24 Hours of Stem Cells—2015 speakers
2015 keynote presentations
Genome editing and stem cell engineering for disease modelling
Senior Director, Synthetic Biology R&D
Thermo Fisher Scientific
The ability to create accurate disease models of human monogenic and complex genetic disorders is very important for the understanding of disease pathogenesis and the development of new therapeutics. Although proof of principle using adult stem cells for disease modeling has been established, induced pluripotent stem cells (iPSCs) have been demonstrated to have the greatest utility for modeling human diseases. Additionally, the latest advances in programmable nucleases have empowered researchers with genome editing tools, such as CRISPR/Cas9, that substantially improve their ability to make precise changes at a defined genomic locus in a broad array of cell types including stem cells. While the utility of these tools is improving, there are several key factors, including design and delivery that should be taken into account to ensure maximum editing efficiency and specificity. Already, these tools have allowed us to efficiently knock out genes and generate single nucleotide polymorphism (SNP) iPSCs. This ability to modify target genomic loci with high efficiency will facilitate the generation of novel genetically modified stem cells for research and therapeutic applications.
Jon Chesnut received a PhD in Cell and Molecular Biology from UC Davis (1994), and did postdoctoral studies at the University of Colorado in recombinant antibody technology development (1994-95). Prior to graduate school, he was employed at the Salk Institute (1983-84) and Hybritech Inc. in La Jolla, CA (1984-88). After his post doctoral research at Colorado he joined Invitrogen as a research scientist (1995). Since then he has led the development of various prokaryotic and mammalian cell cloning and expression systems. He has led groups focused on development of TOPO and Gateway cloning as well as the human embryonic cell engineering technologies. He now leads the Synthetic Biology Research and Development Team in the Life Sciences Solutions Group of Thermo Fisher Scientific in Carlsbad, CA. His group is focused on developing tools for the entire Synthetic Biology and Cell Engineering workflow, specifically Bioinformatics, Genome Editing, and Cellular Analytics.
Probing disease mechanisms in ALS and ion-channel epilepsy using iPSCs, reprogramming and optogenetic approaches
Assistant Professor of Neurology & Physiology
The lack of easy accessibility to the cells of the nervous system has hampered progress towards the discovery of degenerative mechanisms as well as more effective treatments for neurological diseases. The groundbreaking technology of reprogramming, which allows for the generation of patient specific induced pluripotent stem cells (iPSCs) has created an unprecedented opportunity for a new approach towards developing cellular models of human disease. We employ this approach to generate cortical excitatory and inhibitory neurons as well as spinal motor neurons and astrocytes from individual patients. We use gene-editing techniques to introduce or fix mutations and then study the neurons we make by classical methods including immunocytochemistry, biochemistry, global genomic analysis, live cell imaging as well as by non invasive electrophysiological recording techniques. During this talk I will present our progress towards developing models of motor neuron disease as well as pediatric forms of epilepsy.
In 2008, Dr. Kiskinis moved to the US to carry out postdoctoral research training in the Lab of Professor Kevin Eggan at the Department of Stem Cell and Regenerative Biology at Harvard University. While at Harvard, Evangelos acquired expertise in human stem cell biology and explored the possibility of using patient-specific stem cells to investigate neurodegenerative diseases with a particular focus on Amyotrophic Lateral Sclerosis (ALS). His research efforts lead to the discovery of molecular pathways that become dysfunctional in the motor neurons of ALS patients as well as to the discovery of a small molecule therapeutic that is currently being tested in clinical trial for ALS patients. Dr. Kiskinis earned a degree in Molecular Biology from the University of Surrey and a Masters and PhD in Molecular Endocrinology from Imperial College, London, England. During his graduate work he established a mechanism by which transcriptional co-repressors define cellular function by regulating the metabolic profile of muscle and neurons. Dr. Kiskinis also spend a year in Basel, Switzerland working as a research associate at the headquarters of Novartis Pharmaceuticals, setting up novel high-throughput screening assays for evaluating cytotoxicity of candidate therapeutic compounds. In January 2015, Evangelos was appointed as an Assistant Professor of Neurology and Physiology at the Feinberg School of Medicine, Northwestern University. Dr. Kiskinis’ lab is a core member of the newly founded Les Turner ALS Research and Patient Centre. Evangelos is focusing his research efforts on the fight against neurological diseases and is developing novel stem cell based models of ALS and ion-channel forms of epileptic syndromes. He has been the recipient of prestigious fellowships from the European Molecular Biology Organization as well as from the New York Stem Cell Foundation and the Charles A. King Trust Medical Foundation. His research has also been supported by the Project ALS and Target ALS initiatives. At Northwestern University, Dr. Kiskinis serves as the Scientific Director of the Stem Cell Core Facility.
Biomaterials for assembly of stem cell-derived human tissues
William L. Murphy
Harvey D. Spangler Professor of Biomedical Engineering and Co-Director of Stem Cell and Regenerative Medicine Center
University of Wisconsin
The need for human, organotypic culture models coupled with the requirements of contemporary drug discovery and toxin screening (i.e. reproducibility, high throughput, transferability of data, clear mechanisms of action) frame an opportunity for a paradigm shift. The next generation of high throughput cell-based assay formats will require a broadly applicable set of tools for human tissue assembly and analysis. Toward that end, we have recently focused on: i) generating iPS-derived cells that properly represent the diverse phenotypic characteristics of developing or mature human somatic cells; ii) assembling organotypic cell culture systems that are robust and reproducible; iii) translating organotypic cell culture models to microscale systems for high throughput screening; and iv) combining genomic analyses with bioinformatics to gain insights into organotypic model assembly and the pathways influenced by drugs and toxins. This talk will emphasize recent studies in which we have explored biologically driven assembly of organotypic vascular and neural tissues. These tissues mimic critical aspects of human tissues, and can be used for predictive neurodevelopmental toxicity, and for discovery of vascular disrupting compounds.
Bill Murphy is the Harvey D. Spangler Professor of Biomedical Engineering, Professor of Orthopedics & Rehabilitation, and Co-Director of the Stem Cell and Regenerative Medicine Center at the University of Wisconsin. He received his B.A. in Physics from Illinois Wesleyan University, Ph.D. in Biomedical Engineering from the University of Michigan, and postdoctoral training in Chemistry at the University of Chicago. His research interests focus on creating new biomaterials inspired by the materials found in nature. Murphy’s research group is using new biomaterials to understand stem cell behavior and to induce tissue regeneration. He has published more than 140 scientific manuscripts, filed over 30 patents, co-founded multiple start-up companies, and received awards that include the National Science Foundation Career Award, the Wisconsin Vilas Associate Award, and induction as a Fellow in the American Institute for Medical and Biological Engineering.
2015 panel discussions
Standardizing stem cell research
Alexander Meissner, Ernst Wolvetang, Tennielle Ludwig
Key characteristics of pluripotent stem cells are the ability to self-renew and the ability to differentiate towards downstream lineages. Traditional methods to evaluate these properties rely on the measurement of cellular markers associated with self-renewal or differentiation. As stem cell research moves towards clinical applications, there is an increased need to establish as standards the markers that must be measured, and to quantitatively assess these markers using molecular analyses. In this panel discussion, experts will discuss their approaches to characterizing pluripotent stem cells and share how the stem cell research community is evaluating different approaches in the hopes of creating more robust standards for evaluating stem cells.
The path towards scalable, xeno-free applications for regenerative medicine
Tiago Fernandez, Daniel Paull
The promise of pluripotent stem cells lies in their ability to enable better, more physiologically relevant disease model systems. Recently, there has been a noticeable progression in stem cell research towards more scalable, xeno-free media systems to fully evaluate the potential of these disease model systems. Panelists in this live session will discuss their paths to deriving PSCs in scalable and xeno-free formats and how these initial steps will support more scalable, physiologically relevant disease models.
Generation of effective disease models
Katherine Santostefano, Kristen Brennand, Bronwen Connor
The development of robust, reproducible disease models is a key step towards recognizing the full potential of pluripotent stem cells in the understanding of human diseases. Here our panelists will describe optimized approaches for generating effective disease models through the interrogation of genetic variants, directed differentiation, and new screening methodologies. Listen as they discuss their current approaches and what opportunities still lie ahead for disease modeling.
Dental pulp of the third molar: a new source of pluripotent-like stem cells
Director of UIC Regenerative Medicine Research Institute
Universitat Internacional de Catalunya
Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However, no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work, we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF, EGF and PDGF. These cells are SSEA4+, OCT3/4+, NANOG+, SOX2+, LIN28+, CD13+, CD105+, CD342, CD452, CD90+, CD29+, CD73+, STRO1+ and CD1462, and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly, DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm, endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4, GATA6, MIXL1, NANOG, OCT3/4, SOX1 and SOX2 to determine the degree of similarity between DPPSCs, EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs, hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages, they represent an easily accessible source of stem cells, which opens a range of new possibilities for regenerative medicine.
Modeling eye diseases using human adult retinal pigment epithelium stem cells
Icahn School of Medicine at Mount Sinai
The Retinal Pigment Epithelium (RPE) is a monolayer of cells underneath the photoreceptor layer of the retina and above the Bruch’s membrane. The RPE layer performs many functions for and phototransduction and if any of these functions break down, the ultimate result is blindness, which is why there are many RPE-related blinding diseases. We recently discovered a stem cell population in the monolayer we term the RPESC. We are now studying this population to understand its regenerative potential for rescuing vision. We are approaching RPE-related blinding diseases in a number of ways: 1) Transcriptomic and epigenetic analysis of the RPE in normal and diseased states; 2) Developing in vitro RPE disease models; 3) Developing transplantation therapies to replace diseased RPE with normal RPE.
During his PhD at NYU, under the supervision of Dr. Lang, directed by Dr. Llinas, Dr. Blenkinsop conducted in vivo multielectrode recordings of multiple regions of the cerebellar system, in order to show how important gap junctions in the inferior olive and their effects on the rhythmicity and synchrony of the cerebellum are on motor coordination. This electrophysiological background benefited him greatly when he joined the Laboratory of Dr. Sally Temple as a Post-doctoral fellow. Immediately, Dr. Blenkinsop realized that while the RPE should be a cobblestone, pigmented cell, the cells in culture were fibroblastic and not pigmented, and that in order to effectively study how defects in the RPE cause various disorders, including Age-related Macular Degeneration, it was first necessary to develop a cell culture system which preserves the electrophysiological characteristics of RPE in vivo. Both by studying the literature and through clues from development, he was able to establish a reproducible and efficient protocol for isolation of RPE from human adult cadaver donor eyes, which produces pigmented monolayer of functional RPE. Dr. Blenkinsop then helped discover a stem cell in the RPE layer with Dr. Enrique Salero in Dr. Temple’s lab. Since starting his lab in May 2014, Dr. Blenkinsop has concentrated on developing models of RPE-related eye diseases using adult human RPE from patients with those diseases. He is also investigating the role the histone code plays in establishing cell identity along with cell plasticity and under disease states. Moreover, Dr. Blenkinsop is part of a consortium to translate these adult human RPE cultures into a cell transplantation therapy for RPE-related eye diseases. His research is moving the field of RPE-related eye diseases towards new therapies and towards a deeper understanding of the epigenetic foundation and regenerative potential of RPE stem cells.
Muscle stem cells harboring basal levels of pluripotency genes are amenable to pluripotent conversion without using reprogramming factors or small molecules
Assistant Professor and Principal Investigator
Developmentally upstream muscle-derived stem cells (MDSCs) have been explored for their multilineage potential in humans and mouse. MDSCs, although derived from muscle are different from the much known counterpart, satellite cells, the latter being committed muscle stem cells. In the present topic, I will focus on MDSCs from myostatin null mice (Mstn-/-). Myostatin (GDF8) is a muscle inhibitory cytokine and hence Mstn-/- is a favourable condition for muscle growth. Myoblasts from Mstn-/- mice have been reported to be myogenically vigorous, as compared to its wild-type counterparts. Contrarily, MDSCs from Mstn-/- mice behaved weakly myogenic in vitro. Interestingly, microarray data of Mstn-/- MDSCs exhibited upregulation of pluripotency-related markers such as Leukemia Inhibitory Factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) as compared to wild-type MDSCs (WT-MDSCs) (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39765). Impressed by the high expression of LIF and LIFR in Mstn-/- MDSCs, we had cultured Mstn-/- MDSCs in mouse ESC media containing LIF for 95 days and obtained ~50% of pluripotent cells as assessed by live cell staining for SSEA-1 pluripotency marker. We termed the FACS-sorted SSEA-1+ cells as culture induced pluripotent stem cells (CiPSC). CiPSCs expressed all the core pluripotency markers by qRT-PCR and immunofluorescence, formed embryoid bodies in non-adherent conditions, differentiated into ecto-meso-endoderm and also formed teratomas when injected into immunocompromised mice. As the CiPSCs were derived from the Mstn-/- mice, and Mstn being TGFβ family member, we probed the status of TGFβ family of genes and identified BMP2 hypermethylation in CiPSC clones and downregulation of BMP2 protein. Hence, we linked the downregulation of BMP2 with pluripotency induction in Mstn-/- MDSCs. At this stage, pluripotent conversion of MDSCs from Mstn-/- mice is a unique phenomenon. It is unclear that pluripotent conversion is restricted only to MDSCs of Mstn-/- or else, other adult stem cells, as well. However, further investigations involving the core pluripotency circuitry in Mstn-/- MDSCs are required for providing insights into this phenomenon. Taken together, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.
Dr Bipasha Bose obtained her Ph.D. degree in Applied Biology (Molecular Carcinogenesis) from Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, India in 2004. She has a more than 11 years of post-PhD research experience working in Stem Cell Biology, in academia and industry in India, Belgium and Singapore. Dr Bose holds key skills in the field of clinical, as well as, R and D grade stem cells. Her noted contributions to the field of regenerative medicine involve her co-work with the scientists for deriving human embryonic stem cells (hESC) for the first time from the Indian subcontinent, differentiation of hESC into functional insulin producing β islets and their pre-clinical translation, differentiation of hESC into functional hepatocytes. She has also contributed in the field of adult stem cell biology and proven the myogenic potential of aged muscle stem cells and perivascular mesenchymal stem cells, hepatocyte differentiation from Multipotent Adult Progenitor cells. Her contribution in the field of basic biology of stem cells has proven the pluripotent conversion of myostatin- null muscle stem cells without using reprogramming factors. She has contributed to several publications and book chapters as the lead author in the peer-reviewed journals and books of international repute. Dr. Bose also has two patents to her credit. Currently, Dr Bose is working in Yenepoya Research Centre, Yenepoya University. Her present focus is on establishing R and D scale human and mouse stem cell laboratories, guiding Ph.D students in her area of expertise of basic biology of stem cells and stem cell therapy for diabetes, liver diseases and muscle disorder, and also various aspects of ocular stem cells.
Modeling predisposition to schizophrenia using hiPSCs
Icahn School of Medicine at Mount Sinai
Schizophrenia (SZ) is a debilitating neurological disorder. Though postmortem studies have revealed reduced neuron size and spine density in SZ brain tissue, the molecular mechanisms underlying the disease state remain unclear. To address this, we directly reprogrammed fibroblasts from SZ patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated these disorder-specific hiPSCs into neural progenitor cells (NPCs) and neurons. Gene expression comparisons of our hiPSC-derived neural progenitor cells (NPCs) and 6-week-old neurons to the Allen BrainSpan Atlas indicate that our hiPSC neural cells, from controls and patients with SZ, most resemble fetal rather than adult brain tissue, indicating that hiPSC-based models may not yet be suited for the study of the late features of this disorder. We have previously reported aberrant migration, increased oxidative stress, abnormal WNT signaling and elevated global protein synthesis in SZ hiPSC NPCs, together with diminished neuronal connectivity, decreased neurite number, and impaired synaptic morphology in SZ hiPSC neurons. Now, we wish to investigate which genetic variants identified in SZ patients are causal contributors to disease, investigating the link between genotype, gene expression and in vitro phenotype. From two related individuals with large (289kb) heterozygous deletions in CNTNAP2 and discordant clinical outcomes, we generated hiPSC neural cells, observing exon-specific changes in CNTNAP2 expression in both carriers as well as allele-biased expression in CNTNAP2 that was consistent with both clinical outcome and neural migration in vitro. Ultimately, we intend to explore the relationship between genotype and neuronal function by restoring defined mutations in SZ hiPSC neurons and recapitulating them in controls, in order to assess whether they are necessary and sufficient for disease across a range of genetic backgrounds.
Kristen Brennand, PhD is an Assistant Professor of Psychiatry at the Icahn School of Medicine at Mount Sinai, in New York, New York. She trained in developmental and stem cell biology at Harvard University and in neurobiology during postdoctoral at the Salk Institute for Biological Studies. By combining expertise in stem cell biology and neurobiology, she has pioneered a new approach by which to study psychiatric disease. Dr. Brennand’s work is funded by the National Institutes of Health, the New York Stem Cell Foundation and the Brain and Behavior Research Foundation.
A novel PSC-based myogenic platform to explore treatment strategies for muscle diseases
Department of Pathology, Brigham and Women’s Hospital
Harvard Medical School
There is currently no efficient treatment for Muscle dystrophies, where patients suffer of muscle wasting that can lead to permanent immobilization and death. Progresses to find a cure have been slow due to the absence of relevant cellular models of the disease for drug screening. Moreover, while cell therapy is also a promising avenue of investigation to replace the defective tissues in patients, production of muscle cells in the dish is so far very inefficient. During embryonic development, skeletal muscles arise from somites, which derive from the presomitic mesoderm (PSM). Based on our understanding of PSM development, we report in this study that we have now developed efficient directed differentiation culture protocols to differentiate pluripotent stem cells (PSCs) into both contractile muscle fibers and muscle stem cells in the dish, by way of mimicking muscle formation in the embryo. Going further, we established models for myopathies, such as Duchenne Muscular Dystrophy, to characterize diseased muscle cells and which allows to test strategies to correct the pathology. Furthermore, we show that the muscle stem cells produced in the dish can be transplanted into the muscle of myopathic animal model. The engrafted cells can contribute to muscle regeneration and correct some of the disease hallmarks. Therefore, our differentiation method offers an attractive platform to develop treatments for muscle diseases.
Dr. Jérôme Chal is currently Research Scientist in the Department of Pathology at Brigham and Women’s Hospital, Harvard Medical School. His research focuses on harnessing PSCs differentiation toward skeletal muscle and other tissues to develop novel stem cell-based assays and their biomedical applications. Dr. Chal holds a Ph. D in stem cell and developmental biology from the University Pierre et Marie Curie in Paris. He graduated from the Ecole Normale Supérieure and completed his research training at the Stowers Institute for Medical Research in Kansas city with O. Pourquié. While at the IGBMC (Strasbourg, France), Dr. Chal supervised a successful research program aiming at differentiating muscle from PSCs. This work led to the first description and characterization of contractile skeletal muscle fibers derived from pluripotent stem cells (Chal et al, Nature Biotechnology 2015) and promoted the creation of a start-up company, Anagenesis Biotechnology, specializing in the production of muscle cells in vitro for cell therapy and drug screening.
The use of modified mRNA to enhance the generation of induced neural precursor cells from adult human fibroblasts
Associate Professor and Head of the Neural Reprogramming and Repair Lab
University of Auckland
Reprogramming technology has recently provided the capability to directly generate specific neuronal lineages, such as dopaminergic neurons or motor neurons. However, the ability to only generate a specific neuronal population may be advantageous or limiting, depending on the research goals. Recently we developed an efficient system for directly generating neural stem/precursors from adult human fibroblasts. We have shown that transient ectopic expression of neural-promoting transcription factors, SOX2 and PAX6, in human fibroblast cells was sufficient to reprogram them to neural stem/precursor cells, which could then be further differentiated to region-specific neuronal populations. Characterization of the induced neural stem/precursor cells (iNPs) showed they express a range of neural positional markers and their progeny included glutamatergic, GABAergic and dopaminergic neurons. The methodology initially used to generate iNPs involved transfection of fibroblasts with SOX2 and PAX6 cDNA plasmids. Whilst this approach is desirable in that it reduces potential genomic integration of ectopic factors, plasmid transfection efficiency is relatively poor compared to traditional viral transduction. To advance this technology, we have optimized a novel modified mRNA gene delivery system for direct iNP reprogramming. Modified mRNA has the benefit of being extremely stable and non-immunogenic, allowing us to co-transfect adult human fibroblasts with our reprograming factors SOX2 and PAX6 with an efficiency of >80%, significantly higher than the 10-20% transfection efficiency obtained with plasmids. Cell survival was >85% post-transfection, also significantly greater than 20-40% survival with plasmid transfection. Most importantly, co-transfection with SOX2 and PAX6 mRNA increased the rate of iNP reprogramming to ~14-21 days compared with ~45-65 days required using plasmid transfection. Expression of neural positional genes was observed through qPCR, and differentiation of mRNA-derived iNPs generated TuJ1-positive cells co-expressing phenotypic markers including GAD65/67, vGlut and tyrosine hydroxylase. These results represent the first time an mRNA approach has been used to directly reprogram adult human fibroblasts to neural precursor cells.
Bronwen Connor is an Associate Professor in Pharmacology and head of the Neural Reprogramming and Repair Lab at the University of Auckland. Her specific interest is in the identification and development of novel protective or regenerative strategies to treat neurological disease, with particular focus on the potential use of stem cell therapy and gene transfer techniques for the treatment of brain injury and disease. Bronwen is a graduate of the University of Auckland, graduating with a BSc in Pharmacology and Physiology in 1994 and a PhD in Neuropharmacology in 1997. She then spent three years as a Post-Doctoral Fellow at Northwestern University in Chicago, USA studying the potential use of gene therapy for the treatment of Parkinson’s disease. Bronwen has worked on a number of strategies for the treatment of neurological disorders including gene therapy for Parkinson’s disease and Huntington’s disease, the development of stem cell replacement therapy for Parkinson’s disease, Huntington’s disease and stroke, and the identification of novel agents for the treatment of depression. She also has an interest in the re-direct use of current pharmacological agents, such as anti-depressant or anti-psychotic agents for the potential treatment of other neurological disorders and is currently involved in a clinical trial for the use of clozapine for the treatment of multiple sclerosis. Bronwen’s current research program focuses on the direct reprogramming of human somatic cells to neural precursor cells using non-viral gene delivery approaches. She developed a reprogramming protocol using transient overexpression of the neural developmental genes SOX2 and PAX6 to generate induced neural precursor cells, and subsequently mature GABAergic, glutamateric or dopaminergic neurons from adult human skin. Her research group is using this technology to model neurological diseases such as Huntington’s disease, Parkinson’s disease and Alzheimer’s disease using skin cells from affected patients. The objective of this program is to enhance knowledge regarding the pathogenesis of these diseases and to identify new therapeutic targets. Future objectives include the use of these disease models for high throughput drug screening.
Eliminating daily feeding in a feeder-free, xeno-free PSC culture system
Thermo Fisher Scientific
Pluripotent stem cells (PSCs) are powerful tools for developmental biology, regenerative medicine, and the study of debilitating human diseases. While the development of feeder-free culture systems such as Essential 8 Medium has simplified and standardized routine PSC culture, additional hurdles continue to challenge today’s stem cell scientists. Prominent among these is the need for daily media exchanges to maintain healthy PSC cultures, a requirement largely driven by the loss of activity in critical media components at 37°C. Here, we introduce Essential 8 Flex Medium, a xeno-free PSC culture medium that eliminates the need for daily feeding without requiring very low seeding densities or other significant changes to current PSC culture protocols. Our data show that sensitive PSC medium components in Essential 8 Flex Medium experience virtually no loss of bioactivity for periods of up to 72 h at 37°C, thereby enabling PSC culture that fits into a standard five day workweek. Long-term, weekend-free culture experiments further demonstrate that Essential 8 Flex supports stable pluripotency marker expression, robust trilineage differentiation potential, and normal karyotypes through at least 30 passages. Taken together, Essential 8 Flex Medium allows for routine weekend-free maintenance and expansion of PSCs without negatively impacting culture quality and without substantial protocol adjustments.
Matt is currently a staff scientist in Cell Biology R&D within the Life Sciences Solutions Group at Thermo Fisher Scientific, formerly Life Technologies. He obtained his Ph.D. in Chemical and Biomolecular Engineering from the Whiting School of Engineering at Johns Hopkins University under the direction of Dr. Konstantinos Konstantopoulos. His graduate training, focused on the role of glycoproteins in pancreatic and colorectal cancer metastasis, also 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 Life Technologies Cell Culture Essentials group in 2012 and has since acted as the technical lead on various Cell Biology programs involving oncology, primary human tissue culture, and stem cell biology.
HOXA gene expression defines definitive fetal hematopoietic cells differentiated from human embryonic stem cells
Murdoch Children’s Research Institute
The production of definitive haematopoietic lineages from human embryonic stem cells (hESCs) remains a significant challenge. To dissect the requirements for definitive blood, we tracked haematopoiesis from hESCs using Green Fluorescent Protein (GFP) reporter lines targeting the haematopoietic isoform of RUNX1. Under conventional culture conditions in serum free medium, we found that GFP identified the subset of CD34+ cells that harbored clonogenic activity. These GFP+CD34+ progenitors also homed to the bone marrow, a prerequisite for haematopoietic stem cells, but did not engraft. We compared their transcriptome with cord blood CD34+ cells, discovering that hESC-derived progenitors lacked expression of HOXA genes, indicating that hematopoietic mesoderm, from which blood cells arise, was not patterned normally. Using HOXA expression as a guide, we found that a pulse of WNT activation and ACTIVIN antagonism (SB/CHIR) was sufficient to produce HOXA+ mesoderm from which endothelial and haematopoietic precursors derived. HOXA+ precursors maintained higher viability, formed complex hemogenic vascular structures and generated late fetal-liver like erythroid burst forming colonies in which embryonic globins were suppressed. RNA sequencing confirmed upregulated HOX gene expression in SB/CHIR treated cultures in haematopoietic, endothelial and stromal cell types. Our findings show that HOXA codes established early in differentiation predict cellular potential and highlight the importance of appropriate early cell patterning events.
Andrew Elefanty trained as a physician and completed a PhD in leukemogenesis at the Walter and Eliza Hall Institute of Medical Research. He subsequently worked on globin gene regulation with Prof Frank Grosveld at the National Institute for Medical Research in Mill Hill, London before returning to the Hall Institute to pursue interests in developmental haematopoiesis and the differentiation of embryonic stem cells. He moved to Monash University in 2002 to initiate studies with human embryonic stem cells. In July 2012, his laboratory relocated to the Murdoch Childrens Research Institute. His work has focused on human pluripotent stem cell differentiation, with a special interest in haematopoietic lineages. His laboratory wishes to generate cells to model blood diseases in vitro and for transplantation. The laboratories of Andrew Elefanty and Ed Stanley have generated genetically modified human stem cell lines in which lineage-specific fluorescent reporters allow monitoring of differentiation.
The laminin protein family – key to stem cell culture
Sales and Science Manager
Three major problems in stem cell research and cell therapy are due to challenges in growing cells. First, expansion of pluripotent stem cells has been considered difficult. Second, differentiating stem cells to specific phenotypes while correctly mimicking their extracellular matrix niche has not been possible. Third, keeping adult cells in their differentiated state in vitro for prolonged periods is basically impossible. With the help of the laminin protein family, we have solved all these problems. This presentation will focus on the novel BioLamina platform with cell-type specific human recombinant laminins that have unique physiological relevance of critical importance for stem cell culture. By successfully recreating specific in-vivo cell niches in the dish, the functionality and quality of cells are increased while cell cultivation problems and labor time is reduced.
Jesper Ericsson is an engineer by training and obtained his Master of Engineering degree from the Royal Institute of Technology in Stockholm, Sweden. He then continued to perform a PhD within basic neuroscience under the supervision of Professor Sten Grillner at the Karolinska Institute in Stockholm. Jesper studied the physiology of the neuronal microcircuit of the basal ganglia in the brain and how this region governs selection and action of movements. During his PhD he also worked part time at the Unit for Bioentrepreneurship at the Karolinska Institute on how to commercialize science based ideas. After defending his PhD thesis, Jesper joined BioLamina that a spinout biotech company from the Karolinska Institute in Sweden. BioLamina offers cell culture matrices that establish more physiological environments in vitro by growing cells on their biorelevant human recombinant laminins. Jesper currently works as a sales and science manager at BioLamina.
Scalable xeno-free culture system for human induced pluripotent stem cells
Institute for Bioengineering and Biosciences
Human iPSC culture using Essential 8 medium and vitronectin enables iPSC cultures under adherent monolayer conditions. Vitronectin supports the expansion of human iPSCs either in coated plates or on polystyrene-coated microcarriers, while maintaining cell functionality and pluripotency. The scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was carried out to identify the optimal conditions in terms of a) initial cell density and b) agitation speed, to achieve the highest cell yield in a spinner flask culture and c) to minimize the shear force. A maximum cell expansion of 3.5-fold was achieved by inoculating 55,000 cells/cm2 and using an agitation speed of 44 rpm, which yielded a final cell density of 1.4x106 cells/mL after 10 days of culture. At the end of the dynamic culture, cells maintained their typical morphology, pluripotency-associated marker expression as well as their tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid bodies. Microcarrier expanded iPSCs were further subjected to directed differentiation to neural lineages and cardiac lineages successfully. In conclusion, a limited xeno-free scalable culture system with Essential 8 was successfully developed for the large-scale production of human iPSCs. Beyond the scale of 50 mL spinner or aggregate cultures, the culture media would need modifications to achieve further high scalability such as bioreactor type of cultures.
Dr. Tiago G. Fernandes is currently a Post-doctoral research fellow at the Institute for Bioengineering and Biosciences, Lisbon, Portugal, a national center of excellence in biotechnology and bioengineering field. He received his PhD degree from Instituto Superior Técnico, Technical University of Lisbon, in 2009. His research interests are focused on the development of artificial cellular niches for studying the mechanisms that affect human stem cell pluripotency and differentiation. During his doctoral studies he also worked at the Rensselaer Polytechnic Institute, NY, USA, in the development of microscale platforms for high-throughput studies of stem cell fate. His work has earned him the prestigious Malcolm Lilly award at the 7th European Symposium on Biochemical Engineering Science (2008). He was also involved in the production of several book chapters and has been invited to write one book on the topic Stem Cell Bioprocessing. Dr. Fernandes is currently involved in several research projects financed by the Portuguese Foundation for Science and Technology, as well as by the European Community related to the development of different systems for pluripotent stem cell culture envisaging further applications in regenerative medicine and drug screening.
Efficient generation of footprint-free patient-specific iPS cells and the application for drug screening
Project Associate Professor of Ophthalmology
Keio University School of Medicine
Sendai virus (SeV) vectors are cytoplasmic replicating RNA virus vectors that carry no risk of altering host genome. We have previously reported that SeV vectors carrying the reprogramming factors were highly efficient solution for generating safer induced pluripotent stem cells (iPSCs) not evoking viral gene integration. In this presentation, the mechanism of ‘zero-footprint’ and application of the vectors to disease-specific iPS cells and drug screening will be shown.
Noemi Fusaki, Ph.D., is a Project Associate Professor of Keio University. She received her Ph.D. in Molecular Immunology from University of Tokyo in 1995. She began her independent research in signal transduction in the immune system as an assistant professor at Kansai Medical University and Science University of Tokyo, and then continued as an associate professor at Tokyo Medical and Dental University. Her research then switched to a more clinical direction after 2005, focusing on immune therapy of cancer at Keio University, and then to regenerative medicine at DNAVEC (present name: ID Pharma), a virus-vector company in Japan. She established highly efficient method for generating transgene-free induced pluripotent stem cells (iPSC) using non-integrating Sendai virus RNA vectors in 2009. She was also the PRESTO researcher supported by Japan Science and Technology Agency (JST) from 2009 to 2015. She moved to Keio University in 2013, and has been studying patient-specific iPSCs to analyze disease and find new drugs.
PSCs to cardiomyocytes in three steps
Senior Product Manager
Thermo Fisher Scientific
A simplified and reliable media system for generating cardiomyocytes from donor- or disease-specific human pluripotent stem cells (PSC) would provide a valuable source of cells for basic and translational research. Current protocols have led to heterogeneous results with varying purity and long lead times for generation of cardiomyocytes. As a result, we developed, tested and manufactured a GMP-grade culture media system that is scalable and can be used to generate large numbers of continuously maintained or cryopreserved cardiomyocytes.
Alex Hannay is a Senior Product Manager with Thermo Fisher Scientific’s Cell Biology Business. He has a Bachelor’s degree in Biology from Rutgers University and over 10 years’ experience in industry, holding positions in laboratory operations, technical services, and product management. Alex is responsible for development and commercialization of neural cell culture and stem cell differentiation products, including the B-27and Neurobasal line of products.
Combined stem cell transplantation and gene therapy on the regeneration of corticospinal axons after spinal cord injury
Research Associate Professor and Director of the Spinal Cord Repair Laboratory
University of Western Australia
The importance of corticospinal tract (CST) projections in fine manipulatory motor control has warranted the focus of many experimental repair strategies aimed at restoring function following spinal cord injury (SCI). Most studies in rodents have attempted this by delivery of purified neurotrophic growth factors and/or by cell transplantation using donor cells engineered to over-express the growth factors, the factors usually applied to the injury site itself. In such studies, functional improvements usually reflect sprouting and some plasticity in collateral and/or intraspinal pathways, rather than axonal regeneration per se. Here our focus is adeno-associated viral vector-mediated targeted expression of ciliary neurotrophic factor (CNTF) in the motor cortex and in corticospinal neurons (CSN) to enhance axonal plasticity and regeneration, and promote functional improvements following moderate thoracic contusion SCI (in line with our previous work). Additional experiments aimed to further improve morphological and functional outcomes by combining cortical gene therapy with our proven mesenchymal precursor stem cell (MPC) transplantation into the injury site. In our previous work, transplantation of human MPCs have been shown to markedly improved both functional and morphological outcomes after both acute and chronic moderate T10 SCI in our nude rat model. In the present studies, MPC transplantation is believed to provide an even better terrain for plasticity and regeneration of CST axons after targeted CNTF expression in motor cortex. Human CST projections are not completely homologous to the descending CST in rodents (which in these species projects mainly to the forelimbs), so our studies are now being extended into cervical hemisection and hemi-lateral contusion SCI to specifically assess CST recovery in forelimbs.
Stuart Hodgetts is currently Director of the Spinal Cord Repair Laboratory in the School of Anatomy, Physiology & Human Biology, at the University of Western Australia (UWA). He has extensive knowledge and expertise in cell based transplantation therapies and has been devoted to this research since 1998. Previously, he obtained his Ph.D. at the University of Essex, UK and has worked on nuclear matrix proteins involved in immunoglobulin gene transcription at the Oklahoma Medical Research Foundation, USA.
Highly efficient genome editing and cell engineering in stem cells using CRISPR/Cas9
Thermo Fisher Scientific
Senior R&D Manager, Synthetic Biology
Thermo Fisher Scientific
Advances in genome editing has empowered researchers with highly efficient and versatile gene editing tools like Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system thereby making it relatively easier to target user defined endogenous genes in a sequence specific manner. Stem cells have been a preferred platform for various applications including gene function analysis, drug screening disease modeling, and tissue engineering. Therefore novel tools that enable rapid and precise gene manipulation in stem cells are required. Presented here are CRISPR/Cas9 tools and workflows that allow accurate design and rapid synthesis of gRNA along with delivery of Cas9 protein/gRNA RNP complexes into a variety of cells through optimized transfection reagents or electroporation. Discussed here are the results from different CRISPR/Cas9 formats tested in stem cells. Using these formats we have edited mouse embryonic stem cells (ESCs) and human iPSCs with up to 80% to 60% genomic cleavage efficiencies, respectively. The methods described here facilitate efficient disease model generation thereby accelerating research in the field of gene therapy and regenerative medicine.
Shantanu Kumar is currently a Scientist III at Thermo Fisher Scientific. He received his Ph.D. in microbiology from the Tamil Nadu Agricultural University in Coimbatore, India in 2004. Since he has acted as a Research Associate at the Center for Cellular and Molecular Biology (CCMB) and The Scripps Research Institute as well as a Postdoctoral researcher at booth the Moores Cancer Center and the School of Medicine at the University of California San Diego. Shantanu's research background includes genome editing, iPSC generation and differentiation, mouse ESCs, virology, viral vecotrs and gene therapy, epigenetics, cancer biology, and cell signaling.
Namritha Ravinder is a Senior scientist and R and D manager at Thermo Fisher Scientific in Carlsbad, California. She leads product development activities within the Synthetic Biology and Sample Prep team with primary focus on building products, screening tools and workflows for genome editing and cell engineering applications. Prior to her current role she was a technical lead for a wide variety of Synthetic Biology custom service offerings including cDNA library generation; RNAi design and screening; High throughput sample prep, gene expression and miRNA profiling for biomarker discovery; lentivirus production; in vitro transcription and Next Generation sequencing. She did her Postdoctoral research at Children’s hospital in Los Angeles in HIV Virology and Doctoral research in Plant Molecular biology and Biotechnology at University of Alabama in Huntsville.
Tissue engineering of hiPSC for endothelial cell regeneration and cardiac repair
National Heart Centre Singapore
Human induced pluripotent stem cells (hiPSCs) have attracted great interest for therapeutic application because of their ability to generate a broad range of patient-matched, clinically relevant cell types. Cardiomyocytes (CMs), endothelial cells (ECs) and smooth muscle cells (SMCs) differentiated from hiPSCs could be useful for patients with heart disease for autologous cellular therapy, for disease modeling and for testing new drugs in vitro. Here, a new and remarkably more efficient EC differentiation protocol of hiPSCs that incorporates a three-dimensional (3D) fibrin scaffold is presented. hiPSCs are dissociated into single cells and seeded into 3D fibrin scaffolds made of fibrinogen and thrombin. hiPSCs are committed into mesodermal progenitor cells. Following that, cells are further induced into ECs. With this new protocol, the differentiated efficiency increased to 60±2.6% for PCBC16 cell line, and 73.7±8.5% (the highest was 83.3%) for GRiPSC cell line (both are hiPSC cell lines reprogrammed from dermal fibroblasts). Differentiated hiPSC-ECs have typical EC phenotypes: significantly up-regulated gene and protein expression levels of CD31, CD144 and von Willebrand factor-8. They have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. They have high gene expression levels of Nocth-1, Nocth-4, and EphrinB2, which suggests this differentiation protocol promotes arterial ECs differentiation from hiPSCs. The hiPSC-ECs continued to display EC characteristics for 4 weeks in vitro. The functional impact of tri-lineage cardiac cells (hiPSC-Ms, hiPSC-ECs, and hiPSC-SMCs) is studied in an immune-suppressed porcine heart model of ischemia reperfusion (I/R). hiPSC-ECs and hiPSC-SMCs were detected in vascular structures and hiPSC-CMs were found integrated into the myocardium and exhibited organized sarcomeric structure. Tri-lineage of cardiac cell transplantation resulted in significant improvements in infarct size, myocardial apoptosis, arteriole density, LV function, and cardiac metabolism without evidence for adverse effects. Large-scale label-free quantitative proteomics studies identified proteins that were associated with muscle contraction, embryo and muscle development and regeneration in pig heart after tri-lineage transplantation. This large animal model results encourage further development of hiPSCs for cardiac repair applications.
Dr. Ye was conferred his medical degree from Shanghai Medical University, China in 1996 and PhD from National University of Singapore in 2005. After completion of his PhD, Dr. Ye continued his research at National University of Singapore and became Principal Investigator in 2008. In 2010, Dr. Ye was appointed as an Assistant Professor at the University of Minnesota, USA. Dr. Ye returned to Singapore and joined the National Heart Research Institute Singapore/National Heart Centre Singapore In Nov. 2014, Dr Ye’s research interests are 1). human induced pluripotent stem cells (hiPSCs) therapy for cardiovascular regeneration and repair; 2). Tissue engineering for cardiac cell and tissue regeneration; and 3) Endothelial cell (EC) biology in cardiac diseases.
Dr. Ye developed protocols of cardiomyocytes (CMs) and ECs differentiation from hiPSC. The myocyte differentiation protocol yields a high percentage of CMs differentiated from hiPSCs that are reprogrammed not only from dermal fibroblasts, but also from blood mononuclear cells. A remarkably efficient EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold was developed. With this protocol, up to 75% of the differentiated hiPSCs assumed an EC phenotype. The purified ECs continue to display EC characteristics for 4 weeks in vitro.
Dr. Ye received numerous national as well as international awards during various prestigious scientific meetings, including European Society of Cardiovascular Surgery, European Society of Cardiology, and American Heart Association. Dr. Ye has more than 60 papers published in peer-reviewed journals, including Tissue Engineering, Biomaterials, Circulation Research, Circulation, and Cell Stem Cell.
Identification of key regulators of stem cell renewal and differentiation by high throughput functional screens
Principal Investigator, Genomic Research Center
My research interest is identifying key factors govern stem cell renewal and differentiation through gain-of-function and loss- of-function high throughput screens in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) that hopefully can benefit regenerative medicine or the treatment of bone related diseases. ESCs are different from other somatic cells in its immortalizing ability, pluripotency, and teratoma formation ability. To reveal the pathways that determine ESC renewal or differentiation, shRNA screens in performed with around 5200 shRNA, and we identified 143 candidate genes essential for both ESC expansion and pluripotency, 156 genes affect only cell number, and 9 genes block stem cell expansion and pluripotency. At least 12 genes were confirmed to promote or inhibit ESC renewal or pluripotency. Among these genes, non-metastatic cells 6 (Nme6) and non-metastatic cell 7 (Nme7) are essential for ESC self-renewal, oncogenesis, embryoid body formation, and the expression of eight stem cell key regulators such as Oct4, Nanog, Klf4, c-Myc, telomerase, Dnmt3B, Sox2 and ERas. In addition to Nme, by the high throughput screen, we found dual leucine-zipper bearing kinase (DLK), restrained by AKT activity, can inhibit ESCs renewal. In addition, miR200c promote ESC renewal and block ESC differentiation by the regulations of GATA4. These findings may help the applications of ESCs or induced pluripotent cells (iPSCs) in regenerative medicine. MSCs can differentiate into bone. Bone fracture which may cause by osteoporosis, cancer, or accident, takes a long time to recover. About 1/3 of women and 1/5 men have major fracture in the life time which includes 15% of hip fracture which has a 25% mortality rate and half of them need long term care in nursing home. Thus there is an urgent need to develop drug to promote the bone healing process and prevent and treat osteoporosis. By an unbiased high throughput screen with 12380 full length genes in primary human bone marrow MSCs, we identify at least nine novel proteins are both essential and sufficient for bone formation. Among them, MSC02, a soluble factor, can significantly enhance the osteogenesis of primary human MSCs (6-40 fold) and fully prevent and treat osteoporosis in mouse ovariectomy model. We hope our findings can enhance the efficiency of ESC/iPSC renewal and develop new treatment for bone diseases.
Dr. Lu is a PI at genomic research center of Academia Sinica, one of the best research institute in Taiwan. Dr. Lu is the co-PI of the national RNAi core. She visited Broad Institute to perform array-based shRNA screen in stem cells. Afterward, Dr. Lu setup shRNA screens in both mouse and human embryonic stem cells (ESCs), which pinpoint hundreds/dozens of activators or inhibitors of ESC pluripotency or expansion. Dr. Lu published the first shRNA screens of ESCs. Her worked was published in Stem Cells, Cell Cycle, and Current Protocols in Stem Cell Biology. In addition, with a high throughput screen with 12380 genes, at least 9 genes essential and sufficient for the osteogenesis of mesenchymal stem cells (MSCs) were revealed. One of the soluble factors, MSC02, can promote the bone formation upto 40 folds and can prevent osteoporosis in the mouse model. Part of these results were under revision of Nature communication and is published in BioScience Trends. Dr. Lu also first revealed the unexpected finding that GATA4, a key transcriptional factor to regulate endoderm, can regulate the ectoderm, endoderm, and mesoderm and is required for the embryoid body formation. And miR-200c can directly target GATA4 3’ UTR and translation. This paper was publish in Stem cell research. Dr. Lu joined Michael Snyder’s laboratory as a postdoc at Yale University. She initiated the hESC culture and performed a functional screen. She found signals important for renewal and developed a xeno-free defined medium for human ESC cultured. She published the paper in PNAS, which is the top downloading paper in PNAS. Dr. Lu obtained her master and Ph.D degree from National Taiwan University under the supervision of Dr. Ching Hwa Tsai and Dr. Jen Yang Chen. She studied the relationship of EBV and carcinoma and published three first author papers and one second author paper in Cancer Research, Journal of Biological Chemistry, and Journal of Virology.
Antibodies for stem cell research
Senior R&D Manager
Thermo Fisher Scientific
Kara Machleidt (formerly Kun Bi) is a Senior R&D Manager in the Antibodies and Immunoassays business unit at Thermo Fisher Scientific. She manages collaborations with academic labs in developing and validating antibodies for stem cell research. Prior to this role, she and her team in collaboration with the Parkinson’s Institute developed iPSC-based disease models for Parkinson’s disease. She also led the team developed and launched a large number of high-throughput cell-based assays for kinases and signaling pathways. She holds a PhD in Cell Biology from University of Texas Southwestern Medical Center and completed a postdoctoral fellowship at La Jolla Institute for Allergy and Immunology, where she studied T cell receptor signaling.
With the recent technological advances, diverse stem cell lines are derived and cultured under different conditions. Additionally, various differentiation protocols have been developed and optimized to generate mature functional differentiated cells from these stem cells. There is a huge need for reliable characterization methods to confirm the quality of the pluripotent stem cells and adult stem cells, as well as their differentiated derivatives. Current characterization practices consist of panels of assays primarily testing fundamental properties such as potency, analyzing the expression of key markers for cell identity, and detecting abnormalities that can affect cell behavior and safety. Antibody-based detection methods such as immunocytochemistry and flow cytometry are commonly used. High quality of the antibodies is one of the key factors contributing to the success and rapid progress of stem cell research. We offer a comprehensive library of primary antibodies for stem cell research. This presentation will provide an overview of stem cells and introduce some of our antibodies for the characterization of pluripotent stem cells, various adult stem cells such as mesenchymal stem cells and neural stem cells, and downstream functional mature cells.
Epigenetic regulation in stem cells and reprogramming
Professor of Stem Cell and Regenerative Biology and Principal Faculty
Harvard University / Harvard Stem Cell Institute
Human pluripotent stem cells (PSCs) can give rise to all cell types in the body and therefore hold enormous potential for tissue engineering and disease modeling. Here I will summarize our more recent advances in understanding the role of epigenetic mechanisms in regulating this unique cell state.
Dr. Meissner is a Professor of Stem Cell and Regenerative Biology at Harvard University, Principal Faculty at the Harvard Stem Cell Institute, and Senior Associate Member at the Broad Institute, which is a collaborative, multidisciplinary initiative of Harvard University and the Massachusetts Institute of Technology (MIT), and a Robertson Investigator at the New York Stem Cell Foundation. He received his degree in medical biotechnology at the Technological University of Berlin, and his PhD at MIT’s Whitehead Institute, where he also completed a postdoctoral fellowship. Dr. Meissner’s laboratory uses genomic tools to study stem cell biology with a particular focus on epigenetic reprogramming. His laboratory applies next generation sequencing technologies to study the epigenome in early development, stem cells and cancer. Dr. Meissner has received several awards including being named a 2009 Pew Scholar and 2012 Robertson Investigator. Support for his research includes funding from the National Institutes of Health’s and the NYSCF
Human oocyte as source of information to study cell pluripotency and reprogramming
Elena González Muñoz
Centro Andaluz de Nanomedicina y Biotecnología (BIONAND)
Reprogramming of somatic cells by transferring their nuclei into enucleated oocytes pioneered by John Gurdon and colleagues in the 1950s and followed by Blau et al. fusion experiments in the 80s revealed for the first time the remarkable plasticity of the differentiated state of the cell. The new factor based reprogramming approach described by Yamanaka and Thomson in 2007, to generate induced pluripotent stem cells (iPSCs), revolutionized stem cell and regenerative medicine field. Even several years after this milestone, a clear understanding of how this cellular reprogramming process takes place remains incomplete, and the relative low efficiency of the process constitute a challenge for the scientific community. Growing evidence suggests that reprogramming capacity of the mammalian metaphase II oocyte yield superior results. We hypothesized that by understanding the functions of genes present in the MII oocyte, we will be able to identify intra- and extracellular oocyte factors responsible for the oocyte’s reprogramming capacity that may also have a role in dedifferentiation events such as generation of iPSCS. Here we describe as the oocyte specific histone chaperone ASF1A is crucial for reprogramming process and pluripotency of the cells.
Elena Gonzalez-Muñoz is a researcher from the Program for Cell Therapy and Regenerative Medicine of Andalucia, Foundation “Progreso y Salud”, in Malaga, Spain. She has recently (2015) been awarded with “Ramon y Cajal” emergent PI Spanish grant. She studied Biology at the University of Sevilla and she did her PhD at the University of Barcelona (2002-2007). During her first international postdoc at University of California San Francisco she studied the role of asymmetric cell division of neuro stem and progenitor cells in the generation/suppression of brain tumors using mouse oligodendroglioma models and human brain tumors from the Neurosurgery department at USCF. In 2010, she moved to Michigan State University to work under Jose Cibelli supervision in his lab “Cellular Reprogramming Laboratory”. Here she focused her effort in the cellular reprogramming field, optimizing this reprogramming process from human somatic cell to pluripotent cells (iPSCs). In LARCEL (Andalusian Laboratory for Cell Reprogramming) (Malaga, Spain), now she works on deep analysis of human somatic cell reprogramming and pluripotency mechanisms and also applies all this knowledge about stem cells and iPSCs biology and technology to the other branch of the projects that can be called “Neurodegenerative disease modeling: disease in a dish”. To develop neurodegenerative disease models using pluripotent cells obtained directly from patient somatic cells. Using this technology she tries to understand the origin and cause of this kind of pathologies, that will allow us to find new efficient therapies against them.
Pluripotent stem cell culture systems: Identification of appropriate medium, matrix, and passaging reagent for your stem cell workflow
Senior Staff Scientist
Thermo Fisher Scientific
Pluripotent stem cells (PSCs) provide much promise in development of cellular models to understand the fundamental basis for disease, as well as providing tools to facilitate drug discovery and development of cellular therapies. While stem cells have a tremendous proliferative capacity, proper selection of medium, matrix, and associated passaging reagent is critical to ensure optimum survival and maintenance of pluripotency and trilineage potential of PSCs in long term culture. Here we will review the feeder-dependent and feeder-free culture systems available from Thermo Fisher Scientific and discuss the advantages and disadvantages of each system. In addition, we will provide helpful tips and tricks to assist in successful transition of feeder-dependent PSCs to feeder-free culture in Essential 8 Medium. Please join us for this informational seminar to help you in identifying the optimum PSC culture system for your experimental needs.
Dr. Newman joined Thermo Fisher Scientific in 2010 and is currently working on next generation stem cell culture and differentiation systems, enabling researchers to efficiently culture, expand, cryopreserve, and differentiate their stem cells to various cellular lineages. She received a Ph.D. at the University of Iowa from the lab of Dr. Madeline Shea. Subsequently, she completed postdoctoral training in the lab of Dr. Ken Prehoda at the University of Oregon, studying the role of intramolecular interactions in regulating cell signaling cascades in the process of asymmetric stem cell division.
Thermo Fisher Scientific’s cell therapy capabilities
Business Development Leader, Cell Therapy
Thermo Fisher Scientific
Thermo Fisher Scientific is a world leader in life sciences tools and technologies. We are constantly innovating and working to bring better technologies to support cutting edge science and the application of science in medicine. While cell therapies are moving towards commercialization, we are continuing to evolve our products, services and capabilities to better support the scientific, quality and regulatory demands of the industry. In this talk we will discuss the ways in which Thermo Fisher is using its resources to create simpler, more effective and efficient workflows in cell therapy application.
Brian Newsom joined Thermo Fisher Scientific (legacy Invitrogen/Life Technologies) in business development after having worked in the development of cell therapies for over 15 years. His most recent prior post was as the Head of Research and Development at Opexa Therapeutics where he led the development of a T cell vaccination for Multiple Sclerosis into a phase IIb clinical trial. Prior to Opexa, Brian spent five years at Baylor College of Medicine, Center for Cell and Gene Therapy developing gene therapy applications for pediatric oncology. Brian began his career in cell therapy at Aastrom Biosciences, one of the pioneering companies in the industry, in 1992. Brian received his degree in Microbiology from Texas Tech University and an MBA from Texas A&M University.Brian’s current role is to support the commercial development of cell therapy client companies and growing the breadth and impact of Thermo Fisher Scientific’s portfolio of products in relation to cell therapy research, development and industrialization. Brian works across all divisions of Thermo Fisher Scientific to help find solutions to pain points in cell therapy workflows and to help create new solutions where current offerings are not available.
Functional dissection of disease-associated chromosomal deletions with human iPSCs
Associate Professor, Department of Oncological Sciences
Icahn School of Medicine at Mount Sinai
Chromosomal deletions associated with human disease are increasingly being catalogued in normal and cancer genomes and may constitute an important component of the “missing heritability” of complex diseases and the “dark matter” of cancer genetics. Unlike balanced translocations or point mutations, chromosomal deletions are more difficult to study; physical mapping in primary patient material is limited by the rarity of informative cases and synteny issues complicate their modeling in the mouse. We harnessed cellular reprogramming and genome engineering to functionally dissect the loss of one copy of the long arm of chromosome 7 [del(7q)], a cytogenetic abnormality found in Myelodysplastic Syndromes (MDS). MDS are clonal hematologic disorders characterized by ineffective hematopoiesis and progression to acute leukemia. Chr7q deletions are typically very large, no consensus commonly deleted region (CDR) has been identified and the critically lost genes remain unknown. First, by taking advantage of the somatic nature of the deletion, we derived del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and characterized disease-relevant cellular phenotypes. We found that these are rescued by spontaneous dosage compensation and recapitulated by engineered (through Cre-loxP and CRISPR/Cas9 systems) hemizygosity of defined chr7q segments overlapping in a 20 Mb region. We thus provide the first definitive evidence that the del(7q) abnormality confers a profound loss of hematopoietic differentiation potential through a dosage effect and that the critical region lies within cytobands 7q32.3–7q36.1. Finally, we identify candidate haploinsufficient genes mediating the del(7q)- hematopoietic defect through a phenotype-rescue screen. Our study provides a new approach for the study of the phenotypic consequences of segmental large-scale chromosomal deletions and a novel application of human pluripotent stem cells as tools for reverse human genetics and gene discovery.
Eirini Papapetrou earned an MD and PhD from the University of Patras in Greece and did postdoctoral training at Memorial Sloan-Kettering Cancer Center, where she developed technologies for the derivation of patient-specific induced pluripotent stem cells (iPSCs) and contributed some of the earliest proof-of-principle studies for the use of iPSCs in disease modeling and regenerative medicine. She started her independent laboratory in 2012 at the University of Washington and joined Mount Sinai in NYC in 2014 as an Associate Professor. Research in the Papapetrou lab focuses on modeling malignant and pre-malignant myeloid disorders with human iPSCs and genome editing technologies. The lab has established the first iPSC models of Myelodysplastic Syndromes (MDS), which offer exciting new opportunities for studies into the disease mechanisms and for drug screens.
Toward large-scale functional genomic studies through the automation of induced pluripotent stem cell derivation, expansion and differentiation
Director of Automation Systems and Stem Cell Biology
New York Stem Cell Foundation
The use of induced pluripotent stem cells (iPSCs) have become an invaluable tool for modeling how cellular function is disrupted by genetic variants. Using such disease models we have gained an unprecedented understanding of, largely, highly penetrant mutations. However, polygenetic traits are stilly poorly understood and large gaps in understanding the biology behind variants identified in GWAS studies remain. The opportunity now exists to be able to interrogate the genotype:phenotype associations with large-scale in vitro studies using stem cells derived from individuals harboring these variants. Unfortunately, the preparation of somatic cells, the reprogramming process and subsequent differentiations are all laborious, manual processes that limit the scale and level of reproducibility of these experiments. We recently developed a robotic platform that enables iPSC reprogramming in a high-throughput manner requiring only minimal human intervention and demonstrating a significantly reduced variation in cell lines produced. Here we will describe this process as well as ongoing development of techniques to allow parallel differentiation of these cell lines, which will be required for downstream studies. This automated, high-throughput approach to stem cell biology allows for scalable experiments required for the application of iPSCs to population-scaled biomedical problems.
Daniel Paull is currently Director, Automation Systems and Stem Cell Biology at the New York Stem Cell Foundation. Daniel joined NYSCF in 2011, as a postdoctoral fellow in the lab of Dr. Dieter Egli where he worked across multiple fields including diabetes, somatic cell nuclear transfer and in the development of a novel technique for the prevention of mitochondrial disease inheritance. This technique has since gone on to be allowed in the United Kingdom following a law change in early 2015, with plans for human trials now underway. Upon the completion of his post-doctoral fellowship, Daniel joined the NYSCF Global Stem Cell Array team helping to develop the biological workflows that underlie its function. Daniel revived his PhD from University College London and his BSc from the University of Sheffield.
Optimizing retinal cell differentiation of human pluripotent stem cells for large-scale disease modeling
Associate Professor, Department of Ophthalmology
University of Melbourne
Human induced pluripotent stem cells (iPSCs) are valuable cell models for retinal disease modeling, as these cells are of patient origin and can be differentiated into cell types of interest. Here we discuss optimized protocols for the differentiation of human pluripotent stem cells into homogenous population of Retinal Pigment Epithelium (RPE) cells or Retinal Ganglion Cells (RGCs) that can be adapted to automation, hence allowing protocols for the generation of retinal cells on automated platforms for the large scale study of patient samples.
Associate Professor Pébay is an Australian Research Council Future Fellow. She obtained her PhD in Neurosciences from the University of Paris VI in 2001 and subsequently joined Professor Martin Pera at Monash University to undertake research on human embryonic stem cells (hESCs). She then continued her research in this area at the University of Melbourne where she commenced in 2007. Since 2012, Associate Professor Pébay has been appointed to both the Centre for Eye Research Australia and The University of Melbourne. More specifically, Associate Professor Pébay heads research projects on the identification of signaling mechanisms involved in the maintenance of pluripotency, neural differentiation and more recently cardiac and ocular differentiation of pluripotent stem cells and diseased iPSCs. Her long-term research goal is the establishment of well characterized and efficient protocols for maintenance and differentiation of pluripotent stem cells, suitable for drug screening.
Human pluripotent stem cells for antibody discovery
Professor and Chair of Stem Cell Sciences
University of Melbourne
Human pluripotent stem cells (hPSC) provide windows into stages of human development that have not previously been accessible to experimentation. Target populations of tissue progenitor cells derived from hPSC can be used to develop and screen monoclonal antibodies with interesting specificity. We describe the use of the hPSC platform in the development of a panel of antibodies reactive with a large cell surface glycoprotein complex on endodermal progenitor cells. Progenitor cells in liver and pancreas that express this antigen complex are rare in normal tissues, but expand in numbers during regeneration, repair, and neoplasia. This novel antigen complex is a potential serum biomarker for pancreatic ductal adenocarcinoma and liver cancer. The hPSC platform is a powerful tool for antibody discovery and development in oncology and other fields.
Martin Pera is Professor of Stem Cell Sciences at the University of Melbourne, the Florey Neuroscience Institute, and the Walter and Eliza Hall Institute for Medical Research. He serves as Program Leader for Stem Cells Australia, the Australian Research Council Special Research Initiative in Stem Cell Sciences. His research interests include the cell biology of human pluripotent stem cells, early human development, and germ cell tumours. Pera was among a small number of researchers who pioneered the isolation and characterisation of pluripotent stem cells from human germ cell tumours of the testis, work that provided an important framework for the development of human embryonic stem cells. His laboratory at Monash University was the second in the world to isolate embryonic stem cells from the human blastocyst, and the first to describe their differentiation into somatic cells in vitro. He has provided extensive advice to state, national and international regulatory authorities on the scientific background to human embryonic stem cell research.
Generation of induced pluripotent stem (iPS) cell-derived cardiomyocytes for disease modeling and drug discovery
Director, Scientific Partnerships
Stem Cell Theranostics
Induced pluripotent stem cell-derived cardiomyocytes (CMs) offer great promise in the study cardiac disorders for disease modeling, drug discovery and toxicity screening. While many research advancements have been made in the process of cardiomyocyte differentiation, current protocols still lack the ability to generate high purity CMs in a consistent, standardized and scalable manner. This webinar will focus on a new media system that enables researchers to generate CMs that display key functional properties for genetic cardiac diseases such as hypertrophic cardiomyopathy and dilated cardiomyopathy. Patient specific CMs can therefore serve as powerful models by recapitulating cardiac disease traits in vitro.
Dr. Nirupama Pike is the Director of Scientific Partnerships at Stem Cell Theranostics, Inc. She has over 12 years of experience in pluripotent stem cell research and has authored numerous publications, book chapters and patents. She has held the positions of Senior Stem Cell Scientist and Global Training Manager at Thermo Fisher Scientific and Director of Education and Outreach at WiCell and Morgridge Research Institutes at Madison Wisconsin. Dr. Pike holds a Ph.D. from Harvard University and a Masters degree from Loyola University. She has extensive experience in areas of human embryonic and induced pluripotent stem cells, cell-based assays for drug discovery, technology transfer and designing and executing training programs.
Variability of cardiomyocyte differentiation among human pluripotent stem cells: A practical screening approach
University of Florida Center for Cellular Reprogramming
Human pluripotent stem cells (hPSC) have the ability to differentiate into all tissues in the body. For this reason they are a valuable resource to model human diseases. While significant progress has been made in differentiation to various lineages, individual hPSC clones display significant variability to successfully generate lineages including cardiomyocytes. Numerous protocols for achieve cardiomyocyte differentiation have been generated. In this talk I will discuss one particular differentiation protocol and how it can be practically implemented across a number of hPSC lines. We utilize a screening approach to determine optimal single cell seeding density and confluency prior to cardiomyocyte induction. We have found these significantly impact overall purity of cardiomyocytes.
Dr. Santostefano received her PhD from the University of Florida, Gainesville, FL. During her PhD she studied basic biology of how mouse embryonic stem cell pluripotency is lost upon differentiation to definitive endoderm. Dr. Santostefano learned the techniques of human induced pluripotent stem cell reprogramming and culture in Japan in 2010. After completing her PhD, she stayed at the University of Florida to promote iPSC research in the institute. The Center for Cellular Reprogramming was founded in 2013 at the University and she now leads iPSC generation efforts to model human diseases.
Differentiation of midbrain floor plate progenitors and dopaminergic neurons from human pluripotent stem cells
Thermo Fisher Scientific
Midbrain dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) provide an excellent source for disease modeling and drug screening for Parkinson’s disease. During brain development, midbrain floor plate (mFP) is formed during 21-28 days of gestation along the ventral midline of developing neural tube and it has been shown that midbrain DA neurons are differentiated from mFP cells. Recent reports have focused on identifying the appropriate in vitro conditions to differentiate hPSCs to properly regionalized floor plate precursors, rather than a more general neural stem cell population, in order to create authentic DA neurons. However, published protocols are quite lengthy and complicated leading to increased variability in differentiation efficiencies. Also, few reports describe whether specified progenitors can be expanded and cryopreserved. Our objective is to develop a culture media system designed to simplify and standardize this process while compressing timelines and adding increased flexibility in this complex differentiation workflow. Here we describe our results which have broken the process down into 3 distinct steps: (1) specification of hPSC to midbrain floor plate (mFP) cells, (2) expansion and cryopreservation of derived mFP cells, and (3) maturation to DA neurons. Characterization of floor plate cells and mature DA neurons was performed by immunostaining for the presence of specific markers including Lmx1, Otx2, FoxA2 and TH, additional qPCR analysis included expanded lists of genes to help define these cell populations. Electrophysiological characteristics of differentiated neurons were assessed by Multi-electrode array and spontaneous and depolarization induced dopamine release was measured with HPLC. In comparison to published protocols, our new system has several advantages including ease of use, significant expansion and preservation of progenitors in relatively short culture duration. This efficient system will benefit researchers with increased scale and flexibility in targeted studies.
Currently, Soojung serves as a Senior R&D Scientist in the Primary and Stem Cell unit in the Cell Biology group within Thermo Fisher Scientific. Soojung has an undergraduate and MS at Seoul National University and holds a Ph.D. in regenerative medicine from the University of Georgia, with post-doctoral work at NIA/NIH.
Pluripotent stem cell characterization methods
Thermo Fisher Scientific
Pluripotent stem cells (PSCs) have the ability to self-renew and, with the right cue, differentiate into a wide variety of cell types representative of the three germ layers. This makes PSCs like embryonic stem cells (ESCs), powerful tools for drug screening and cell therapy, more so with the development of reprogramming technologies that enable the creation of induced pluripotent stem cells (iPSCs) from donor-derived somatic cells. With all of the recent technological advances, iPSCs can now be derived from various somatic cells using different reprogramming methods and can be cultured with different media and matrices. As diverse PSC lines are derived and cultured under different conditions, there is a need for reliable characterization methods to confirm the quality of the PSCs. Current PSC characterization practices consist of a panel of assays primarily testing functional pluripotency and detecting abnormalities that can affect cell behavior and safety. Here we describe the basic and common PSC characterization practices in the context of reprogramming and the derivation of a new iPSC line.
Deborah Tieberg is a product manager in the Cell Biology business unit (formerly Primary & Stem Cell Systems), responsible for the reprogramming, culture matrices and characterization portfolios. Previously she developed products for the DAS (Discovery and ADME/Tox Solutions) portfolio. Prior to joining Life Technologies, Ms. Tieberg performed vaccine research at the University of Minnesota and Wyeth. Ms. Tieberg graduated from the University of Wisconsin-Madison with a B.S. in Chemical Engineering and a Masters of Business Administration. She also holds an M.S. in Microbial Engineering from the University of Minnesota.
Standardized generation of patient-specific iPSC lines and scalable production of PSC-derived cardiomyocytes
Project Manager and Development Scientist
Centre of Commercialization of Regenerative Medicine (CCRM)
CCRM is a translational centre based in Toronto, Canada, focused on the development and commercialization of regenerative medicine and cell therapy technologies. An early achievement was the establishment of an iPSC production facility focused on generating high quality pluripotent cell lines from patient samples for academic researchers and clinicians. Fully operational for three years, CCRM has delivered over 75 patient iPSC lines that are being used for disease modelling, and in drug screening initiatives, at Institutes across Canada. Specializing in non-integrative reprogramming technologies, CCRM has developed SOPs to reprogram many common cell types in feeder-free conditions, including dermal fibroblasts, bone marrow stromal cells, cord and peripheral blood, and endothelial cells. Considering that many iPSC- or hESC- based cell therapies or drug-screening programs will require routine expansion and differentiation of billions of cells, another core focus has been to translate cultivation of pluripotent cells from static, adherent culture to an agitated suspension system. Suspension expanded pluripotent cells can then be differentiated in the same scalable culture system in which we have demonstrated production of over 1 billion cardiomyocytes in a 500 mL volume. CCRM’s goal is to further refine methods to produce an array of PSC-derived differentiated cells in closed-system automated stirred tank bioreactors in a robust and cost-effective manner. These methods represent opportunities to translate CCRM technologies to GMP facilities (including a facility operated by CCRM and anticipated to open in 2017) for manufacture of cell therapy products.
Emily is Manager, Technology in the Product and Process Development labs at CCRM, with a focus on cell reprogramming and genome engineering. She obtained her PhD from the Institute of Biomaterials and Biomedical Engineering at the University of Toronto, where she used a combination of laboratory and bioinformatics approaches to define and interpret gene regulatory networks controlling embryonic stem cell fate decisions. At CCRM, Emily develops and evaluates technology related to iPSC derivation, directed differentiation and genome engineering, and works on projects that advance the commercialization of stem cells.
Improved T cell function and in vivo engraftment of CAR-T cells expanded ex vivo with CTS Immune Cell SR
Senior Staff Scientist
Thermo Fisher Scientific
If you use serum in your T cell research, chances are you evaluate it in advance of your experiments to ensure lot-to-lot consistency. And, if you have plans to scale-up your process commercially, you may wonder how you’ll be able to secure enough supply for the long-term. In this session we share primary T cell data obtained ex vivo and in vivo that compares the performance of human AB serum to a xeno-free, defined serum-replacement supplement. Experiments we will discuss include:
- CAR-engineered T cell expansion
- CAR lentiviral transduction efficiency
- CAR T cell efficacy in a mouse model of leukemia
Our findings support a strategy to substitute human AB serum with a xeno-free supplement (CTS Immune Cell SR) in T cell experimental protocols without compromising performance, while also reducing concerns of experimental variability and process scalability. Because this new supplement meets USP 1043 Requirements for Ancillary Materials for Cell, Gene and Tissue Engineered Products and is manufactured with a scalable cGMP process to ISO13485 standards, the implication of these results is that scale-up and risk-mitigation could become lesser concerns for our fast-growing immunotherapy industry.
Angel is technical lead for research and development of cell culture media for cellular therapies and viral vaccine production. Before joining Gibco R&D in 2011, Angel had an academic R&D career. He received a B.S. in Biological Sciences at the University of Puerto Rico and a Ph.D. in Immunology from the University of Pennsylvania; followed by post-doctoral fellowships at UPenn and the University of Michigan. His background includes research and development of T cell-based immunotherapies for cancer and infectious diseases as well as recombinant vaccines and viral vectors for gene therapy.
In vitro quantification of the terminal differentiation potential of pluripotent stem cells
Group Leader, Stem Cell Engineering Group
University of Queensland
The efficiency of human pluripotent stem cells (hPSC) to differentiate into cells representative of the three germlayers can vary greatly between lines. Quantitative assays to functionally assess differentiation bias and the quality of hPSC are highly desirable, particularly for cell lines destined for clinical applications. Currently, the terminal differentiation ability and tumorigenicity of human stem cells is assessed by the in vivo teratoma assay where undifferentiated hESCs or hiPSCs are injected into immune-deficient mice, or inferred from gene expression data. Although teratomas can qualitatively assess the lineage specific differentiation ability of a pluripotent stem cell line it is not quantitative. Here we report on the development and validation of a simple, cost-effective, high-through-put quasi-teratoma assay that quantitatively assesses the pluripotency and tumorigenic propensity of hESC and iPSC lines in vitro. We show that this assay can distinguish lineage differentiation bias amongst human and mouse ESC and iPSC and constitutes a cost effective, standardized, animal ethics unencumbered platform for quantification of the terminal differentiation potential of pluripotent stem cells.
Prof Wolvetang obtained his PhD from the University of Amsterdam, where he investigated metabolite transport in peroxisomes. After investigating the role of chromosome 21 transcription factors in Down syndrome during his post-doctoral studies he joined the laboratory of Prof. Martin Pera to help pioneer human embryonic stem cell research. In 2009 He took up a group leader position at the Australian Institute for Bioengineering and Nanotechnology at the University of Queensland to interface with emerging microfluidic, nano- and “smart” surface-technologies at the Institute. His laboratory employs iPSC as in vitro disease models and uses CRISPR genome editing tools to interrogate the underlying gene regulatory networks and epigenetic bases of in particular complex neurological diseases. He leads “Cell reprogramming Australia” and is a principal investigator in the ARC Centre of Excellence “Stem Cells Australia”.