American Heart Association, and NIH.

 

Pluripotent stem cells (PSCs) have differentiation potentials into any type of cells in our bodies and therefore hold great promise for regenerative medicine. The conventional PSCs are embryonic stem cells (ESCs) derived from the inner cell mass (ICM) of a pre-implantation embryo. The clinical application of ESCs is hampered by issues of ethical concern, technical limitation, a limited supply of human embryos, and non-autologous nature. PSCs can be induced from fibroblasts or other somatic cells by ectopic expression of a few transgenes, e.g. OCT4, SOX2, KLF4, and C-MYC. Induced pluripotent stem cells (iPSCs) have removed many problems associated with the use of ESCs. However, iPSC reprogramming still has problems of low efficiency, incomplete reprogramming, epigenetic memory of the starting cells, stochastic nature, immunogenicity, and reprogramming-associated mutagenesis. The molecular mechanisms of iPSC reprogramming remain poorly understood. My lab found that proteins of bromodomain extra-terminal (BET) family play different roles in iPSC reprogramming process. By screening a human cDNA library, we discovered that a short isoform of human BRD3, BRD3R, displayed reprogramming activity while other BET proteins lack reprogramming activity. BRD3R also gains mitotic activities, which may be partially responsible for its gained reprogramming activity. Interestingly, our further detailed study showed that chemical inhibition of BET proteins at low concentration enhanced iPSC reprogramming by three distinct BET inhibitors, while a high concentration of these BET inhibitors impairs iPSC reprogramming. At the same time, our RNA-seq data showed that such mild chemical inhibition of BET proteins erased fibroblast transcriptional program. Our published and unpublished data indicate sophisticated roles of BET protein in cellular reprogramming.

 

CRISPR-directed human gene editing in somatic cells and stem cells is rapidly transitioning from bench to bedside with clinical trials beginning to appear throughout the world. Our laboratory and others have demonstrated that the repair of a point mutation can be facilitated by the combined activity of a single-stranded oligonucleotide and the CRISPR/Cas9 system. Using K562 cells, we found that the presence of the appropriate CRISPR/Cas9 complex leads to an enhancement in the frequency of gene editing. While gene editing activity does in fact increase in a dose-dependent fashion, we fi nd a heterogeneity of modifi ed genetic sequences that are created and maintained even when repair at the sickle cell disease mutation in the human beta-globin gene is successful. Our data suggest that CRISPR complexes leave a genetic footprint at the target site by creating a DNA junction heterogeneity as a byproduct of the reaction. Th is heterogeneity could be called on-site mutagenesis and one of the most interesting is a curious pattern of DNA nucleotides adjacent to the double-stranded break site; footprints of the human delta-globin gene. A targeted population of cells contains one allele of apo appears to be a chimera of the HBB and the hemoglobin delta gene (HBD). Our results suggest the genes with similar sequences (related family members) such as HBB and HBD could be involved in template repair of double-strand DNA breaks independent of exogenously added donor DNA. We further evaluated the relationship among cellular delivery, nuclear uptake in CD34+ progenitor cells, oft en viewed as the benchmark metric of successful gene editing, and single base repair. We took a similar combinatorial approach using single-stranded oligonucleotide and a CRISPR/Cas9 ribonucleoprotein to convert wild-type HBB into the sickle cell genotype. Confocal microscopy data show that the CRISPR/Cas9 ribonucleoprotein tends to accumulate at the outer membrane of the CD34+ cell nucleus when NEON Transfection System is employed, while the ribonucleoproteins do pass into the cell nucleus when nucleofection is used. When the correct delivery system is maximized, we once again a similar genetic pattern of on-site heterogeneity created by the activity of the CRISPR Gene editing complex. Our results indicate that more stringent criteria must be established to facilitate the clinical translation and scientifi c robustness of gene editing for sickle cell disease, most critical in stem cell therapy.

 

Naresh Kumar Rajendran has completed his Ph.D. (Medical Biochemistry) in 2015 from University of Madras, India and currently working as a Postdoctoral Reseach Fellow in Laser Research Centre, Faculty of Health Sciences and University of Johannesburg, South Africa. He has two years of research experience at the University of Tokyo, Japan. He has published more than 15 papers in peer-reviewed journals and has been serving as a reviewer for 5 international journals. He has presented his research outcomes in various international and national conferences. He is engaged in national and international collaborations and co-supervising postgraduate students.

 

Delayed wound healing is considered one of the most common and serious complications of diabetes mellitus. High glucose levels induce oxidative stress and it leads to the central pathogenesis of chronic complications associated with diabetes. Controlling the factors leading to oxidative stress outbursts may emerge as an effective step to treat diabetic chronic wounds. Photobiomodulation (PBM) is a technique based on the use of low-powered light to regulate various pathophysiological cellular effects, including inflammation and chronic wounds. However, the exact mechanism of action behind PMB remains unclear. The present study aimed to determine if PBM at 660nm attenuates oxidative stress by inhibiting the FOXO1 signaling pathway. Four experimental group’s namely normal, wounded, diabetic and diabetic wounded WS1 fibroblast cells were exposed to laser irradiation at a wavelength of 660nm and a fluence of 5J/cm2; non-irritated cells served as experimental controls. Migration of cells was determined by inverted microscopy at 0, 24 and 48h. Cells were harvested at 0, 12, 24 and 48h. Cell viability was determined by the trypan blue exclusion assay and adenosine triphosphate (ATP) luminescence assay. Indirect ELISA was used to determine the levels of HMOX1, SOD, and CAT in cultured cells. Western blotting and immunofluorescence were performed to analyze AKT and FOXO1. RT-PCR was used to determine gene expression of TGF-β, PDGF, and VEGF. Irradiation of cells resulted in a significant decrease in FOXO1 and increase in AKT protein. The treatment also upregulated the expression of TGF-β1, PDGF and VEGF genes. The level of antioxidants such as HMOX1, SOD, and CAT was also increased in irradiated groups. The present study confirmed that PBM at 660nm enhances wound healing through attenuation of oxidative stress by regulating the FOXO1 signaling pathway.

 

Yoo-Hun Suh is now Chaired Professor and President of the Neuroscience Research Institute of Gachon University. He was the founding president of Korea Brain Research Institute. Furthermore, he is a Professor Emeritus of College of Medicine at Seoul National University. He won Korea’s Most Distinguished Scientist Award, the National Government Medals, and many other prizes. He was selected one of 20 outstanding Korean Medical Scientists and one of 21 outstanding Korean Scholars of the 21st Century. He is an Editor and Editorial Board Member for 6 SCIs. He fi first cloned the gene for epinephrine synthesizing enzyme, PNMT and has greatly contributed to the discovery of a new potential gene and factors for the AD, the development of potential stem cell and drugs for AD and PD. He has published more than 200 papers and over 50 books. Presently, he is a Member of Board of Trustee, HFSP, a Member of Korean National Academy of Science and Technology, a Member of Korean National Academy of Medicine and a Member of Korean National Science and Technology Council. In addition, he is a Member of International Scientific Advisory Board of AD & PD and a Member of International Scientific Advisory Board of International Conference of Alzheimer’s Disease and related disorders.

 

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid plaques and neurofibrillary tangles accompanied by cognitive dysfunction. We examined whether intracerebrally or intravenously transplanted human adipose-derived stem cells (hASCs) could have therapeutic or preventive effects in AD/PD mice model. We demonstrated that intracerebral or intravenous injection of hASCs rescued memory deficit and gave benefits of blocking the pathogenesis in the brain of AD Tg mice by reducing the number of plaques and neuropathology. Among stem cells, autologous human adipose-derived stem cells (hASCs) elicit no immune rejection responses, tumorigenesis, or ethical problems. We found that intravenously transplanted hASCs passed through the BBB and migrated into the brain. The e-learning, memory, and pathology in an AD mouse model (Tg2576) mice greatly improved for at least 4 months after intravenous injection of hASC. Th e number of amyloid plaques and Aβ and APP-CTs levels decreased signifi cantly in the brains of hASC-injected Tg mice compared to those of the Tg-sham mice. Intravenously or intracerebrally transplanted hASCs significantly rescued memory defi cit and neuropathology in the brains of Tg mice by upregulating IL-10 and VEGF and be a possible use for the prevention and treatment of AD. We are currently conducting clinical phase I & II study of hASCs for an AD in the USA. Here, we demonstrated that transplantation of neural stem cells into 12-month-old Tg2576 brains markedly improved both cognitive impairment and neuropathological features by reducing β-amyloid, secretion of anti-inflammatory cytokines, endogenous neurogenesis, as well as synapse formation. In contrast, the stem cell transplantation did not cover cognitive dysfunction and β-amyloid neuropathology in Tg2576 mice aged 15 months when the memory loss is manifest. However, the optimal stage of the disease for stem cell transplantation to have a therapeutic effect has yet to be determined. Overall this study underscores that stem cell therapy at an optimal time frame is crucial to obtain maximal therapeutic effects that can restore functional deficits or stop the progression of the AD. The treatment of Parkinson’s disease (PD) using stem cells has long been the focus of many researchers, but the ideal therapeutic strategy has not yet been developed. The hASC were intravenously injected into the tail vein of a PD mouse model induced by 6-hydroxydopamine. The behavioral performances were significantly improved at 3 weeks after the injection of hASC. Additionally, dopaminergic neurons were rescued, the number of structure-modified mitochondria was decreased, and mitochondrial complex I activity was restored in the brains of the hASC-injected PD mouse model. Overall, this study strongly indicated that intravenously transplanted hASC may have therapeutic potential for PD by recovering mitochondrial functions.

 

Matthew Fischer is a Former Government Attorney with a passion for the emerging stem cell industry. Prior to joining the fi rm, he served as a Senior Attorney Advisor in the US Department of Health and Human Services, Office of Medicare Hearings and Appeals. He advised Federal Administrative Law Judges (ALJs) on reimbursement appeals, including Medicare contractor overpayment audits (i.e., RAC, ZPIC, and PSC), statistical sampling extrapolation issues, Medicare Part D exceptions, and secondary payer appeals. His current practice is concentrated in healthcare with a strong focus on stem cell regulatory development and enforcement.

 

There are no off the shelf solutions when it comes to starting a new business or adding a new component to a practice. Between navigating regulations, receiving training, and marketing the service, there's a lot to address in a short time. Trying to do it all yourself? You may be a highly trained clinician, but given healthcare’s ever-changing regulatory environment, it is highly encouraged for providers to seek experienced counsel first. To get started, here is a short summary of what to expect. The first issue is always protection when starting a business or adding a new service. Take the case of an orthopedic physician that wants to add stem cell treatments (e.g. PRP) to his or her practice. The initial inclination is usually to create a new entity separate from the medical practice. What the physician is unaware of is that this may create exposure to state self-referral laws. Typically, under these types of laws, the intent is not a requirement. Therefore, is it important to determine if your state has this type of law and if so, how to structure the new venture before moving forward? The FDA has the authority to regulate stem cell products. Most products are regulated by the FDA as an HCT/P while others are not. Many direct-to-consumer providers are unsure about what this means and need to know what they are able to do and say. Thus, it is imperative to be up to date on the issues and know what boundaries have been set by the FDA. With the rise of stem cell science and treatments, many state boards have warned physicians that they could face potential disciplinary action for failing to meet the prevailing professional standard of care or for performing an experimental procedure without first obtaining full, informed consent. Many physicians would like to have a mid-level practitioner (i.e. NP or PA) assist or provide injections. Once again, state regulations need to be reviewed to determine who can provide treatments and what level of supervision is required.

 

Stanca A Birlea MD has completed her Ph.D. at the age of 34 from the University of Medicine Cluj-Napoca Romania and postdoctoral studies on the Genetics of Vitiligo and Other Autoimmune Diseases in Professor Richard Spritz lab from University of Colorado School of Medicine. She is an Associate Professor in the department of dermatology at the University of Colorado. She has published more than 25 papers in reputed journals and has been serving as an editorial board member of repute Pigment Cell & Melanoma Research Journal. Her projects focus is on melanocyte regeneration in vitiligo, a collaboration between Department of Dermatology–Chair Professor David Norris- and Gates Center for Regenerative Medicine-Director Dr Dennis Roop.

 

Vitiligo is an autoimmune depigmentation disorder characterized by white spots on the skin that cause profound social and psychologic stigma in patients. Vitiligo is caused by CD8+ T cell-mediated destruction of epidermal melanocytes (MCs). Vitiligo repigmentation requires proliferation and migration of MC precursors from the hair follicle (HF) bulge to repopulate the interfollicular epidermis, and the strongest stimulus for this process is Narrow Band UVB (NBUVB). To better understand this process, we developed a research platform that used skin biopsies collected from 6 vitiligo patients treated with NBUVB and 6 untreated vitiligo patients, using rapid fluorescent immunostaining combined with laser capture microdissection to collect RNA from bulge MC precursors and mature MCs from the epidermis of the regenerated vitiligo skin. The total RNA captured from MCs was subjected to whole transcriptome RNA sequencing, followed by gene expression analysis. We found upregulation of TNC, GJB6 and THBS1 transcripts in the bulge MC precursors of NBUVB-treated vitiligo skin as compared with epidermal MCs of regenerated NBUVB-treated vitiligo skin, and of β-catenin as being the top upstream transcription regulator of this process. We also identified that GLI1, a candidate stem cell-associated gene, was significantly modulated by NBUVB in the bulge MCs. The above pathway and signals are potentially key-players in the activation of bulge MC precursors during vitiligo repigmentation.

 

Renee Cottle is an Assistant Professor of Bioengineering at Clemson University. She earned her Ph.D. in Biomedical Engineering from the Georgia Institute of Technology and Emory University in 2015. She completed a T32 Postdoctoral Fellowship in the Cardiovascular Research at MUSC in 2016 and started her faculty position at Clemson in August 2016. Her expertise is in gene editing, gene therapy, and non-viral delivery strategies. Her research group is focused on cell-based gene therapies for inherited metabolic diseases of the liver, including familial hypercholesterolemia. Her research addresses technical barriers for advancing novel gene therapies for genetic disorders.

CRISPR and associated protein (Cas9) system have proven to be the most promising gene editing tool available for therapeutic applications due to its facile design and robust targeting activity in mammalian cells. When coupled with a donor template, CRISPR-Cas9 nucleases trigger the homologous directed repair pathway to precisely incorporate new gene sequences into the genome, which can be leveraged for the correction of genetic diseases in the patient’s cells. Despite the advantages of this approach, there are several barriers to its application in humans. One major hurdle is delivering a sufficient amount of CRISPRCas9 complexes and donor templates into target cell types. In previous research, we investigate microinjection, traditionally applied for in vitro fertilization, for direct, controlled delivery of nucleases and donor template into human hematopoietic cells as a novel therapeutic strategy for sickle cell disease. We characterized a microinjection system, investigated the effects of microinjection on cell functionality, and demonstrated proof-of-principle of gene editing in human hematopoietic K562 cells microinjected with TALENs and CRISPR-Cas9 along with donor template. We found that injection negligibly affects the cell proliferation potential, provides high cell viability, and can be used to control the exposure of nucleases in injected cells. A major drawback of microinjection is the low throughput. In contrast, nucleofection is amenable for therapeutic applications. Currently, we are optimizing nucleofection of CRISPR-Cas9 nucleases and donor templates into hepatocytes as a therapeutic strategy for familial hypercholesterolemia. We show >90% on-target CRISPR-Cas9 activity in primary hepatocytes, spurring further development into a novel cell-based gene therapy for metabolic liver diseases.

 

Alessandra Giuliani is Permanent Researcher and Aggregate Professor in Physics Applied to Cultural Heritage, Environment, Biology and Medicine at the Polytechnic University of Marche, Clinical Science Department. Within the Physics Group, she coordinates the research in Physics Applied to Biomaterials, Tissue Engineering and Regenerative Medicine. The purpose of her research is to study, using advanced physical techniques (such as microdiffraction, computed microtomography, holotomography), based on synchrotron radiation, all the structural changes of various biological tissues (mice bone under conditions of micro and macro gravity and / or of transgenic type, dental implants of various origins, tendons treated with collagen membranes, infarcted rat hearts treated with cardiac progenitor cells, mice dystrophic muscle injected with human AC133+ cells). A particular attention is paid to the vascularization issue of the regenerated tissue using an innovative imaging technique - the computed holotomography. She is author of around 54 peer-reviewed journal papers, chapters on 7 books internationally distributed and numerous works and abstracts related to National and International Congress presentations. Her researches in Physics applied to Tissue Engineering and Regenerative Medicine have been the subject of >45 presentations to Congresses and Schools, the most of them as invited speaker.

Devyani Joshi obtained her bachelor’s degree from the University of Mumbai, India. She is a graduate student in Mercer University College of Pharmacy, Atlanta, GA. As a graduate student, her area of research is particulate vaccines against infectious diseases and novel treatments for neurodegenerative diseases.

 

Parkinson’s disease affects millions of people the world over and the incidence and severity of this disease continue to increase. This disease is characterized by decreased levels of catecholamines including dopamine in the brain. PC-12 cell, a pheochromocytoma cell line from Rattus norvegicus, is a cell line that is capable of producing catecholamines. These cells can synthesize, store and be stimulated to release dopamine. Additionally, a PC-12 cell has the capacity to undergo neuronal differentiation in response to a nerve growth factor and can be a useful and important feature of PC-12 cells for Parkinson’s Disease (PD) studies. The neuronally differentiated cells directly model sympathetic neurons which are one of the neuron types affected by Parkinson’s disease. PC-12 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Horse Serum, 5% Fetal Bovine Serum and 1% Penstrip at 37°Ð¡ and 5% CO2. For fabrication of the microcapsules, 1% w/v solution of sodium alginate was prepared, and PC-12 cells were added to it and allowed to stir for fifteen minutes. After stirring, the alginate–cells suspension was sprayed through a 1.40mm nozzle using a Buchi spray dryer B-190 into the calcium chloride solution (1.5% w/v). The microcapsules in calcium chloride solution were allowed to stir for fifteen minutes and washed with phosphate buffered saline (PBS) and centrifuged twice at ×285g (1200 RPM) to remove excess of calcium ions. The suspension of microcapsules was then transferred to the chitosan glutamate solution (0.5% w/v), stirred further for fifteen minutes and washed with PBS, centrifuged twice at ×285g (1200 RPM) to remove the excess chitosan glutamate. The suspension of the microcapsules was then transferred to the media and kept in the incubator at 37°Ð¡ and 5% CO2. The parameters affecting the size of the microcapsule were air flow rate, pump speed controlling the flow rate of the alginate cell suspension, the distance between the nozzle and calcium chloride solution, nozzle diameter, and spray rate. In this study, we emphasized the air flow rate through the nozzle to alter the size of the microcapsules and kept all the other parameters constant. We found that maintaining the higher flow rate leads to the reduction in the size of the microcapsules due to high shear at the tip of the nozzle. The size of the microcapsules was in the range of 250–350μm. The alginate microcapsules were spherical in shape and no deformities were observed. The short-term stability studies showed that the cells were viable in the capsule for up to 7 days. The microcapsules containing these cells can be delivered into the brain through the intra-cranial injection for the treatment of the Parkinson’s Disease (PD). These encouraging primary results will lead the way for further research in the area and testing in murine models of Parkinson’s disease.

 

Alessandra Giuliani is Permanent Researcher and Aggregate Professor in Physics Applied to Cultural Heritage, Environment, Biology and Medicine at the Polytechnic University of Marche, Clinical Science Department. Within the Physics Group, she coordinates the research in Physics Applied to Biomaterials, Tissue Engineering and Regenerative Medicine. The purpose of her research is to study, using advanced physical techniques (such as microdiffraction, computed microtomography, holotomography), based on synchrotron radiation, all the structural changes of various biological tissues (mice bone under conditions of micro and macro gravity and / or of transgenic type, dental implants of various origins, tendons treated with collagen membranes, infarcted rat hearts treated with cardiac progenitor cells, mice dystrophic muscle injected with human AC133+ cells). A particular attention is paid to the vascularization issue of the regenerated tissue using an innovative imaging technique - the computed holotomography. She is author of around 54 peer-reviewed journal papers, chapters on 7 books internationally distributed and numerous works and abstracts related to National and International Congress presentations. Her researches in Physics applied to Tissue Engineering and Regenerative Medicine have been the subject of >45 presentations to Congresses and Schools, the most of them as invited speaker.

 

The evaluation of engineered tissues is usually performed by light microscopy on one or more histological sections. This conventional analysis provides only bi-dimensional (2D) information with the consequent risk that the selected sections do not properly represent the entire biopsies. In recent years there has been an increasing interest in a novel approach to evaluate different engineered tissues by means of synchrotron micro-tomography (SCT). Using SCT, tissue regeneration subsequent to grafting hosting sites with different types of biomaterials (with or without stem cells seeding) was recently explored. SCT was shown to be fundamental to explore the dynamic and spatial distribution of regenerative phenomena, also in complex anatomic structures. Traditionally, absorption imaging with SCT is conducted with almost no distance between sample and detector. Homogeneous materials with a low attenuation coefficient (like collagen, unmineralized extracellular matrix, vessels, nerves, etc.) or heterogeneous materials with a narrow range of attenuation coefficients (like the case of heterologous bone scaffolds or graded mineralized bone) produce insufficient contrast for absorption imaging. For such materials, the imaging quality can be enhanced through the use of phase contrast tomography (PCT), oft en achieved with an increased distance between sample and detector (propagation-based imaging). In the present lecture, the most recent breakthroughs in regenerative medicine will be shown, demonstrating the unique capabilities of the SCT in offering not only an advanced characterization of different biomaterials (to understand the mechanism of their biological behavior as tissue substitute) but also to investigate the growth kinetics of regenerated tissues in different environments.

 

Sarah K Steinbach completed her Ph.D. in 2008 from the University of Saskatchewan. Before that, she completed her undergraduate degree with a specialist in Human Biology and Major in Microbiology at the University of Toronto. She is currently working as a scientist for a top 5 pharmaceutical company. She has published 9 articles in high impact peer-reviewed journals and has presented over 39 abstracts in national and international conferences.

 

Objective: Few methods enable molecular and cellular studies of vascular aging or type 2 diabetes (T2D). Here, we report a new approach to studying human vascular smooth muscle cell (VSMC) pathophysiology by examining VSMCs differentiated from progenitors found in the skin. Approach and results: Skin-derived precursors (SKPs) were cultured from biopsies (N=164, 1 cm2) taken from the edges of surgical incisions of older adults (N=158; males 72%; mean age 62.7±13 years) undergoing cardiothoracic surgery, and differentiated into VSMCs at high efficiency (>80% yield). The number of SKPs isolated from subjects with T2D was 50% lower than those without T2D (cells/g:0.18±0.03, N=58 versus 0.40±0.05, N=100, P<0.05). Importantly, SKP-derived VSMCs from subjects with T2D had higher Fluo-5F-determined baseline cytosolic Ca2+ concentrations (AU: 1,968±160, N=7 versus 1,386±170, N=13, P<0.05), and a trend toward greater Ca2+ cycling responses to norepinephrine (NE) (AUC: 177,207±24,669, N=7 versus 101,537±15,881, N=20, P<0.08) despite a reduced frequency of Ca2+ cycling (events s−1 cell−1: 0.011±0.004, N=8 versus 0.021±0.003, N=19, P<0.05) than those without T2D. SKP-derived VSMCs from subjects with T2D also manifest enhanced sensitivity to phenylephrine (PE) in an impedance-based assay (EC50 nM: 72.3±63.6, N=5 versus 3,684±3,122, N=9, P<0.05), and impaired wound closure in vitro (% closure: 21.9±3.6, N=4 versus 67.0±10.3, N=4, P<0.05). Compared with aortic- and saphenous vein-derived primary VSMCs, SKP-derived VSMCs are functionally distinct, but mirror defects of T2D also exhibited by primary VSMCs. Conclusion: Skin biopsies from older adults yield sufficient SKPs to differentiate VSMCs, which reveal abnormal phenotypes of T2D that survive differentiation and persist even after long-term normoglycemic culture.

 

Maria Giovanna Francipane has completed her PhD from the University of Palermo, Palermo, Italy, and postdoctoral studies from the University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. She is a Research Assistant Professor in the Department of Pathology, University of Pittsburgh School of Medicine, and an affiliated faculty member of the McGowan Institute for Regenerative Medicine in Pittsburgh. She also holds a position as Principal Investigator at Fondazione Ri.MED in Palermo. She has broad expertise in the fields of cancer and regenerative medicine, and extensive technical skills, as evidenced by more than a dozen first author publications in reputed journals.

 

The need for alternative therapies to replace dialysis and transplantation for renal failure is imperative. However, despite some promising research developments, rebuilding a kidney is still a long way from clinical application. Vascularization is one of the greatest challenges that tissue engineering faces in order to achieve functional kidney substitutes. In our lab, we have pioneered an in vivo vascularized tissue-engineering model, in which target cells/tissues are implanted into a mouse lymph node (LN). Upon transplantation into the LN, fragments of mouse and human embryonic kidneys acquire markers of mature renal structures, have excretory, homeostatic, and endocrine functions. The LN also acts as an innovative bioreactor to organize kidney organoids into vascularized kidney elements. Here, we identified the lymphotoxin-beta receptor (LTβR) as a new pathway that the lymphoid stromal microenvironment utilizes to enhance angiogenesis of the transplanted tissues. These findings will guide our future translational efforts to engineer renal functions.

 

Nadja Zeltner completed her Master's degree in Zoology at the University of Zurich, in her native Switzerland. She then earned her Ph.D. in gene therapy and virology at the Icahn School of Medicine at Mount Sinai, followed by a postdoc in stem cell biology and neurodevelopment at the Sloan Kettering Institute in New York. She established her research group as a junior faculty member at the Center for Molecular Medicine at the Universtiy of Georgia in the spring of 2018, where she is also affiliated with the Cellular Biology Department and the Department of Biochemistry and Molecular Biology. She has published several high-impact papers describing the in vitro modeling of various diseases as well as pioneered the establishment of in vitro differentiation protocols for a variety of neural cell types.

 

Functional and molecular aspects of the human genetic disease can be recapitulated in vitro using patient-specific pluripotent stem cells (PSCs). Familial Dysautonomia (FD) is a debilitating developmental and degenerative disorder that primarily affects derivatives of the neural crest (NC), such as the peripheral nervous system (PNS). For unknown reasons, FD patients present with mild or severe disease despite carrying the identical, homozygous point mutation in IKBKAP. We present in vitro phenotypes at various stages of development that capture severe and mild FD in human PSC-derived cellular lineages. Patient-specific cells only from severe but not mild FD display an impaired capacity of developing into NC derivatives, such as autonomic and sensory neurons, thus they have neurodevelopmental defects. Interestingly, however, both severe and mild FD cells show defects in peripheral neuron survival, indicating neurodegeneration as the primary culprit in mild FD. Importantly, we found that neuronal degeneration in mild FD can be halted by treatment with candidate therapeutic compounds. Genetic rescue of the FD mutation in severe FD iPSCs reversed NC, but not sensory neuron lineage phenotypes, implicating that the known FD mutation does not account for all symptoms. Employing whole-exome sequencing, we identified candidate mutations that were only found in severe but not mild FD patients, providing evidence that FD may constitute two genetic sub-diseases. Our study demonstrates that human iPSC-based disease modeling is sensitive in recapitulating disease severity. This paves the road for applications in personalized medicine and raises the prospect that individual patient’s disease could be studied in vitro.

 

High-volume clinical biobanks face challenges balancing time-intensive chain of custody (CoC) measures with timing, space, and personal demands. We at MD Anderson have developed a fully integrated clinical trial, lab and sample management system. The system, Prometheus, accelerates the pace of translational research by eliminating error-prone data entry and ensuring CoC. In addition to the software, we integrated a liquid handling robot to automate sample preparation (Vacutainer to cryo-storage) allowing for the dramatic decrease in the effort required to gather these samples for analysis. By striving to create a single source of truth, namely the one abstracted from the clinical record in the clinical trial data record, we clarify the exact nature of the sample and prevent artificial reclassification. By automating the preparation steps, we remove human variance in processes and provide a rich source of metadata to help explain why we may get unanticipated results in downstream assays. This was achieved by the development of trial management platform that handles information from trial design, patient acquisition, sample acquisition, processing, storage, and analysis. It utilizes best in class automation tools and storage products to ensure data and sample integrity. By redesigning processes, improving software and implementing automation, we have enabled a small team of three staff members to generate 140,000 samples per year, while maintaining high quality in both samples and work-life balance. In conclusion, in the rapidly expanding demand for samples to perform translational assays to understand the fundamental mechanics of disease, having an automated sample acquisition pipeline is required to meet the demand of modern research.

 

Nashwa AM Mostafa has completed her Ph.D. at the age of 35 years from Faculty of Medicine, Assiut University, Egypt and she had a postdoctoral fellowship from Bern University, Switzerland. She has published 15 papers in reputed journals and has been serving as an Editorial Board Member of repute. She is paying a major concern to basic researches that may lead to possible/defi nite improvement in health services and decreasing disabilities, morbidity, and mortality. Also, interested in stem cells and regenerative medicine.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammatory synovitis and production of auto antibodies and several pro-inflammatory cytokines, which lead to joint destruction. Mesenchymal stem cells (MSCs), exert profound immunomodulation which encourages a possible use of these cells in the treatment of autoimmune diseases. Berberine is an isoquinoline alkaloid with anti-inflammatory activities. This work aimed to evaluate the efficacy of stem cells alone or combined with berberine versus methotrexate in rheumatoid arthritis treatment. Forty-two rats were used, divided into six groups, with 7 rats each. GI: control group. GII: RA group. GIII: RA was given mesenchymal stem cell derived from bone marrow by single intravenous injection. GIV: RA treated with berberine orally. GV: RA treated with mesenchymal stem cells combined with berberine. GVI: RA treated with methotrexate intraperitoneal injection. Rheumatoid arthritis will be induced in rats by intravenous injection of Complete Freund’s adjuvant (CFA) at day 0 and 21 as immunization boost dose. All the treatment was given after 21 days of induction of RA for one month. At the end of the experiment blood samples were collected from all groups then animals sacrificed. Whole knee joints were excised and the specimens were processed for histological, immunohistochemical examination. Treatment with stem cells significantly reduced clinical symptoms and joint pathology. Interestingly, stem cells decreased periarticular and systemic bone loss in RA by maintaining trabecular bone structure more than the other groups.

 

Background: Gastric ulcer is one of the most irritating health problems. The commonly used drugs for peptic ulcers have the danger of drug interaction, adverse effect and increased incidence of relapses during ulcer therapy. Adipose tissue-derived stem cells (ADSCs) are considered to be ideal for application in regenerative therapies. Objective: Study the role and mechanism of action of adipose-derived-mesenchymal stem cells in accelerating healing of indomethacin-induced gastric ulcer in rats. Methods: The ulcer was induced by 200mgl/kg indomethacin, and then after 5 hours, adipose-derived mesenchymal stem cells (from lipectomy operation) are processed and injected intraperitoneally in rats with an ulcer. Flow cytometric analysis has been done on isolated cells. Hematoxylin & Eosin stain has been done to detect signs of healing at 1,2,3,4 days. Also, the expression of VEGF & PCNA immunohistochemical reactions has been carried out to confirm the results. Results: The results show that healing is accelerated in rats injected with stem cells at 2, 3, 4 days compared to +ve control (auto healing) model; also an expression of VEGF and PCNA is increased in stem cells injected specimens. Detection of human leukocytic antigen (HLA) after 4 days of injection confirmed migration of stem cells to the site of the ulcer. Conclusion: Adipose-derived-mesenchymal stem cells has a role in accelerating healing of gastric ulcer in rats through stimulating angiogenesis at ulcer base and proliferation of cells at ulcer margin.

 

Satish Kumar has expertise in Assisted Reproduction Technology. He is Assistant Professor at Ambala College of Engineering and Applied Research Ambala, Haryana, India. He is actively engaged in research and teaching in Department of Biotechnology. He has guided eleven M-Tech students in different research areas of Biotechnology and has been member and coordinator of various committees at college and university level. He has MSc degree in Biotechnology and Molecular Biology, Haryana Agricultural University, Hisar and PhD in Animal Biotechnology from Chaudhary Devi Lal University, Sirsa, and Haryana, India. He has authored one book and three book chapters and research articles in Journal of International and National Repute. He also serves as a member of the scientifi c advisory board of International Journal of Animal Biotechnology, India. He is a popular speaker who delivered lectures on the role of Biotechnology in human welfare and worked with scientists of International repute from top institutions across India. He has delivered many lectures in different institutions in India. He has also delivered a lecture via webinar on the topic “Advances in Biocatalysis and its impact on the early and late development of small molecules” to 12 PhD scientists and over 20 Research Associates to Merck Research Laboratories USA.

 

Cryopreservation is an important tool for conservation of biological materials. This study reports effects of vitrification on the morphology of ovine immature cumulus oocytes complexes, and mRNA content to observe the expression of key apoptotic genes, BAD, BAK, BAX, BID, BOK, BCL2A1, BCL2, MCL1, P53, and GAPDH on immature oocytes. For morphological evaluation using Zoom stereo-microscope, 853 oocytes were divided into 3 groups for vitrification using 40% Glycerol or 40% DMSO or 20% Glycerol + 20% DMSO. After thawing, a total of 819 oocytes were recovered, resulting in a loss of 34 oocytes during handling. The morphological examination of the oocytes revealed that the percentage of oocytes recovered in a morphologically normal state was minimum 84.2±2.3% for glycerol 40% and maximum 94.09±1.1% for 20% Glycerol + 20% DMSO. The proportion of oocytes recovered in a morphologically normal state was significantly lower (P<0.01) for 40% Glycerol (84.2±2.3%) compared to that for these groups. For scanning electron, microscopic studies 451 oocytes were used in this experiment for vitrification using a combination of 20% Glycerol + 20% DMSO and percentage of oocytes recovered in a morphologically normal state was 95.7±2.07 recorded. The real-time PCR results for the comparative expression of the apoptotic gene using mRNA abundance showed the down-regulation BAD, BCL2A1 and MCL1. The expression of BCL2, BAX and BOK are similar in both the groups. However, BID and BAK were up-regulated. In addition, P53 was also downregulated proves vitrification safe for the preservation of immature oocytes in ovine.

 

Background: A case report of 20-year-old female diagnosed through EMG and nerve conduction study with Duchenne muscular dystrophy. The patient was presented with severe pain in lower limbs that made mobility almost impossible, cardiomyopathy and muscle weakness. Rehabilitation and medical treatment was combined since the age of 17 and was gaining some results till the age of 19. After this age, all treatments failed and the patient becomes almost immobile with attacks of fainting. Objective: The aim of this study is to extensively investigate the effect of the platelets rich plasma injections in a try to control the muscle fibrosis to its test effect in all patient's symptoms. Methods: A high resolution 10 MHz musculoskeletal ultrasonography was done to all major lower limb muscles showed profound muscle fi brosis especially in Tibialis anterior muscle and Vastus medialis obliques, most probably they are the main reason for gait deviation and severe pain. Bilateral Ultrasound guided PRP intramuscular injection was done to these muscles under sterile technique. Cardiac enzymes CBC, ESR, CRP, visual analog scale for pain, manual muscle testing, ultrasound scan, patient's vitals and pictures of the gait. All of this was collected pre and after 15 days of the procedure. Follow up was done for 21 days. Results: Mobility was independently regained with very mild deviation, 95% reduction in pain 60% increase in muscle power with evidence of less fibrosis in US scan and moderate improvement in cardiac markers happened with no single attack of fainting happened along the period of follow up. All patient's vitals were within normal limits.

 

Heidi Ulrichs is a senior undergraduate student at the University of Georgia with a major in Biology with a Neuroscience emphasis and minor in Spanish. She has been working in the Zeltner Lab in the Center for Molecular Medicine since June 2018.

 

Our goal is to develop a protocol to differentiate sympathoadrenal progenitor (SAP) cells from human pluripotent stem cells (hPSCs). The hPSCs’ ability to self-renew and differentiate into any cell type of the body within the embryo and in vitro makes them a powerful tool to study early embryonic development, model diseases affecting specified cell types, conducting drug screening, and drug testing. During development, hPSCs diff erentiate into ectoderm, which then gives rise to neural crest (NC) cells that further diff erentiate into SAPs. SAPs can then give rise to sympathetic neurons, chromaffin cells, and small intensely fluorescent (SIF) cells. Generating these cells from patients PSCs is relevant to study diseases that affect them including neuroblastoma, adrenal medulla malignancies, and different genetic disorders affecting sympathetic neurons, like Familial Dysautonomia (FD). In our approach, we initiate hPSC diff erentiation in a monolayer. From there, SOX10-expressing NC cells are induced by TGFβ inhibition and activation of WNT and BMP4. Then they are differentiated towards SAP using WNT, BMP4, neuregulin, SDF1, and retinoic acid. The SAP is compared to cranial- and vagal-NC as controls. We identified robust SOX10 and HNK1 expression followed by expression of medium to lower HOX genes and SAP-specific gene markers. In conclusion, we developed a protocol to generate SAPs in vitro from hPSCs. Overall, the SAP protocol will allow researchers to investigate diseases affecting those cell types.

 

Divya Desai is a Ph.D. Research Scholar, 2nd year, from SDSOS, NMIMS University from Mumbai, India. She is currently working on stem cell biology and epigenetics.

 

Pluripotent stem cells have the capacity to self-renew and differentiate into multiple cell lineages- ectoderm, mesoderm, and endoderm. Progression from undifferentiated to differentiated state needs stable restricted chromatin compaction which enables the cell to retain memory without altering the DNA sequence. Epigenetics modulators like DNMTs, NuRFs and histone modifiers are critical for establishing and maintaining heritable genetic changes and maintain cellular memory during development. Polycomb group (PcGs) proteins are one of the histone modifiers which regulate transcription by forming large protein complexes, notable amongst these complexes are Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2) which add ubiquitin and methyl molecules respectively on specific histone proteins. PRC1 has two core components-RING1B and BMI1, where RING1B is the catalytic unit which performs monoubiquitylation of lysine 119 on Histone H2A. Earlier studies have highlighted the function of PcG proteins using mouse models of mammalian development and these were found to play an essential role in determining the fate of neuronal development. We used human pluripotent stem cells as an in vitro model to study the role of these PcG proteins during early human neural development. The stem cells were differentiated by forming embryoid bodies (EBs) via the hanging drop technique and later they were plated on gelatin-coated dishes; these EBs were observed to display cells of varied morphologies. The RING1B and BMI1 expression were checked over a span of two weeks, it was seen that the RING1B and BMI1 expression was upregulated in differentiated cells compared to undifferentiated cells. Subsequently, these PcG proteins were inhibited to determine the fate of diff erentiation and for this, we used two commercially available inhibitors. PRT-4165 was used to inhibit RING1B expression and PTC-209 was used to inhibit BMI1 expression, acute treatment of inhibitors was given to EBs for 24 hours. It was observed that with inhibition of RING1B and BMI1, EBs showed a higher expression of neuroectodermal markers such as NESTIN, PAX6, and SOX1 compared to its control. We also checked for expression of endoderm and mesoderm specific markers such as SOX17, BRACHYURY, TBX5, and HAND1, but there was no significant change in expression of endoderm and mesoderm markers compared to the control. Thus, our results indicate that PcG proteins are crucial for neuronal differentiation of human pluripotent stem cells and the inhibition of RING1B and BMI1 leads to the generation of more neural lineage cells. Thus, our results show the importance of PRC1 components in neurogenesis and understanding the functioning of RING1B and BMI1 proteins may be important in the context of neurodevelopmental diseases.

 

Nashwa AM Mostafa has completed her Ph.D. at the age of 35 years from Faculty of Medicine, Assiut University, Egypt and she had Postdoctoral Fellowship from Bern University, Switzerland. She has published 15 papers in reputed journals and has been serving as an Editorial Board Member of repute. She is paying a major concern to basic researches that may lead to possible/defi nite improvement in health services and decreasing disabilities, morbidity, and mortality. Also, interested in stem cells and regenerative medicine.

 

Epilepsy is a chronic disorder characterized by recurrent seizures. Phenytoin is the most common and effective antiepileptic drug prescribed for a prolonged period to achieve seizure control but is known to induce cognitive dysfunction, cerebellar dysfunction, and degeneration. Young green barley leaves are one of the richest sources of antioxidants and contain the flavones C-glycoside, saponarin, and lutonarin. This work aimed to investigate the protective effect of barley compared to phenytoin on the cerebellum of mice with electrically induced seizures. Twenty-five adult male mice about 25-30g were used. The animals were divided into five groups: GI: control group. GII: treated with phenytoin for three months. GIII: treated with barley + phenytoin for three months. GIV: treated with barley for 1 month. GV: treated with barley for 3 months. Phenytoin was administered intraperitoneally (i.p.) in a volume of 1ml/100 g of the animal. Barley was given orally (200gm barley grains/kg/ day). Electroconvulsions was induced in all animals by means of constant current stimulation delivered to saline-wetted eyes via corneal electrodes from a Rodent Shocker Generator. All groups were sacrificed after three months by decapitation except for group IV was sacrificed after one month. Th e cerebellum from all animals was dissected out immediately and processed for light and electron microscopic examination. In group V, histological examination revealed the normalized appearance of the cerebellum which was more observed than the other groups at the cellular level. Conclusion: Barley had a promising cytoprotective effect on the cerebellum of mice with epilepsy.

 

Nashwa AM Mostafa has completed her Ph.D. at the age of 35 years from Faculty of Medicine, Assiut University, Egypt and she had a postdoctoral fellowship from Bern University, Switzerland. She has published 15 papers in reputed journals and has been serving as an Editorial Board Member of repute. She is paying a major concern to basic researches that may lead to possible/defi nite improvement in health services and decreasing disabilities, morbidity, and mortality. Also, interested in stem cells and regenerative medicine.

 

Konan Kouassi Martin a Ph.D. student in Functional and Molecular Biology at the Félix Houphouët-Boigny University in Côte d'Ivoire. As part of this research, he joined the Cell Biology Unit of the Institute Pasteur Côte d'Ivoire. His research work focuses on the impact of environmental pollutants on male fertility. He also worked as a co-author of several scientifi c publications.

 

Introduction: Many authors have reported adverse effects of environmental pollutants on sexual function, such as tobacco pesticides and heavy metals. Exposure of heavy metals has been associated with adverse effects on the development of gonads. In animals, exposure to lead could damage. Several experimental studies have reported impairment of the spermatogenesis but the mechanisms implied in the pathogenesis are not yet completely understood. Therefore, the present study was undertaken in albino rats to investigate the effects of lead on spermatogenesis on the one hand and testicular and serum gonadotropins and testosterone levels on the other.

Methods: For this study, ten male pubescent rats were randomly divided into two groups (n=5 in each group). The control group received distilled water and the experimental groups received the lead acetate solution (0.3%) while 90 consecutive days. After 90 days, the rats were euthanized. The blood and the testes were sampled for carrying out of the different tests. Results: The results indicate hypertrophy of the testes in the exposed rats. In addition, we have observed a significant reduction in sex hormones and a highly disturbed spermatogenic process.

Conclusion: The present study demonstrates that lead accumulation in the blood affects male fertility by disrupting the biosynthesis of gonadotropins and testosterone as well as the process of spermatogenesis.