24 Hours of Stem Cells

Keynote presentations

Jun-An Chen

Assistant Research Fellow
Institute of Molecular Biology
Academia Sinica

Title:
Utilization of stem cells to crack motor neuron development and degeneration

Abstract:
Regulatory non coding RNAs, including microRNAs (miRNAs) and long non coding RNAs (lncRNAs), have shown to be essential for animal development and viability, yet dissecting the relevance of individual miRNA or lncRNA has been challenging for a given cell context. In addition, the role of ncRNAs for neurodegeneration is still obscure. To identify ncRNAs participated during motor neuron development and degeneration, we used human embryonic stem cell and motor neuron disease iPSCs as paradigms and robustly harnessed them into different motor neuron subtypes to perform strand specific RNA-seq and small RNA-seq simultaneously. We identified several novel MN signature miRNAs and lncRNAs, and systematically analyzed their functions by generating knockout mice. I will present the current progress of the characterization of these MN-ncRNAs by gain-of-function and loss-of-function studies. Collectively, these results will provide critical information for ncRNAs function and will fill in the information on how ncRNAs mediate MN development in vivo.

Biography:
The focus of research in my laboratory is to elucidate how neurons establish individual identity in the developing nervous system and why only specific neuron subtypes are vulnerable in the neurodegenerative diseases. We tackle these questions via studying non-coding RNAs and their roles during motor neuron generation and degeneration. My lab employs motor neurons generated from mouse and human embryonic (ES) and induced pluripotent (iPS) stem cells, as well as mouse animal models to investigate motor neuron development and disease. We have developed and generated a series of stem cells and animal models to study functions of microRNA and lncRNA by “gain-of-function” and “loss-od-function” approaches. Besides elucidating the basic molecular mechanisms underlying specification of neuronal diversity during CNS development, I apply the stem cell system to study motor neuron diseases. In particular, I am engaged in the establishment and study of patient specific iPS cell based models of spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS). iPS cell derived motor neurons are used in my lab to perform deep sequencing from healthy and ALS motor neurons and to functionally characterize non-coding RNA pathology in motor neuron. I am also a core member of Neuroscience Program and RNA program in Academia Sinica, which provides strong and solid consortium to give advice and exchange the ideas for our projects. In summary, I have multidisciplinary approaches, from in vitro stem cells to in vivo mouse models, to study motor neuron development and degeneration.

Matt Dallas

Senior R&D Manager
Thermo Fisher Scientific

Chris Scanlon

Market Development Manager
Thermo Fisher Scientific

Title:
Gibco Sera - Driving quality and innovation in a dynamic market

Abstract:
In today’s volatile Fetal Bovine Sera market, it is critical for researchers to understand the complex nature of the market especially with demand escalating every year. The demand for sera products has no direct impact on the supply of upstream raw materials, but it does influence cost. Supply is finite because FBS is a byproduct of harvesting cattle for the meatpacking industry. Over the last few years, the costs of raw materials needed to produce FBS have increased significantly due to limited supply in most geographical regions. As a result, FBS suppliers were forced to increase pricing. The FBS market is very dynamic, and changes can happen quickly. Learn how Thermo Fisher Scientific the leading globe supplier of FBS is committed to investing in this the Sera business to ensure we can continue to provide the high quality and innovative products, with the service that our customers have come to expect.

Biography:
Matt Dallas is currently a Senior Manager in Cell Biology Research and Development at Thermo Fisher Scientific. He obtained his Ph.D. in Chemical and Biomolecular Engineering from the Whiting School of Engineering at Johns Hopkins University, training that included a fellowship within the Johns Hopkins Institute for NanoBioTechnology, a multi-disciplinary institution with efforts spanning basic biological science, clinical science, and public health. Matt joined the Gibco R&D team in 2012 and has led development programs spanning oncology, primary human tissue culture, and stem cell biology. His current team is responsible for new product and process development in basal cell culture media, fetal bovine and other animal sera, and recombinant proteins.

Chris Scanlon is a Global Marketing Development Manager in Cell Biology Research Sera at Thermo Fisher Scientific. He obtained his Bachelor of Science Business Administration majoring in Marketing from West Virginia University. Also, in 2013 he received an Associate of Applied Science in Nursing from Trocaire College. Chris joined the company back in 2006 as a Senior Marketing Program Manager and is currently based at the Grand Island, NY location which is home to the Gibco manufacturing site. He is responsible for all Gibco Sera marketing and programs that communicate the benefits of our Gibco cell culture products to researchers around the world.

Helmuth Gehart

Postdoctoral Researcher
Hubrecht Institute for Developmental Biology and Stem Cell Research

Title:
Organoid technology—from adult stem cells to miniature organs

Abstract:
Adult stem cell research is one of the most dynamic and fast growing fields of our time. Investigating how organs maintain and repair themselves has led to the discovery of cell populations throughout the body that hold immense regenerative capacity. For a long time, research on adult stem cells has been held back by the inability to grow and study these cells in vitro. This limitation has finally been overcome with the development of organoid technology, a system that enables us to grow single adult stem cells into self-organizing miniature organs. These organoids perform many functions of their parent tissue and are excellent tools to study organ regeneration, organ function and disease development. This presentation provides an overview of the many contributions the team around Hans Clevers has made to the development of organoid culture and delves into applications of the technology in disease modeling, pharmacological screening, regenerative medicine and cancer research.

Biography:
Helmuth Gehart received his PhD from ETH Zurich (Zurich, Switzerland) in 2013. During his PhD training with Prof. Romeo Ricci, Helmuth studied the mechanisms behind type II diabetes and the complex cellular checkpoints that maintain balance to prevent disease. Thereafter, Helmuth aimed to combine his knowledge of signaling and metabolism with the fascinating field of stem cell biology. Thus, he joined the group of Prof. Hans Clevers at the Hubrecht Institute (Utrecht, Netherlands). There he investigates adult tissue stem cells of the liver and their use in clinical research and regenerative medicine.

Alexander Meissner

Director
Max Planck Institute for Molecular Genetics

Title:
Mammalian DNA methylation landscapes and human cancers

Abstract:
Concerted efforts over past decades have established a thorough understanding of the canonical somatic DNA methylation landscape as well as its systematic misregulation across many cancers. However, the underlying mechanisms that may direct this genome-scale transformation remains elusive, with no clear model for its acquisition or understanding of its potential developmental utility. I will first summarize work from the past decade that provided key insights into our general understanding of DNA methylation and then discuss recent data that provide evidence for a developmental origin of the shared, erratic cancer methylome.

Biography:
Alexander Meissner studied Medical Biotechnology at the Technical University Berlin before starting his PhD studies with Rudolf Jaenisch at the Whitehead Institute/MIT in 2002. He completed his PhD in 2006 and spent the next year and a half working with Rudolf Jaenisch and Eric Lander before starting his own lab as an assistant professor in the Department of Stem Cell and Regenerative Biology at Harvard University and as an associate member of the Broad Institute in 2008. He was promoted to associate professor in 2012 and full professor with tenure in 2015. In 2016 he has been appointed as Director and Head of the Department of Genome regulation at the Max Planck Institute for Molecular Genetics in secondary employment and changed it to his principal employment in 2017.

Christine Mummery

Professor of Developmental Biology, Chair Dept. of Anatomy & Embryology
Leiden University Medical Center

Title:
Human pluripotent stem cells in understanding genetic cardiovascular disease and effects of drugs

Abstract:
Derivation of many different cell types from human pluripotent stem cells (embryonic stem cells or HESCs and induced pluripotent stem cells or hiPS cells) is an area of growing interest both for potential cell therapy and as a platform for drug discovery and toxicity. Most particularly, the recent availability of methods to introduce specific disease mutations into human pluripotent stem cells and/or to derive these cells as hiPS cells by reprogramming from any patient of choice, are creating unprecedented opportunities to create disease models “in a dish” and study ways to treat it or slow down its rate of development. Understanding the underlying developmental mechanisms that control differentiation of pluripotent cells to their derivatives and mimicking these in defined culture conditions in vitro is now essential for moving the field forward. We have used these methods to produce isogenic pairs of hiPSC lines to compare diseased and corresponding control cardiomyocytes and vascular endothelial cells and identify disease related phenotypes and mechanisms. The use of isogenic pairs has proved crucial since variability between “healthy control” hiPSC lines is often greater than the difference between a diseased cells and its isogenic control. We have also examined drug responses of hESC-derived cardiomyocytes to a variety of cardiac and non-cardiac drugs and shown that iPSC derived cardiomyocytes with mutations in ion channel genes can accurately predict changes in cardiac electrical properties and reveal drug sensitivities also observed in patients. Similar studies will be described using vascular endothelial cells from hPSC. Relevant in all cases is the development of appropriate bioassays in which to measure disease phenotypes which may be highly cell type specific dependent. For heart cells, this might be electrical activity or contractions force; for vascular cells, responses to fluid flow and inflammation. Various approaches to this will be presented.

Biography:
Christine Mummery is Chair of Anatomy and Embryology and Professor of Developmental Biology at Leiden University Medical Center. Her research concerns cardiovascular development and disease models based on human pluripotent stem cells. Immediate interests are on developing biophysical techniques for characterization and functional analysis of cardiovascular cells from hPSC. In 2015 she became guest professor at the Technical University of Twente to develop organ-on-chip models.

Dr. Mummery is a member of the Royal Netherlands Academy of Science, on the board of the Netherlands Medical Research Council and holds a European Research Council Advanced Grant to study cardiac development and disease in humans based on stem cell models. She wrote a lay-guide on stem cells “Stem Cell: Scientific Facts and Fiction” (Elsevier 2014) and is Editor-in-Chief of Stem Cell Reports, the journal of the International Society of Stem Cell Research. She is also on the editorial boards of Cell Stem Cell, Cardiovascular Research and Stem Cells.

Nina Tandon

CEO/Co-Founder
EpiBone

Ben Shepherd

Director
Therapeutics at Organovo

Title:
On the road to realizing tissue engineering’s transformative potential: multidisciplinary approach to accelerate bench-to-clinic transition

Abstract:
A panel of experts will discuss recent advances and current challenges for the successful development and application of human tissue for drug screening, tissue replacement and eventually organ replacement.

Biography:
Nina Tandon is CEO and co-founder of EpiBone, the world’s first company growing living human bones for skeletal reconstruction. She is the co-author of Super Cells: Building with Biology, a book that explores the new frontier of biotech. She is a TED Senior Fellow, Adjunct Professor of Electrical Engineering at the Cooper Union and a former Staff Associate Postdoctoral Researcher in the Laboratory for Stem Cells and Tissue Engineering, Columbia University. She has a Bachelor’s in Electrical Engineering from the Cooper Union, a Master’s in Bioelectrical Engineering from MIT, a PhD in Biomedical Engineering, and an MBA from Columbia University. Her PhD research focused on studying electrical signaling in the context of tissue engineering, and has worked with cardiac, skin, bone, and neural tissue.

Benjamin Shepherd, PhD has more than 17 years of experience in Tissue Engineering and Regenerative Medicine research. Dr. Shepherd’s research in vascular biology and microvascular tissue engineering has been focused on the ability to use creative approaches to generate microvascular networks to support the fabrication and surgical implantation of prevascularized neotissues. In addition to these research interests, he has successfully led early-stage research programs in liver biology, oncology and directed differentiation of stem cells within 3D and bioprinted tissues. Prior to joining Organovo, he was an Associate Research Scientist at the Yale School of Medicine, where he also conducted postdoctoral training. In addition to his appointment at Yale, he was a staff perfusionist at the University of Connecticut Health Center. Dr. Shepherd is a board certified clinical perfusionist (C.C.P.), received his Ph.D. in Physiological Sciences from the University of Arizona and a B.S. in Zoology from the University of Washington.

Erik Willems

Staff Scientist
Thermo Fisher Scientific

Title:
Eliminating inherent genome editing bottlenecks in iPSCs to build physiologically relevant disease models

Abstract:
Therapeutic development for human diseases continues to face obstacles, particularly in translating targets or compounds identified by in vitro screening campaigns to valid targets or efficacious and safe compounds once tested in humans. Here we discuss strategies that leverage induced pluripotent stem cells (iPSCs) to increase the relevance of human cell models for these in vitro approaches. We review current advances in genome engineering to pivot from challenges with delivery, identification and selection and subsequent clonal outgrowth by leveraging tools to consistently and reliably generate knock-out and knock-in models for use in target or compound identification. Specifically, we demonstrate this approach with iPSCs to build isogenic disease models, which can be further differentiated to various cell types of interest such as cardiomyocytes and dopaminergic neurons to model disease and are more directly related to a disease area than commonly used immortalized cell lines. We expect strategies combining genome engineering and stem cells to provide platforms for more robust disease models that will provide more predictable translation of in vitro to in vivo results.

Biography:
Dr. Erik Willems was trained as a stem cell biologist in Brussels, Belgium where he obtained his PhD in 2007, after which he soon relocated to San Diego, California to develop his expertise in the use of pluripotent stem cells in high throughput screening assays for understanding the basic biology and disease of the developing heart in the Sanford Burnham Prebys Medical Discovery Institute. Dr. Willems then pursued his passion for the development of biotechnology tools and applications and joined Thermo Fisher Scientific in Carlsbad, California where he - as a Manager in the Cell Biology Business - currently leads pluripotent stem cell-based customer driven projects and product applications, including characterization, reprogramming, genome editing, differentiation and disease modeling with a focus on drug discovery applications. Now in the stem cell field for over 15 years, Dr. Willems published numerous peer reviewed articles including in high impact journals such as Cell Stem Cell. His key expertise encompasses pluripotent stem cell biology, differentiation, genome editing, high throughput screening and drug discovery.

NEW Career development presentations

Sarah Gibson

Senior Manager of Talent Acquisition
Thermo Fisher Scientific

Title:
Preparing for what’s next – keys to a successful job search

Abstract:
Thinking about a starting a job search? Join us to listen to some tips to help you prepare for starting a successful career/job search. Topics covered: resume writing tips, preparing for general and behavioral interview questions, developing your personal brand and the benefits networking and mentoring.

Biography:
Sarah Gibson is a Senior Manager in the talent acquisition at Thermo Fisher Scientific, leading recruitment delivery for Genetic Sciences and Clinical Next-Generation Sequencing. Sarah has over 18 years of recruiting and human resources experience including many years of management experience. Currently, she manages a full life cycle recruiting team while championing employment branding campaigns, fostering a customer-oriented culture of collaboration, striving for excellence and building meaningful relationships. She is focused on attracting, identifying and welcoming talent whose groundbreaking discoveries and innovations enable our customers to make the world healthier, cleaner and safer.

Prior to this role, Sarah was a Recruiting Manager with Korn Ferry Healthcare Sector RPO business working at Stanford Hospitals & Clinics located in Palo Alto, CA. Prior to this role, Sarah spent 12 years in consulting and management with Accenture, where she focused on leading Talent Acquisition efforts for the Information Technology and Outsourcing practice.

Sarah holds a Bachelor of Science, from California State University Chico. She is active in her San Francisco community volunteering for philanthropic organizations and local charities.

Kevin McCormack

Communications Director
California Institute for Regenerative Medicine

Title:
How to do an elevator pitch that will win you friends, admirers and maybe even funding

Abstract:
How often are you asked what you do and when you start to explain you see the person you are talking to start to twitch, their eyes glaze over and they are desperately trying not to check their phone for something more interesting. One way to avoid that is to have an “elevator pitch” explanation ready. This is simply a plain English, well-rehearsed explanation about what you do, why it’s important and why people should care. It sounds easy but it’s not. This brief section will explain the key things you need to keep in mind when developing an “elevator pitch” and the things you definitely need to avoid.

Biography:
Kevin McCormack is the Communications Director at CIRM, the state’s stem cell agency. He considers himself to be the official translator for the agency, working to turn complex language about equally complex science into everyday English that anyone can understand. Before joining the agency he spent more than 20 years working as a journalist, most of that in TV news in San Francisco.

Rebecca Shumbata

Special Sales Account Manager
Mary Ann Liebert, Inc. Publishers

Title:
Journal publishing tools that make YOUR research stand out

Abstract:
There are a number of free and independent scholarly publishing tools available that save journal authors time and ensure research is found, read and tracked. In this talk, we will explore three of them:

  • ORCID—a persistent digital identifier that distinguishes you from every other researcher and, through integration in key research workflows such as manuscript and grant submission, supports automated linkages between you and your professional activities ensuring that your work is recognized.
  • Digital Science’s Altmetric—thousands of conversations about scholarly content happen online every day. Altmetric tracks a range of sources to capture and collate this activity, helping you to monitor and report on the attention surrounding the work you care about.
  • Kudos makes sure more people find, read, apply and cite your research. Kudos provides you with simple tools and guidance to help you maximize readership and citations for your work.

Biography:
Rebecca Appleby Shumbata received her degree from Connecticut College. Today, Rebecca is a scholarly publishing professional with over 15 years of experience. At Mary Ann Liebert publishers, Rebecca works with organizations to find topics and customize existing content, create new content, or extend planned live content into enduring educational materials that improve branding and generate leads.

Before joining Mary Ann Liebert publishers, Rebecca worked with publishers and institutions to improve the dissemination of research at Kudos. Prior to that, Rebecca served as Senior Global Sales Manager for Hosting Platforms at Ingenta, previously Publishing Technology. Rebecca has 8 additional years of publishing experiencing working for publishers, Cell Press, published by Elsevier, and the New England Journal of Medicine, published by the Massachusetts Medical Society.

Rebecca is also co-Chair of the Society for Scholarly Publishing Development Committee.

Ania Wronski

Field Applications Specialist
Thermo Fisher Scientific

Title:
How to get the most out of LinkedIn

Abstract:
Finding a job is a daunting and stressful process. Although technology has made the job find and application process much easier – but it also brings new challenges with how to manage the new technology involved in job seeking. In many ways, LinkedIn, a powerful professional networking tool has revolutionized the way we find jobs. In this seminar, Ania Wronski, Field Application Scientist for cel biology and former president of the Tufts Postdoctoral Association will provide advice as to how to use LinkedIn for your job search and answer common questions and pitfalls.

Biography:
Ania Wronski, PhD, is the CellModels Field Application Scientist (FAS) for the Cell Biology group within Thermo Fisher Scientific. She is passionate about providing solutions for researchers applying Cell Models such as iPSCs to the world of drug discovery. As an FAS, Ania travels the country, supporting the cell biology sales force and helping life science researchers do amazing work. Ania received her PhD in molecular biology and biochemistry from the University of Queensland in Australia and completed a four year postdoctoral study in the lab of Charlotte Kuperwasser at Tufts University.

On-demand presentations

Mary Kay Bates

Senior Global Cell Culture Specialist
Thermo Fisher Scientific

Title:
Physiological Oxygen: Historical and Molecular Perspective for Stem Cell Culture

Abstract:
For many decades, cell lines have been cultured in standard CO2 incubators at “normal” atmospheric oxygen concentrations of about 21%. But oxygen concentrations in the human or animal body are much lower, varying from as low as 1-2 % up to about 12%, depending on the tissue microenvironment. Oxygen concentration affects metabolism, proliferation, differentiation, disease progression and more. Controlling oxygen in vitro is increasingly important in biological function and disease modeling, pharmaceutical development and production, cell therapy, and of course in culturing stem cells. Creating hypoxia is critical to maintain pluripotency, control differentiation, limit oxidative damage and more. In this talk, we:

  • Review history and advances in understanding oxygen concentration in vivo and how hypoxia-inducible factor-1 (HIF-1) modulates the cell’s response.
  • Present examples demonstrating the effects of hypoxia on stem cells and primary cells, including better cell growth, less differentiation and less oxidative damage.
  • Explain how “tri-gas” CO2 incubators provide a physiological oxygen environment.

Biography:
Mary Kay Bates is a Senior Global Cell Culture Specialist with Thermo Fisher Scientific, where she provides cell culture expertise to colleagues and customers. Her knowledge is based on twenty years of experience in academic and industrial cell and molecular biology labs, focusing on cancer and gene therapy, authoring several publications. Mary Kay holds an M.S. in microbiology from the University of Wisconsin-Madison, and has presented seminars at institutes around the world.

Stuart Chambers

Senior Scientist
Amgen

Title:
Industry uses of human pluripotent stem cells

Abstract:

Human pluripotent stem cells (hPSCs) and their derivatives are increasingly being used in industry to gain a competitive edge in drug discovery and a new wave of biotechnology start-up companies are poised to evaluate hPSC-derived cell therapies.  Over the past decade protocols to a broad repertoire of hPSC-derived cell fates have been identified and published differentiating pluripotent stem cells to generate cells more capable of recapitulating phenotypes seen in primary cells.  Although, commercial uses for these cells are quite similar to academics on the day-to-day, there are stark differences in how industry plans to use pluripotent tissue.  Seen through the lens of industry, approaches to high throughput screening, how one employs disease modeling, and the overall need for greater scale and reproducibility become greater priorities. 

Worldwide hPSC-derived cells are being evaluated in clinical trials, and with great enthusiasm within the United States several industry and academic efforts are underway to determine if a cure can be found.  These endeavors further underscore the importance of keeping the technical limitations of hPSCs and their derivatives in mind.  Numerous hurdles still remain before cells can make it to the clinic.  Cells will have to be thoroughly vetted for safety, engineering solutions need to be identified for large-scale production, and protocols require adaptation to adhere to good manufacturing practices.  Even then, limitations remain as to appropriate disease indications for cellular therapies.  

Lukas Cyganek

Head of Stem Cell Core Facility
University of Göttingen

Title:
Custom-tailored cardiomyocytes: Directed differentiation of human pluripotent stem cells into defined atrial and ventricular cardiomyocyte subtypes

Abstract:
Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) as well as engineered heart muscles offer great potential for regenerative applications by CM transplantation, for the study of cardiac development and disease modeling, as well as for drug discovery and cardiotoxicity screenings in a human physiologically relevant model system. Several optimized protocols are available to efficiently differentiate iPSCs into CMs, which possess structural and functional properties of fetal CMs. However, the current methods produce a heterogeneous population of ventricular, atrial and pacemaker-like CMs strongly limiting their field of application. The generation of homogenous populations of subtype-specific iPSC-CMs and their comprehensive phenotypic comparison is crucial for a better understanding of the predominantly cardiac subtype-restricted disease mechanisms as well as for regenerative and pharmacological applications.

The goals of this study were to develop an efficient method for the directed differentiation of human iPSCs into defined functional CM subtypes in feeder-free culture conditions and to obtain a comprehensive understanding of the molecular, cell biological and functional properties of atrial and ventricular iPSC-CMs on both single cell as well as tissue level.

On the basis of temporal modulation of canonical WNT and retinoid acid signaling throughout differentiation of iPSCs via small molecules, we were able to guide the cardiac progenitor cells towards distinct cell fates resulting in homogeneous populations of either atrial or ventricular CMs. Transcriptome and proteome profiling as well as functional analysis of the CM subtypes via optical action potential screening, calcium imaging as well as engineered heart muscles demonstrate that atrial and ventricular iPSC-CMs highly correspond to CMs from the human atrium and ventricle, respectively.

In summary, this study provides a comprehensive understanding of the molecular and functional characteristics of atrial and ventricular iPSC-CMs and supports the suitability of these cells for its application in more precise disease modeling, drug screening as well as for cell-based therapeutic approaches.

Biography:
Lukas Cyganek is head of the Stem Cell Unit at the University Medical Center Göttingen. The SCU offers support within the scope of generation, cultivation and characterization of patient-specific induced pluripotent stem cells (iPSCs), its genome editing as well as its in vitro differentiation into patient-specific cardiomyocytes. The SCU further offers their expertise in the analysis of iPSC-derived cardiomyocytes and engineered heart muscles on molecular and functional level for detailed phenotyping. Apart from the laboratory services, the SCU acts as biobank for patient-specific iPSCs and iPSC derivatives. During his PhD at the European Neuroscience Institute Göttingen, Lukas Cyganek focused on the neurogenesis of the somatosensory system. In 2013, he joined the stem cell lab of Prof. Kaomei Guan in the cardiology at the University Medical Center Göttingen as a postdoctoral researcher, before he became head of the SCU in 2015. His recent research focusses on the generation of patient-specific as well as engineered iPSCs of and their applications in disease modelling of cardiovascular diseases, drug screening and tissue engineering.

Xavier de Mollerat du Jeu

R&D Director, Cell Biology
Thermo Fisher Scientific

Title:
New viral and non-viral platforms for T-cell engineering

Abstract:
Recent FDA approval of the first Chimeric Antigen Receptor T cell (CAR-T) therapy offers cancer patients more promise than ever for curative effects. However, many technical challenges in T cell gene delivery still remain in order for this therapy to become a standard of care practice. In this webinar, we will highlight the different viral and non-viral delivery approaches used in T cell engineering for cell and gene therapy applications including:

New solution for small-to-large scale serum-free, suspension lentiviral production – LV-MAX Lentiviral Production System:

  • Platform development process using Design of Experiment (DOE) methodologies
  • High-throughput to large scale bioreactor protocols
  • Cost benefits of this system over current methods

Novel gene editing tools for primary T cells:

  • New potent gene editing tools to increase knock-in and knock-out efficiency
  • Addressing non-viral delivery barriers through protocol optimization

Biography:
Xavier de Mollerat du Jeu
, Ph.D. is a Director of R&D transfection group at Thermo Fisher Scientific, working on creating and improving new nucleic acids delivery for both research and therapeutic applications. Xavier identified new DNA delivery approaches for hard to transfect cell lines and primary/stem cells and he is the inventor of Lipofectamine 3000. Xavier's team is also focusing on new delivery solutions for CRISPR delivery, new scalable lentiviral production systems, mechanical delivery approaches for primary T cells and in vivo delivery of RNAi/mRNA for research and therapeutic application. He studied molecular biology and plant physiology at the University of Montpellier II in France, and received his Ph.D. in human genetics in 2003 from Clemson University in South Carolina. His thesis work involved identifying the gene(s) responsible for Split Hand/Split Foot Malformation 3 (SHFM 3). His post-doctoral fellowship research was in the laboratory of Dr. Michael G. Rosenfeld at UCSD, where he studied the roles of microRNAs in pituitary gland development. He joined Thermo Fisher in 2005.

Christian Freund

LUMC hiPSC core facility
Dept. of Anatomy & Embryology
Leiden University Medical Center

Title:
Comparison of the Teratoma assay and in vitro surrogate tests for assessment of pluripotency of human pluripotent stem cells

Abstract:
For optimal use of human induced pluripotent stem cells (hiPSCs) it is essential to identify lines that are fully reprogrammed and of high quality with proven pluripotency in terms of differentiation. The ability to form teratomas in vivo is regarded as functional evidence of pluripotency for human pluripotent stem cells (hPSCs). Since the Teratoma assay is animal-dependent, laborious and only qualitative, there is an ongoing debate whether it is an acceptable tool. This has led to the development of an assay for quantitative analysis of teratomas (TeratoScore) as well as in vitro alternatives such as PluriTest and the hPSC Scorecard. We compared normal hPSCs, hiPSCs with reactivated reprogramming transgenes and nullipotent human embryonal carcinoma (hEC) cells in these assays. Cells were cultured on Vitronectin in TESR-E8 media (hPSCs) or in DMEM/10% FCS (hEC cells). The quality of undifferentiated cells was analysed by FACS for OCT3/4 as well as karyotyping. As assessed by immunohistochemistry and immunofluorescent staining the normal hPSCs gave rise to typical teratomas whereas the xenografts of the hEC cells and the hiPSCs with the reactivated reprogramming transgenes were largely undifferentiated and displayed a malignant phenotype. TeratoScore confirmed typical teratomas and tumors lacking differentiation but was unable to identify partially differentiated tumors. The hPSC Scorecard assay confirmed the line-specific differentiation propensities in vitro. However, when undifferentiated cells were analysed with PluriTest only hEC cells were identified as distinct. All other undifferentiated cells lines including hiPSCs with reactivated transgenes were undistinguishable and resembled normal hPSCs. We propose a combination of PluriTest and hPSC Scorecard for quantitation of pluripotency status and function of hPSCs used for in vitro purposes. By contrast, the Teratoma assay is the only method which is able to determine whether hPSCs could potentially develop a malignant phenotype, resulting in exclusion for any clinical application. 

Biography:
Christian Freund received his PhD at the Max Delbrück Centre for Molecular Medicine in Berlin, Germany, studying the role of transcription factor NF-kB in cardiac hypertrophy in mice. In 2006 he joined the lab of Christine Mummery at the Hubrecht Institute in Utrecht, The Netherlands. He initially worked on the differentiation of human embryonic stem cells into cardiomyocytes and showed that insulin had adverse effects on early specification into the mesoderm lineage. After having moved with the Mummery group to the Leiden University Medical Centre (LUMC) he started working on human cellular reprogramming by exogenous transcription factors. Since 2010 he has been co-heading the LUMC hiPSC core facility which up to date generated human induced pluripotent cells from more than 125 tissue samples for LUMC researchers and external investigators.  

Alessandra Giorgetti

Research Associate
Center for Regenerative Medicine in Barcelona

Title:
Directed differentiation of hematopoietic cells from human pluripotent stem cells

Abstract:
The continuous generation of blood cells throughout life relies on the existence of hematopoietic stem cells (HSC) generated during embryogenesis. Given the importance of HSC transplantation in cell-based therapeutic approaches, considerable efforts have been made toward understanding the developmental origins of embryonic HSC. Adult-type HSC are first generated in the aorta-gonad-mesonephros (AGM) region between days 27 and 40 of human embryonic development. It is relatively well accepted that the HSC emerge in the AGM through a hemogenic endothelium, but the direct precursor of this cell type remains to be clearly identified. The current picture of human hematopoietic development is largely extrapolated from animal models (mouse and zebrafish), as comparable studies on the human embryo are not possible. Human pluripotent stem cells (hPSCs) offer unprecedented opportunity to complement this knowledge, assuming that differentiation in the culture dish can recapitulate the dynamic and complexity of hematopoietic development in vivo. Here we summarize the recent advances made to understand the origins of hematopoietic stem cells in the early embryo and we discuss in detail latest protocols to obtain HSC from hPSCs.

Biography:
Dr. Giorgetti holds a Bachelor of Biology (1998) and PhD in Molecular Medicine from the University of Milan, Italy. She has a long-standing interest in hematopoietic stem cells, which started in her PhD thesis. She continued her studies in Dr. Rafii´s Lab at the Weill Cornell Medical College in New York. In 2008 she joined Center of Regenerative Medicine of Barcelona (CMRB). Her work contributed to the molecular understanding of nuclear reprogramming (Cell Stem Cell 2009; Nature Protocols 2010; Nature 2011, Stem Cells 2011, PNAS 2012, Nature Commun 2013). In 2012, she joined Inbiomed as Head of the Laboratory of Hematopoiesis and Blood Disorders. In 2013, she relocated to the Josep Carreras Leukaemia Research Institute where she contributed to develop new strategies for the in vitro generation of blood cells (Stem Cell Reports 2016; Exp Hematol 2017; Stem Cells 2017). Recently she relocated to the CMR[B] where she has just established herself as Team Leader.

Donna Goodenow

Graduate Student
University of North Carolina at Charlotte

Title:
Examining the negative impacts of bioflavonoids on the DNA damage response and DNA repair mechanisms in mouse embryonic stem cells

Abstract:
Bioflavonoids are dietary agents found in everyday food items such as fruits, vegetables, and legumes. It has been suggested that bioflavonoids be used with chemotherapeutics as an anti-cancer agent due to their anti-inflammatory capabilities and their ability to inhibit the DNA damage response. However most drugs that inhibit the DNA damage response can have secondary effects on normal cells. For instance, chemotherapeutics, such as etoposide, can cause DNA translocations leading to malignant transformations of normal cells to cancer cells. Studies have shown that etoposides and other chemotherapeutics increase the risk of leukemia. Therefore, it is our hypothesis that bioflavonoids inhibit the DNA damage response in normal cells, leading to improper repair of DNA damage, such as chromosomal translocation that can lead to leukemia. To study this we will use mouse embryonic stem cells, to model both acute high-dose exposure and chronic low-dose exposure to bioflavonoids such as genistein, quercetin, luteolin, myricetin etc. After we expose cells to these bioflavonoids, we will be examining them for DNA damage using Immunohistochemistry (IHC) to identify phosphorylation histone variant 2AX, a well-studied marker of DNA damage, with confocal microscopy. We will also be using a translocation reporter construct, which causes cells to produce green fluorescent protein when a translocation has occurred. We have integrated this construct into a mouse embryonic stem cell line, to measure incidences of DNA translocation by flow cytometry. Finally, we will be collecting DNA from these cells for PCR analysis and DNA sequencing to verify any DNA translocations that have occurred.

Biography:
I was born, raised and am currently living in Charlotte, North Carolina. I received my Bachelors of Science in Biology from Queens University of Charlotte. Now I am a third year Biology PhD student at the University of North Carolina at Charlotte (UNCC). Before beginning graduate school, I worked at Carolinas Medical Center in the General Surgery Research Department studying hemorrhagic shock and reperfusion injury in a rat model. At UNCC, I study the effects of dietary bioflavonoids on mouse embryonic stem cells to determine if these flavonoids cause improper DNA repair leading to chromosomal translocations and cancer.

Jacob (Yaqub) Hanna

Assistant Professor
Weizmann Institute of Science

Title:
Mechanisms for assembling and resolving naïve pluripotency

Abstract:
The identity of somatic and pluripotent cells can be epigenetically reprogrammed and forced to adapt a new functional cell state by different methods and distinct combinations of exogenous factors. The aspiration to utilize such ex vivo reprogrammed pluripotent and somatic cells for therapeutic purposes necessitates understanding of the mechanisms of reprogramming and elucidating the extent of equivalence of the in vitro derived cells to their in vivo counterparts. In my presentation, I will present my group’s recent advances toward understanding these fundamental questions and further detail our ongoing efforts to generate developmentally unrestricted human naive pluripotent cells. I will conclude by highlighting new avenues for utilizing epigenetic reprogramming to naïve pluripotency for unraveling critical gene regulatory mechanisms acting during early mammalian development and highlighting prospects for new platforms for human disease and developmental modelling.

Biography:
Dr. Hanna earned a B.Sc. in Medical Sciences (2001, summa cum laude), and a PhD/MD in clinical medicine (2007, summa cum laude), all from the Hebrew University of Jerusalem, Israel. For more than four years, he conducted postdoctoral research with Prof. Rudolf Jaenisch at the Whitehead Institute for Biomedical. In his recently established lab at the Weizmann Institute, Dr. Hanna combines diverse experimentation methods with computational biology to explore topics in embryonic stem cell biology, early embryonic development and the modeling of human diseases. Projects include deciphering the mechanisms by which IPS and germ cells are produced, characterizing unique naïve human pluripotent stem cells and various stages in early human development. In addition to helping elucidate the molecular basis of cell reprogramming, this research offers the promise of creating powerful research models for infertility, cancer, degenerative and autoimmune diseases such as type 1 diabetes.

His numerous honors and fellowships include a Novartis Fellowship by the Helen Hay Whitney Foundation (2007), a Genzyme-Whitehead Fellowship for outstanding postdoctoral fellows (2009), the TR35 award by MIT review magazine for young innovators (2010), a European Research Council early career development award (2011), the Rappaport prize in biomedical research (2013), the Krill prize by the Wolf Foundation for outstanding research achievements (2013), featured among “40 under 40” innovative scientists by Cell press (2014) and recently became an EMBO member (2017).

Jahid Hasan

Senior Scientist
Cell and Gene Therapy Catapult

Title:
Pluripotent stem cell bioprocessing platforms for cell therapy manufacturing

Abstract:
Pluripotent stem cell (PSC) culture at commercial scale requires a shift from the current manual processing methods and costly materials to deliver affordable differentiated cell products in a repeatable and robust manner. The development of automated processing and analytical platforms for manufacturing allogeneic cell therapies derived from pluripotent stem cells is one of the aims of the Industrialisation group of the Cell and Gene Therapy Catapult (CGTC). This is centred around developing starting materials compliant with industry-standards, scalable 2D and 3D expansion systems as well as flexible downstream processing solutions. Here, we describe the current progress of CGTC’s Cell Plasticity platform programme with respect to developing automated processes for controlled expansion of pluripotent stem cells in 2D and 3D systems and strategies for culture intensification and integrated downstream processing solutions.

Biography:
Jahid is a Senior Process Development Scientist at the Cell and Gene Therapy Catapult where he has been working for the last 4 years on various upstream and downstream development projects including the Cell Plasticity platform programme. He holds a doctorate degree from the University of Leeds where he developed a method for decellularising porcine meniscus with bone blocks for use as a meniscal replacement therapy. Jahid completed the Biochemical Engineering undergraduate programme at University College London gaining an MEng degree during which he undertook a year of work at Lonza’s Cell Culture Process Development team in Slough-UK. Here, he assessed medium formulation and stability on CHO cell culture.

Calley Hirsch

Development Engineer / Scientist II
CCRM

Title:
Identification of a high titer lentiviral production alternative to facilitate regenerative medicine therapies

Abstract:
Regenerative medicine therapeutics aimed at restoring normal cell function due to trauma and disease are gaining increasing momentum in clinical trials. For this purpose, lentivirus-based gene delivery is actively being investigated to treat a collection of monogenic and neoplastic diseases. The recent success of CAR-T-cell products further indicate an emerging role for lentiviral therapies, however, scalability and cost-effective production still present manufacturing challenges. To circumvent these problems, transient lentivirus production has been proposed in suspension host cells, yet consistent high titer yields remain difficult to achieve. In this presentation, a lentiviral production screen will be discussed where different cell lines, transfection reagents and media have been assessed to define a baseline process for scalable and cost-effective manufacturing of high titer lentivirus.

Biography:
Calley Hirsch has been a Development Engineer / Scientist II at CCRM in Toronto for 8 months, where she specializes in upstream lentiviral production and gene delivery technologies. Prior to working at CCRM, Calley was a postdoctoral fellow at the Lunenfeld-Tanenbaum Research Institute in Toronto and MD Anderson Cancer Center in Houston working on pluripotent stem cells and somatic cell reprogramming. Calley received her PhD from the University of Saskatchewan.

Navjot Kaur

Senior Staff Scientist
Thermo Fisher Scientific

Title:
New Gibco B-27 Plus Neuronal Culture System-Next Generation Media for Superior Neuronal Survival and Functionality

Abstract:
Since 1993, Gibco B-27 Supplement and Neurobasal Medium has been the trusted standard for a variety of neuronal culture applications, with citations in more than 11,000 publications. However, as the desire for more reliable and biologically relevant models has increased, so too has the necessity for a media system that can maintain and mature functional neurons over long periods of time in culture. To meet this need, we have developed the new Gibco B-27 Plus Neuronal Culture System comprising a neuronal basal medium (Neurobasal Plus) and serum-free supplement (B-27 Plus) that provides significant improvements for long term viability and functionality of primary and PSC-derived neurons in vitro. This next-generation media system features an optimized formulation, upgraded manufacturing process, and more stringent quality control for raw materials and final product.

Performance of this system was evaluated by a number of criteria including neurite outgrowth, neuronal survival and functionality in primary rat, mouse and human PSC-derived neurons for long term cultures at both low and high cell density.  Taken together the results demonstrate that our new B-27 Plus Neuronal Culture System is a superior solution to the current trusted standards used for culturing primary neurons, and maturing and maintaining hPSC-derived neurons.

Biography:
Navjot Kaur is a Senior Staff Scientist at Thermo Fisher Scientific in the Cell Biology business based in Frederick, MD. She is part of a team focusing on research, development and commercialization of next generation tools and reagents for Neurobiology, stem cell culture and differentiation. She has served as an R&D lead for launch of over 8 new products in the market spanning Neurobiology, cryopreservation, and stem cell differentiation areas. Navjot has developed and utilized a range of research tools including media formulation optimization, verification & validation process, development of standardized QC assays, technology transfer, manufacturing protocols, experimental design and statistical analyses. Prior to starting at Thermo Fisher Scientific in 2007 as Staff Scientist, she joined University at Buffalo, NY as a Postdoctoral Fellow and published more than 20 research articles in peer-reviewed journals in the fields of Neurobiology, Cell Signaling, and Protein Biochemistry.

James Kehler

Director of Scientific Alliances
Thermo Fisher Scientific

Title:
New Tools for improved CRISPR gene-editing in stem cells

Abstract:
The development of the CRISPR/Cas9 gene-editing platform enables the rapid generation of new genetically modified stem cell models of human diseases, as well as providing new potential therapeutic treatments. One challenge has been the efficient and consistent delivery of a range of gene-editing tools into human Pluripotent Stem Cells (PSCs) across different culture systems. We developed the Lipofectamine Stem transfection reagent as a complementary option to electroporation to be able to deliver large DNA plasmids, RNAs and Ribonucleoprotein (RNP) complexes into human induced Pluripotent Stem Cells and Neural Stem Cells. The ability to co-deliver Cas9 RNP complexes along with ssDNA templates enables both the disruption of genes, as well as introduction of specific basepair changes by Homology Directed Repair. Data demonstrating improved cutting activity in iPSCs with the next generation of TrueCut Cas9 Protein v2 with synthetic TrueGuide sgRNAs will be presented. In addition to Neon electroporation, optimized transfection and subsequent single-cell clonal isolation in a StemFlex Medium workflow will be reviewed. Scientists interested in gene-editing to create stem cell models for basic research, drug discovery and translational medicine will discover how these new tools can accelerate their research programs.

Biography:
James Kehler VMD, PhD is a comparative stem cell biologist who thrives on developing productive collaborations to translate scientific discoveries into transformative products. He trained at the University of Pennsylvania, where he received his VMD in 2002, and PhD in Cell and Molecular Biology in 2004. James has worked as a visiting researcher at the National Institutes of Health for over 10 years, where he and his collaborators have developed animal and stem cell-based models of human diseases. James has worked for stem cell companies from product development and management to directing custom reprogramming and gene-editing services. James joined MTI-GlobalStem now part of Thermo Fisher Scientific, as Director of Scientific Alliances to foster productive collaborations with academic, biotech and pharmaceutical partners.

Karl Koehler

Assistant Professor
Indiana University School of Medicine

Title:
Building inner ear organoids from human pluripotent stem cells through directed self-assembly

Abstract:
The human inner ear contains ~75,000 sensory hair cells that detect sound or movement via mechanosensitive hair bundles and transmit signals to the brain via specialized sensory neurons. Inner ear sensory cells derived from pluripotent stem cells would provide a valuable model for drug testing, yet a defined differentiation approach has been challenging to identify. Our group has established a step-wise method for generating inner ear organoids, with functioning hair cells, from human pluripotent stem cells. In a 3D culture system, we modulated TGF, BMP, FGF, and WNT signaling to direct the self-organization of multiple otic vesicle-like cysts from a homogenous cell aggregate. Over a 60-day period, the vesicles developed into multi-chambered organoids with sensory epithelia containing hair cells, reminiscent of the membranous organs of the inner ear. This presentation will discuss the process of making inner ear organoids as well as the potential applications of this technique as a powerful tool for investigating human inner ear development and drug discovery.

Biography:
Karl Koehler, PhD is an Assistant Professor of Otolaryngology-Head and Neck Surgery at Indiana University School of Medicine. He joined the faculty after completing his PhD degree in Medical Neuroscience in 2014 under the mentorship of Dr. Eri Hashino. Dr. Koehler began his career studying how embryonic and induced pluripotent stem cells could be used to produce neurons to treat hearing loss patients. His early work, published in Nature and Nature Protocols, detailed a novel culture system for growing mini inner ear organs, known as inner ear organoids, from mouse pluripotent stem cells. He then spearheaded an effort to decode the signaling mechanisms required to coax human pluripotent stem cells to become inner ear organoids. This work was recently published in Nature Biotechnology in June 2017. His research now focuses on using the organoid culture system as a platform to develop regenerative therapies for the inner ear and various craniofacial tissues. His work is funded by grants from the National Institute of Health and the Indiana Clinical and Translational Research Institute.

Alan Lam

Associate Staff Scientist
Bioprocessing Technology Institute (BTI), A*STAR

Title:
Integrated processes for derivation, expansion and differentiation of hiPSC to blood in IPS-Spheres™ cultures

Abstract:
Current methods for human induced pluripotent stem cells (hiPSC) derivation, expansion and differentiation can be limited in scalability and costly (due to their labor intensive nature). This can limit their use in cell therapy, drug screening and toxicity assays. One of the approaches that can overcome these limitations is microcarrier (MC) based cultures in which cells are reprogrammed and expanded as cell-MC aggregates and then directly differentiated in the same agitated reactor. This integrated process can be scaled up and eliminate the need for some culture manipulation used in common monolayer and EBs cultures. Here, this presentation describes the principles of using IPS-Spheres™ based integrated hiPSC derivation, expansion and differentiation process. Finally examples of integrated process for generation blood cells as well as challenges to be solved are described.

Biography:
Dr. Alan Lam graduated with a PhD (Biochemistry) from Chinese University of Hong Kong. He is currently the associate staff scientist in the Stem Cell Bioprocessing Group at the Bioprocessing Technology Institute, A*STAR, Singapore. His research interest focuses on developing scalable platforms using microcarriers for adult and pluripotent stem cell derivation, expansion and differentiation.

Andrew Laslett

Associate Professor
Commonwealth Industrial and Scientific Research Organisation (CSIRO)
Monash University

Title:
New monoclonal antibodies to defined cell surface proteins on human pluripotent stem cells

Abstract:
The availability of well-characterised monoclonal antibodies (mAbs) detecting cell-surface epitopes on human pluripotent stem cells (hPSCs) provides useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. We recently described generation of seven new mAbs that detect cell surface proteins present on primed and naive human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. In addition, we report that subsets of the seven new mAbs are also immunoreactive to specific human somatic cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types.

Biography:
Associate Professor Andrew Laslett is a Research Team Leader with CSIRO Manufacturing and a Research Group Leader with the Australian Regenerative Medicine Institute, where he leads a human pluripotent stem cell biology research group. His laboratory is currently focused on exploiting the basic biology of these cell types to create novel cell lines and tools that enhance human pluripotent cell research translation within CSIRO, Australia and internationally. Dr. Laslett leads a successful independent research program as well as having significant national and international collaborations.

Xiquan Liang

Staff Scientist
Thermo Fisher Scientific

Title:
Highly efficient stem cell engineering via Cas9 protein transfection

Abstract:
CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. The delivery of Cas9 plasmid DNA or mRNA involves in transcription and/or translation. On the other hand, the direct delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) approves to be more effective. In this endeavor, we have developed robust methods to purify and delivery Cas9 RNPs into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Recently, we enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. Under optimal conditions, we achieved precise genome editing rates of up to 24% in induced pluripotent stem cells (iPSCs) and 40% in Cas9-expressing iPSCs for a single nucleotide substitution at multiple genomic loci. One of the keys to high HDR efficiency was placement of the cleavage site in close proximity to the intended site of editing. Secondarily, asymmetric PAM and non-PAM single stranded (ss) DNA donors were also found to enhance HDR efficiency. Taken together, we provide simple and highly efficient approaches for modulation of the mammalian genome and for generation of knock-in and knock-out cell lines.

Biography:
Xiquan Liang graduated from City College of New York in 1999. He joined Thermo Fisher Scientific as a staff scientist in 2004. Since then he has led development of product development leadership in protein analysis, including High Molecular Weight MS standard, SILAC for protein identification and quantification, and Dynabeads-TiO2 for phosphopeptide enrichment. He is now engaged in product development in Synthetic Biology, including Bluegrass, GeneArt seamless DNA assembly tools, site-directed mutagenesis, and CRISPR-mediated mammalian cell engineering in the Life Sciences Solutions Group of Thermo Fisher Scientific in Carlsbad, CA. He is focused on developing tools for the entire Synthetic Biology workflow, specifically DNA cloning, Gene Synthesis and Assembly, and Molecular and Cell Engineering.

Alessandro Magli

Assistant Professor
University of Minnesota

Title:
Genomic profiling reveals novel PAX7 targets CD54, integrin α9β1 and SDC2, as markers for isolation of human ES/iPS cell-derived muscle progenitors

Abstract:
Therapeutic application of pluripotent stem (PS) cell-derived products represents the ultimate goal of stem cell research. In order to apply this technology to patients, it is fundamental to characterize in detail the cell population of interest and identify strategies for its purification from unwanted cells using clinically-compatible methods. In the case of skeletal muscle wasting disorders, we have shown that human PS cell-derived PAX7-induced myogenic progenitors may represent an excellent candidate for cell therapy. To successfully translate this approach toward the clinic, we took advantage of next-generation sequencing techniques to dissect PAX7 function during human myogenesis. Combination of PAX7 genomic target profiling using ChIP-seq and whole transcriptome analysis (RNA-seq), in which we systematically evaluated different time points of the PAX7-dependent myogenic commitment from human PS cells, revealed a subset of genes differentially expressed at various stages of this differentiation process, including a discrete number of surface markers. After Fluorescence Activated Cell Sorting (FACS)-mediated screening, we identified α9β1 integrin, CD54 and Syndecan2 (SDC2), as potential surface markers to be used for the prospective isolation of human PS cell-derived myogenic progenitors. We demonstrate that these surface molecules reproducibly allow for the isolation of myogenic progenitors from multiple human ES/iPS cell lines, in both serum- and serum-free culture conditions, and that α9β1+CD54+SDC2+ (triple+) cells represent a homogenous population of PAX7+ cells endowed with in vivo muscle regeneration potential. Furthermore, we demonstrate that a single marker is sufficient for the magnetic-based isolation of myogenic progenitors, thus enabling adaptation of our differentiation protocol to cGMP standards. These novel findings provide a clinically relevant method for the purification of PS cell-derived muscle progenitors for clinical applications.

Biography:
Dr. Alessandro Magli has worked in the skeletal muscle field for over 10 years. After completing his college studies at the University of Modena and Reggio Emilia (Italy), Dr. Magli earned his doctoral degree in the same institution by studying the molecular regulation of skeletal muscle differentiation. He later moved to the University of Minnesota where he conducted his post-doctoral training in the laboratory of Rita Perlingeiro. During this time, he investigated the mechanisms of cell fate choice during development, with a special focus on the mesodermal lineage derivatives. In particular, these studies recently resulted in the identification of markers for the purification of myogenic progenitors from differentiating human pluripotent stem cells, a finding that may enable the application of these ES-derived myogenic cells in cell therapy of muscle diseases. Dr. Magli is author of multiple scientific papers and, among other achievements, he was recipient of the Minnesota Regenerative Medicine educational award in 2015.

Cécile Martinat

INSERM director at iSTEM
President of French Stem Cell Society

Title:
Pluripotent stem cells to explore mechanisms and treatments of neuromuscular diseases

Abstract:
Disease-specific human pluripotent stem cells (hPSCs) represent a new chance to unravel cellular and molecular mechanisms of neurological diseases. Along this line, we were among the first to demonstrate that PGD-derived hES cells and derivatives offer pertinent cellular models of neuromuscular diseases. Indeed, we showed that PGD-derived hES cells and derivatives which express the causal mutation leading to the neuromuscular disorder Myotonic Dystrophy type 1 (DM1), can be used to model DM1-specific pathological features in vitro. Furthermore these models when combined to a whole genome transcriptomic analysis can unravel new physiopathological mechanisms. In parallel to these mechanistic analyses, we demonstrated that DM1 hESCs-based models can be used for therapeutic screenings leading to the recent launching of a clinical trial on DM1 patients.

Altogether, our DM1 studies participated to the demonstration that disease-specific hPSCs and their derivatives are effective models of neuromuscular diseases to decipher new pathological mechanisms and allow for drug discovery with potential clinical applications. Furthermore failures and successes in the DM1 program was instrumental in defining important roadblocks to overcome in order to successfully model neuromuscular pathologies using hPSCs : 1) the development of a collaborative environment to have access to other models of the pathology that will further validate the results obtained using hPSCs derived cells, 2) the careful analysis of appropriate controls (ideally isogenic ones) and 3) the development of differentiation protocols to obtain with high yield and robustness the cell population most relevant to the pathology of interest.

Our general objective is now to take advantage of this expertise to refine pathological modeling of neuromuscular disorders. Neuromuscular diseases are a heterologous group of pathologies characterized by muscular paralysis due to intrinsic muscular defects and/or impairment of neural circuits controlling movement often through to spinal motor neuron degeneration. In that sense Myotonic Dystrophy Type 1 and Spinal Muscular Atrophy, the two pathologies on which our group is focusing represent paradigmatic neuromuscular disorders. They are both caused by ubiquitously expressed mutations and characterized by defects in the motor neuron/muscle interconnection.

Biography:
Research projects developed by my group, that is implanted in the institute ISTEM, is to better understand physiological mechanisms implicated in the development of neuromuscular diseases and subsequently to develop new therapeutic strategies. We have three main domains of research: i). improve the protocols of differentiation to convert efficiently and reliably cell types of interest, i.e. motoneurons and muscle cells; ii). To improve our knowledge on the communication between motoneurons and muscle cells by developing new cellular systems iii). apply these different tools to neuromuscular diseases with the aim to decipher physiopathological mechanisms and to identify new pharmacological strategies. My group is mainly interested in two neuromusuclar diseases: myotonic dystrophy type 1 and spinal muscular atrophy. I recently took the head of the INSERM Unit located in ISTEM, institute dedicated to the use of human pluripotent stem cells to develop new therapeutic strategies for monogenic diseases. Two main of applications are developed in ISTEM: cell therapy and drug screening. More recently, I have been elected president of the French Society for Stem Cell Research (FSSCR). The main objective of this society, launched in January 2017, is to underpin and federate short and long-term efforts to ensure the place of France as a leader in the domain of stem cell research.

Suman Pradhan

Staff Scientist
Thermo Fisher Scientific

Title:
Comprehensive characterization of pluripotent stem cells

Abstract:
As reprogramming methodologies have become more reliable and efficient, corresponding improvements were needed in the characterization workflow as well; specifically in terms of the speed, efficiency, specificity and sensitivity of the methods used. Molecular analysis platforms offer a quantitative, accurate, and fast alternative to current methods and have recently been utilized to qualify pluripotent stem cells (PSC). When characterizing stem cells, it is important to confirm that a cell line a) has not acquired any genomic abnormalities during derivation or genetic engineering, b) has or (maintains) the capacity to differentiate into all three germ layers, and c) maintains expression of pluripotency associated markers. In addition to the characteristics described above, it is highly desirable to be able to uniquely ID every parental and subsequent derivative cell line; especially in core facilities, stem cell banks, or cell therapy development.

Here we describe three characterization methods: PluriTest, KaryoStat, and ScoreCard,. PluriTest and KaryoStat leverage microarray based platforms that provide a comprehensive gene expression profile to determine the state of pluripotency and whole genome coverage to detect chromosomal abnormalities via copy number variations, respectively. ScoreCard utilizes a focused panel of TaqMan qPCR based assays to assess expression of specifically selected genes that confirm pluripotency and differentiation potential. These three methods provide a complete solution to fully characterize PSC lines for pluripotency, differentiation potential, genomic integrity and sample ID.

Biography:
Dr. Pradhan joined Thermo Fisher scientific in 2016 and is currently working on next generation tools and technologies development with the goal of providing assays and methods for molecular characterization of stem cells and T cells utilizing methylation platform, array-based whole genome and transcriptomic profiling and TaqMan qPCR assays. He earned his Ph.D. in Molecular Biophysics from University of Calcutta, India, investigating the structural and functional consequences of binding of putative anti-cancer small molecules to chromatin in the context of transcription and disease. After graduating Dr. Pradhan moved to United States and did two postdocs – a brief one at UC San Diego followed by a second postdoc at UC Los Angeles. He made a few seminal contributions in unraveling the mechanism of eukaryotic gene expression and silencing leveraging sophisticated biochemistry and extensive next generation sequencing approaches.

Alessandro Prigione

Independent Team Leader
Max Delbrueck Center for Molecular Medicine (MDC)

Title:
iPSC-derived neural cells for drug discovery of mitochondrial brain diseases

Abstract:
Mitochondrial defects represent a common pathogenic mechanism associated with neurodegeneration. At the same time, mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Addressing the mechanisms underlying mitochondrial impairment in patient-derived neural cells may therefore lead to the identification of therapeutic strategies counteracting neurodegeneration. A critical element for cell-based drug discovery is the use of cells that are homogeneous and obtainable in a reproducible manner. We recently showed that NPCs derived from human induced pluripotent stem cells (iPSCs) represent an effective model system for mtDNA disease drug discovery (Lorenz et al, Cell Stem Cell 2017). Here, we present how we derive NPCs and post-mitotic neurons in a robust manner in order to use these cells for therapeutic screenings. Finally, we describe how to analyze the mitochondrial and metabolic properties of the patient-derived cells and how to set up high-content analysis (HCA)-based compound screening strategies.

Biography:
Dr. Prigione is an Independent Team Leader at the Max Delbrueck Center for Molecular Medicine (MDC) in Berlin. His group was established in 2015 with the generous support of a junior investigator grant from the German Ministry of Education and Research (BMBF).

Dr. Prigione received a M.D. from the University of Milan in 2002 and a PhD from the San Raffaele Institute of Milan in 2008. During his PhD, Dr. Prigione worked on neurological diseases at the University of Milan-Bicocca and on mitochondrial disorders at the University of California in Davis, USA. As postdoctoral scientist at the Max Planck Institute for Molecular Genetics in Berlin, Dr. Prigione described for the first time the reconfiguration occurring to mitochondria during the reprogramming of human fibroblasts to iPSCs.

The focus of the Prigione lab is now the application of iPSCs in modeling and treatment discovery of neurological diseases with mitochondrial impairment. His latest work was published in Cell Stem Cell in January 2017 and demonstrated the use of iPSC-derived neural cells for drug discovery of neurological disorders caused by mitochondrial DNA mutations.

Stevens Rehen

Head of Research–D’Or Institute for Research and Education
Professor of Biomedical Sciences–Federal University of Rio de Janeiro

Title:
New insights about Zika virus infection using iPS cells

Abstract:
In the last years, progress has been made regarding the differentiation of human pluripotent stem cells into neural stem cells and astrocytes, growing into neurospheres and cerebral organoids. These models offer an exciting new range of opportunities to investigate developmental changes associated with Zika virus (ZIKV) infection in human neural cells. Using immunocytochemistry and electron microscopy, we showed that ZIKV targets human brain cells, reducing their viability and growth as neurospheres and brain organoids. Combining transcriptomics and proteomics, we showed that ZIKV alters the molecular fingerprint of neural stem cells by activating responses to viral replication, DNA damage targets, cell cycle arrest, apoptosis, as well as the downregulation of neurogenic programs. Our recent results will be discussed during the presentation.

Biography:
Stevens Rehen received his Bachelor’s degree, Master’s degree, and Ph.D. at the Federal University of Rio de Janerio in Brazil. He later completed postdoctoral training at the University of California San Diego and The Scripps Research Institute. Over the past five years, Stevens has published over 76 peer-reviewed publications. Currently, Stevens is a Full Professor at the Federal University of Rio de Janerio. Additionally, he is the Head of Research at D’Or Institute for Research and Education (IDOR) and Regional Committee Member of the Pew Latin American Program in the Biomedical Sciences.

Meritxell Rovira

Research Associate
Center for Regenerative Medicine in Barcelona

Title:
Pancreatic progenitors: from the organ to the plate

Abstract:
Ductal cells share several markers with pancreatic embryonic progenitors, suggesting that ductal cells could be the closest lineage to an adult progenitor in the pancreas, thus their stem-like capacities in vitro have been under intense investigation. A commonly used assay for identifying stem or progenitor cells involves the isolation and culture of candidate cells under specific culture conditions to test their ability to generate colonies/spheres or organoids that can differentiate into multiple cell lineages either in vitro or following transplantation in vivo (Deleyrolle and Reynolds, 2009). Using such strategies, several cell types from the exocrine pancreatic compartment have been shown to acquire stem/progenitor-like properties in vitro (Rovira et al., 2010, Seaberg et al., 2004, Huch et al., 2013, Baeyens et al., 2005).

These research efforts have demonstrated that ductal/centroacinar cells, from human or mouse origin, can form spheres in suspension culture as well as organoids that under specific growth factor conditions or after kidney capsule engraftment can differentiate into β cells (Rovira et al., 2010, Huch et al., 2013, Boj et al., 2015, Seaberg et al., 2004, Jin et al., 2013, Lee et al., 2013). These organoid cultures represent a useful tool to study pancreas biology in an untransformed system in vitro, from human and mouse pancreatic ducts. This culture system together with new tools for genome editing holds promise for the study of pancreatic diseases and their progression (Huch et al., 2013, Huch and Koo, 2015, Hindley et al., 2016, Broutier et al., 2016).

Moreover, knowledge gained from studying the acquisition of stem-like/progenitor properties by some terminally differentiated pancreatic cell types in vitro can provide invaluable insight into harnessing these cells for regenerative therapy. Such studies can lead to defining the signaling pathways or growth factors whose activation in vivo is required to generate the proper niche that allow such population to act as dedicated or facultative progenitors.

Biography:
Meritxell Rovira holds a Bachelor in Biology (2001) from University of Barcelona and a PhD in Health and Life Science (2007) from University Pompeu Fabra. After her PhD she moved to USA in 2008 for her postdoctoral training at Johns Hopkins/School of Medicine to work in Steve D Leach and Mike Parsons laboratories, where she acquired expertise in adult pancreatic progenitors in mouse and zebrafish models. She came back to Barcelona in 2011 as a Marie Curie postdoctoral fellow at Jorge Ferrer laboratory (IDIBAPS) where she gained expertise in the field of epigenetics. After her postdoctoral training, she joined Núria López-Bigas laboratory at IRB as a research associate to establish and supervise the experimental part of Núria’s lab. She joined the CMR[B] in 2017 after being awarded with a Jovenes Investigadores fellowship from the Spanish Ministry.

Meritxell's main research interest is the study of pancreatic progenitors in zebrafish, mouse and human. Embryonic pancreatic multipotent progenitors and adult pancreatic duct cells express common markers in all animal species studied thus far; suggesting that adult duct cells might be exploited to create a pool of β-cell progenitors. Thus my main research interest is to understand the transcriptional and epigenetic changes that occur between embryonic progenitors and adult cells, as well as the influence of the extrinsic signals from the surrounding mesenchymal cells. This knowledge will allow the identification of the molecular mechanisms that restrict the lineage potential of duct cells and could be used to create a model of β-cell regeneration for the treatment of Type 1 Diabetes.

Jens Christian Schwamborn

Chief Scientific Officer (CSO)
Braingineering Technologies SARL

Title:
Braingineering’s human midbrain organoids as novel model for neurodegenerative

Abstract:
Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Here, we describe a robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model for the study of Parkinson’s disease. Midbrain organoids are characterized in detail for neuronal, astroglial, and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.

Biography:
Since 2013 Jens C. Schwamborn, PhD, is head of the Developmental and Cellular Biology group at the Luxembourg Centre for Systems Biomedicine (LCSB) as well as Professor at the University of Luxembourg. Additionally, since 2016 Jens is Chief Scientific Officer of the biotech company Braingineering Technologies. In 2002 Jens obtained is diploma in Biochemistry from the University Witten/Herdecke in Germany and in 2005 his PhD in Biology from the University Muenster in Germany. He worked as a postdoctoral researcher at the Institute for Molecular Biotechnology in Vienna / Austria. The focus of his work over the last years was on Neurobiology, Stem Cell research and Parkinson’s disease. In particular he is interested in using human induced pluripotent stem cells for the development of advanced approaches for in vitro disease modeling. Jens published more than 50 papers in peer reviewed journals, holds several patents and severs as reviewer for numerous journals and funding agencies.

Adrian Teo

Independent Fellow, IMCB, A*STAR
Adjunct Assistant Professor, SBS and LKCMedicine, NTU, Adjunct Assistant Professor, NUS Medicine, NUS

Title:
Patient-specific hiPSCs for understanding diabetes disease mechanisms

Abstract:
Diabetes is a debilitating chronic disease that is spirally out of control. Fundamentally, the progressive failure of pancreatic beta cells results in decreased insulin secretion, ultimately giving rise to hyperglycaemia and overt diabetes. Given that there is a lack of access to pancreatic islets from diabetic patients with defined gene mutations or variants, the use of diabetic-patient-specific human induced pluripotent stem cells (hiPSCs) and their differentiation into pancreatic beta-like cells will provide an inexhaustible source of material for 1) in vitro disease modelling to study diabetes-related mechanisms, 2) developing small molecules that can enhance human beta cell replication and even 3) transplantation therapy.

Here, I will highlight our efforts in recruiting various types of diabetic patients, obtaining skin biopsies or peripheral blood mononuclear cells (PBMCs), deriving hiPSCs from these somatic cells and differentiating them into pancreatic cells. I will also provide an example of subjecting these diabetic-hiPSCs through a pancreatic differentiation protocol for in vitro disease modelling of diabetes. Overall, it will be evident that disease modelling of human diabetes via the use of diabetic-hiPSCs will provide novel insights into the development of diabetes and its complications.

Biography:
Adrian Teo is an Independent Fellow at IMCB and an Adjunct Assistant Professor at NTU and NUS. He obtained his B.Sc. (1st Class) from NUS and then worked on human pluripotent stem cells (hPSCs) with Ray Dunn, PhD, and Alan Colman, PhD, at ES Cell International Pte. Ltd. followed by IMB. In April 2008, he joined the laboratory of Ludovic Vallier, PhD, at the University of Cambridge to pursue his PhD, under the AGS(O) scholarship. Concurrently, he was also an Honorary Cambridge Commonwealth Trust Scholar. He completed his PhD in July 2010 and joined the laboratory of Ray Dunn, PhD, at IMB as a postdoctoral fellow before heading to the laboratory of Rohit Kulkarni, M.D. PhD, at Joslin Diabetes Center, Harvard Medical School. During his fellowship, he obtained HSCI seed grants and a JDRF fellowship to pursue his research interests in using hPSCs for in vitro disease modelling of diabetes. He currently runs the Stem Cells and Diabetes Laboratory with a major focus on differentiating human pluripotent stem cells (hPSCs) into pancreatic cells and cell types affected in diabetic complications to dissect the pathology of diabetes and its complications.