6th annual 24 Hours of Stem Cells™ virtual event

The Neon transfection system enables robust transfection efficiencies in hiPSC for DNA, mRNA and Cas9/gRNA RNP complexes. For hiPSC these improved transfection as well as post-electroporation culture conditions are essential for increased cell survival for downstream applications. This live training will review protocols involving Neon electroporation of hiPSC and post electroporation culture, using delivery of CRISPR/Cas9-mediated genome editing tools as the example.

Neuronal migration defects, including pachygyria, are among the most severe developmental brain defects in humans. Using human genetics approaches, we recently identified bi-allelic truncating mutations in CTNNA2, encoding αN-catenin, in patients with a phenotypically distinct recessive form of pachygyria. Interestingly, two mouse lines harboring nonsense mutations of the ortholog to human CTNNA2 (Catna2) have been characterized, but failed to recapitulate the human cortical lamination phenotype observed in our patients. Therefore, to investigate neuronal phenotypes in a human model, we developed stem cell-derived neuronal assays to assess neuron morphology as well as define the molecular disease mechanism. I will highlight the need for human cell-based models for neocortical gyration disorders as well as discuss the advantages of using stem cell based systems to discover pathogenic mechanisms and novel insight into human brain development, with this case as an example.

Generation of robust iPSC-derived neural stem cell cultures and neurons with shorter maturation times provide opportunities for quick and reliable generation of neuronal in vitro models. This live training will review protocols involving neural induction of hiPSCs into Neural Stem Cells (NSCs) and subsequently passaging of NSCs to plate for maturation into Neurons.

The complexity of the human brain, with thousands of neuronal types, permits the development of sophisticated behavioral repertoires, such as language, tool use, self-awareness, symbolic thought, cultural learning and consciousness. Understanding what produces neuronal diversification during brain development has been a longstanding challenge for neuroscientists and may bring insights into the evolution of human cognition. Human pluripotent stem cells have the ability to differentiate in specialized cell types, such as neurons and glia. Moreover, induced pluripotent stem cells can be achieved from living individuals by reprogramming somatic cells that would capture their entire genome in a pluripotent state. From these pluripotent state, it is possible to generate models of the human brain, such as brain organoids. We have been using brain-model technology (BMT) to gain insights on several biological processes, such as human neurodevelopment and evolution. We also applied BMT to measure the impact of genetic variants in autism spectrum disorders and for evolutionary studies. The reconstruction of human synchronized network activity in a dish can help to understand how neural network oscillations might contribute to the social brain. Our findings suggest a potential bridge to the gap between the microscale in vitro neural networks electrophysiology and non-invasive electroencephalogram.

Watch how to passage feeder-free Pluripotent Stem Cells (PSCs) cultured in Essential 8 medium using EDTA. Learn the best practices to help ensure successful passage of your cells.

CRISPR ribonucleoproteins (RNPs) can generate programmable gene edits, however imprecise editing and efficient delivery to human stem cells are key challenges. Here we describe novel biochemical techniques to assemble various biomolecules and coatings with nanoscale precision around a RNP. First, by modifying the sgRNA with a short S1m RNA aptamer, we developed a modular strategy, termed an “S1mplex,” to assemble Cas9 RNPs with biotinylated moieties. Using S1mplexes with biotinylated short oligonucleotides improves the ratio of precise to imprecise editing up to 18-fold over conventional methods approaching a ratio of 4 precise edits to every imprecise mutation, while assembly with fluorescent molecules allows selection and enrichment for cells with multiplexed gene edits. Second, we developed synthetic coatings for nonviral delivery of RNPs to human pluripotent stem cells. Combined, these strategies, which utilize chemically-defined, off-the-shelf reagents, have significant promise for gene editing applications in vitro (e.g., drug discovery, disease modeling) with human stem cells.

The use of human pluripotent stem cells (hPSCs) for in vitro disease modeling is limited by the lack of robust and efficient protocols for the differentiation of relevant adult cell types. Previously, we have reported a method to generate vascular endothelial cells from hPSCs (Patsch et al. Nat Cell Biol. 2015). This novel and robust protocol in conjunction with use of programmed nucleases allows generation of relevant endothelilal cell disease models. We will show examples of modeling high-barrier resistance endothelial cells in vitro, by generation of CLDN5 reporter and use of chemical profiling, and modeling the effect of a severe metabolic genetic disease (AKT2 loss-of-function or dominant active mutation) on endothelial cells.

Picking stem cells, whether it be for clone selection or general line maintenance, is an important and useful technique with which stem cell researchers should be comfortable.

Development of physiologically relevant cellular models, with strong translatability to human pathophysiology, is critical for identification and validation of novel therapeutic targets. Cell types derived from induced pluripotent stem cells (iPSCs) provide an important resource for modelling native biology in human cells and tissues for drug discovery.  Understanding the phenotypic and functional relevance of these iPSC derived cells and tissues and how well they represent human biology is vital and as such we have developed tool iPSC-lines and techniques to optimize and monitor differentiation. Applying CRISPR/Cas9, Next Generation Sequencing (NGS), functional testing and advanced imaging techniques to iPSC model validation, we have established a platform that enables an accurate assessment of their benefits, maturity, gaps and challenges, as well as application in a drug discovery setting. Data presented will be from a key kidney model system we have developed to examplify our platforms.  This will describe the validation and application of our kidney model, using SIX2 and NPHS1 markers non-invasively in real-time, to trace early nephron commitment and podocyte-maturation in the same cell. In adition, we will also describe how we use transcriptomic analysis by NGS, in addition to functional assays, to get the most from our iPS derived 3D cell models.

In contrast to autologous cell therapies, allogeneic therapies do not require collection of starting material from patients and could: be manufactured in larger batches, be made available “off-the-shelf”, and potentially utilise established biopharma manufacturing technologies. Despite this, allogeneic therapies constitute half of pre-clinical trials and a third of clinical trials in the UK. In an addition to the potential for an immune response, another major barrier to the adoption of allogeneic therapies is current manufacturing cost which increases when using pluripotent stem cells (PSC) as starting material. The manufacturing process is often open and manual, utilising 2D, adherent culture systems and xeno or serum containing materials. As such, these processes are regulatorily challenging and impractical to scale-up to meet market demands. This talk will cover our work to date and future plans in addressing these barriers and realising the potential of PSC-derived allogeneic therapies.

The Cell and Gene Therapy (CGT) Catapult have been working to address some of the challenges of manufacturing processes for PSC derived therapies, towards improving commercial viability. We used regulatorily amenable starting materials, developed invasive and non-invasive methods for process characterisation, developed exemplar processes for the expansion of pluripotent aggregates in stirred tank reactors, and developed concentration and wash processes for large-scale PSC processing. CGT Catapult’s next development focuses are the scale-up and intensification of PSC expansion in stirred tank reactors, translation of differentiation processes to automated and scalable culture systems, and cost of goods reduction.

Human organoid systems recapitulate in vivo organ architecture, yet fail to capture complex pathologies such as inflammation and fibrosis. Here we developed multi-cellular human liver organoids (HLO) from 11 different healthy and diseased PSC lines that display essential features of steatohepatitis. Single-cell-level analysis revealed steatohepatitis organoids exhibited persistent hepatic steatosis, followed by progressive activation of pro-inflammatory and fibrotic lineages. Interestingly, expression of the steatohepatitis phenotype correlates with the presence of clinically significant GWAS factors: PNPLA3, GCKR and TM6SF2. We developed an organoid-level readout with atomic force microscopy, and demonstrated that organoid stiffening reflects the fibrosis severity. Furthermore, organoid model of an iatrogenic parental nutrition associated liver disease (PNALD) identified obeticholic acid as effective in preventing pathology progression, suggesting the potential repurposing for PNALD. The presented key methodology and preliminary results offer a new method for studying human precision for inflammation and fibrosis, facilitating the discovery of effective treatments.

Pancreatic β cell replacement therapy is considered as a potential strategy to treat diabetes. To date, transplantation of pancreatic islets from cadavers is the most effective approach for treating diabetic patients, but this approach has limitations in terms of the necessity of strong immunosuppressive drugs and the shortage of donors. Alternatively, human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and hiPSCs, can be differentiated into insulin-secreting β cells that have a great potential to treat diabetes. However, generation of functional β cells similar to those present naturally in the pancreas is, so far, a work in progress. Indeed, few studies reported the generation of functional β cells from hPSCs, but those cells were low in efficiency and functionality, creating a major obstacle to the use of these cells for cellular therapy. Recent progress showed that patients with diabetes could be transplanted with hPSC-derived pancreatic progenitors co-expressing the two transcription factors (TFs), PDX1 and NKX6.1. Those progenitors are known as the precursors of functional pancreatic β cells.; therefore, after their transplantation, they are converted into functional insulin-secreting β cells inside patient’s body. Recently, we have established an efficient protocol for maximizing generation of PDX1+/NKX6.1+ pancreatic progenitors from hPSCs. We enhanced the generation of PDX1+/NKX6.1+ population, by manipulating in vitro culture conditions. Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (~90%) and upregulation of the key TFs controlling pancreatic β cell development. The improved efficiency of pancreatic differentiation was associated with an inhibition in the hepatic lineage and an increase in the proliferation of NKX6.1+ cells. Further differentiation validated the ability of PDX1+/NKX6.1+ progenitors to generate endocrine progenitors (NGN3+ cells). In addition, we were also able to enrich a novel PDX1-/NKX6.1+ population by manipulating the re-plating densities, that appeared in the form of three-dimensional clusters. Our findings provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ pancreatic progenitors with an increased self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also supports the presence of two distinct NKX6.1+ pancreatic populations during human pancreatic development, which may suggest a new trajectory to generate β cells in humans. In this talk, I will present our recently published work on generating distinct pancreatic progenitor populations from hPSCs.            

Delicate cultures including stem cells and primary cells are increasingly used in cell therapy research and development for applications in oncology, immunology, neurology and more. Commonly, these cells require daily manipulation, potentially exposing them to contamination. Such cells are also uniquely reactive to environmental variation. To address these challenges, we developed the Thermo Scientific™ Cell Locker™ System, a new CO₂ incubator design which includes six individual transparent Cell Locker chambers. Tests show the Cell Locker System provides enhanced stability, contamination control and flexibility for isolation of labile cell types or sensitive projects in a shared incubator. Each Cell Locker chamber features dual 0.2 µm membrane filters which allow air and humidity exchange but prevent transmission of microorganisms including mycoplasmas.

Functional studies show that when one Cell Locker chamber is opened, remaining chambers in the Cell Locker System provide environmental stability for temperature, humidity, and CO₂/O₂ atmosphere, maintaining ideal conditions not possible in a standard incubator. Gas use and media evaporation were both reduced by 50%. Multiple clones of induced pluripotent stem cells from different donors were evaluated and found to be indistinguishable from control incubators where conditions and manipulations had previously been optimized, showing that the Cell Locker System design and materials facilitated optimal cell growth. The Cell Locker System represents a novel approach for culturing stem cells and other sensitive cultures used in cell therapy applications.

Induced pluripotent stem (iPS) cells are a promising source of personalized therapy. These cells can provide immune-compatible autologous replacement tissue for the treatment of potentially all degenerative diseases. We are preparing a phase I clinical trial using iPS cell derived ocular tissue to treat age-related macular degeneration (AMD), one of the leading blinding diseases in the US. AMD is caused by the progressive degeneration of retinal pigment epithelium (RPE), a monolayer tissue that maintains vision by maintaining photoreceptor function and survival. Combining developmental biology with tissue engineering we have developed clinical-grade iPS cell derived RPE-patch on a biodegradable scaffold. This patch performs key RPE functions like phagocytosis of photoreceptor outer segments, ability to transport water from apical to basal side, and the ability to secrete cytokines in a polarized fashion. We confirmed the safety and efficacy of this replacement patch in animal models as part of a Phase I Investigational New Drug (IND)-application. Approval of this IND application will lead to transplantation of autologous iPS cell derived RPE-patch in patients with the advanced stage of AMD. Success of NEI autologous cell therapy project will help leverage other iPS cell-based trials making personalized cell therapy a common medical practice.                                                             

I describe several strategies for writing successful grant proposals.  Each strategy derives from understanding the implications of two basic facts: funders seek to advance their missions through grants, and proposals are offers to sell research projects to funders.  I outline the steps for ensuring proposal content and presentation fit a grant opportunity well.  I also highlight logistic and bureaucratic matters, ways to build skill in developing and writing grant proposals, and sources of feedback and help. 

Thermo Fisher Scientific has developed an advanced assay to qualify FBS for use with ESCs.  This assay is designed to sustain undifferentiated ES cells while maintaining karyotype integrity, LIF responsiveness and pluripotency markers.  ES Cell FBS Qualified, has proven applications in reprogramming, pluripotency marker maintenance, and PSC morphology maintenance.  When compared to competing products, it shows superior performance with regard to morphology and maintenance of pluripotency. The stringency of this assay provides researchers with the highest quality FBS for use in ES cell culture.

This presentation provides a brief introduction to biotech entrepreneurship including factors that are pertinent to biotech entrepreneurship and different myths and facts that exist. You will also learn about factors that many investors consider and find attractive for potential investments specifically in biotech. Finally, we will review what to consider after the startup and into the future.

One would think that getting the diagnosis after fifteen years would end the journey. How does your work at the bench affect patients and how can you change lives through cell therapy?  This talk highlights the power of precision medicine with the story of the ADCY5 gene variant and the journey of a family who went from being n of 1 to having fifty stem cell lines. The mother of a patient shares her perspective and determination to find a diagnosis and advancements in science and technology that will help hasten the path towards treatment.

The isolation of human embryonic stem cells (hESCs) and the discovery of human induced pluripotent stem cell (hiPSC) reprogramming have sparked a renaissance in stem cell biology, in vitro disease modeling, and drug discovery. In general, hPSC-based disease models are well-suited to study genetic variation. Studies commonly compare patient-derived hiPSCs, e.g., with a disease-causing genetic mutation, and (age-matched) control subject-derived hiPSCs, typically differentiated to the disease-affected cell type, e.g., neurons. A major caveat of this disease-modeling strategy is the variability of differentiation propensities and phenotypic characteristics, even in hPSCs derived from the same donor. Still, even if the cellular phenotype of a given mutation is strong and highly penetrant, it may be lost due to confounding effects of differences in genetic background of unrelated hPSC lines. A very powerful approach to overcome this hurdle is to use custom-engineered endonucleases, such as CRISPR/Cas9 that enable precise and programmable modification of endogenous hPSC genomic sequences. In our lab we use hPSC-based disease modeling to study the neurological movement disorder dystonia, in particular X-linked Dystonia Parkinsonism (XDP). In this talk I will show how we use hPSC-based disease modeling in combination with CRISPR/Cas gene editing, to elucidate the underlying molecular pathogenesis of XDP. I will also address some of the potential problems one might face using hPSC-based disease modeling in combination with gene editing.  

Human iPSC-derived neurons have increasingly become a valuable system for the study of neurological disorders.   Robust cell reprogramming and improved differentiation protocols enable scientists to generate patient-specific, disease in a dish models for disorders such as Parkinson's, Alzheimer's, and Autism, among others.  These human models tend to be flexible, scalable and maintain many of the characteristics of found in these disorders, which are key requirements for their use in mechanistic and drug discovery studies.  Further, the development of gene editing technology has spawned intense interest in the use of gene-edited, patient-specific iPSC-derived neurons in cell therapy applications for the treatment of neuro-degenerative disorders.

A critical step in generating useful iPSC-derived neurons is neuronal maturation.  During maturation neurons extend neurites to form highly connected networks, express synaptic markers, and become electrically active.  Typical maturation conditions are inefficient,  generating poorly matured neurons with low levels of functionality over extended periods of time.  Recently we developed a new neuronal neuronal maturation and maintenance system, (B-27TM Plus and NeurobasalTM Plus) and showed significantly improved neuronal survival, maturation, and functionality of primary rodent neurons compared to other culture systems.

Here we expand our studies to PSC-derived neurons, utilizing multiple human lines PSC and different approaches for neural stem cell derivation.  Diverse endpoints were used to interrogate maturation; neurite outgrowth, neuronal maturation marker expression (through quantitative imaging), and functionality through Multi-Electrode Array (MEA) analysis.  We found that human PSC-derived neurons matured in the new “Plus” system showed both accelerated neurite outgrowth and improved activity as compared to other approaches.   Additionally our studies highlight the importance of optimizing several key parameters, including extracellular matrix coating concentrations and delivery conditions for improved reproducibility and quality of stem cell derived neural cultures.

Learning how to leverage your network and enhance your current reputation can boost your career Development path. This workshop is designed to provide tools and strategies to enhance your personal brand.

Pluripotent stem cells can reproduce indefinitely and develop into any cell type in the body.  We are putting pluripotent stem cells to use, for a neuron replacement therapy for Parkinson’s disease, treatment of multiple sclerosis, understanding autism, and rescuing endangered species.

The capacity to generate disease-relevant cell populations from human pluripotent stem cells has tremendous potential for shedding light on human disease mechanisms. I will discuss basic principles of in vitro disease modelling, including the generation of isogenic models with CRISPR/Cas9, the issue of recurrent culture-acquired mutations and how to assess stem cell genomic integrity, and cell type maturity and phenotyping strategies. To frame these issues, I will discuss my research group's interest in obesity, which is largely a disease of excess food intake, which is in turn regulated by neurons in the hypothalamus. We use human stem cell-derived hypothalamic neurons to gain insight into the environmental and genetic factors that alter neuron function.

My talk shall encompass how the stem cell research field has evolved from embryonic stem cells to adult stem cells and currently induced pluripotent stem cells with special reference to the field of neuroscience in a bench to bedside approach. Studying neurological disorders has always been a challenge.  This is compounded by the lack of predictive pathophysiological models to identify and test potential therapeutic targets. Adult stem cells being multipotent and non-teratogenic have been in the limelight of stem cell clinical research. Multiple clinical trials have been conducted across the globe with limited inconsistent results. As we progress to understand how stem cells possibly orchestrate the regeneration process, it has opened up a whole new avenue of questions which I shall elaborate during the course of my talk. While, adult stem cells continue to be the focus of clinical trials at the moment, it is essential to mention that the discovery of induced pluripotent stem cells, has made the field even more exciting. Along with the technological advancements in sequencing and gene manipulation techniques, personalized medicine seems to be the future. Reconstruction of the early developmental stages is now possible by deriving induced human pluripotent stem cells from patient samples using advanced molecular and cellular manipulation methods. For eg: We, have been successful in reprogramming and differentiating human patient derived iPSC into the three common brain cell types- neurons, glial cells and oligodendrocytes of cortical origin, importantly using pathways and trophic factors that can sequentially recapitulate human neural development. This is imperative for accurate understanding of the disease and enhancement of therapeutic strategies.   Upon marker analysis and functional characterization, human iPSCs are able to consistently generate cortical neurons which are electrophysiologically active having intrinsic electrical properties comparable to adult human neurons and capable of generating mature action potentials upon stimulation. However, lack of synaptic activity lead us to further explore an astrocyte -neuronal co-culture system which closely mimics the internal milieu of the brain and responds to drugs. This platform is currently being used to understand pathophysiology of various neurological, neurodevelopmental and neurodegenerative disorders. Importantly, it opens up unlimited access to human tissue material in an unprecedented manner, which can be used for drug testing, neurotoxicity studies, epigenetic changes, gene editing studies and personalized medicine.

The use of pluripotent stem cells is dramatically altering the R&D landscape, providing new insights into both the basic biology of disease progression and novel cell types for pharmaceutical drug screening.  Additionally, pluripotent stem cells are already in clinical use for a number of disease indications. However, one limitation of the work to date is that in-vitro studies have largely focused on highly penetrant point mutations known to directly lead to a disease phenotype. While useful, this approach may not always be applicable to idiopathic versions of the same disease. For the majority of diseases, underwritten by common causal variants, modeling using only a limited number of cell lines is insufficient. To overcome these issues, we have developed a fully automated platform which allows high throughput cell biology to be accomplished: from reprogramming of hundreds of cell line in parallel to high throughput differentiations, large scale experiments can now be performed to interrogate these diseases. Here we report on progress in adapting the NYSCF Global Stem Cell Array technology to encompass high throughput genome editing as well as large-scale differentiations and screens. We will present a number of case studies highlighting the continued development of this automated system for stem cell biology.

For more than four decades, restrictions on research with psychoactive drugs have slowed progress in understanding how such substances impact brain metabolism. Besides the historical restrictions, the impacts of drug exposure in human neural cells have been compromised by limitations of adequate models. I will present the effects of the β-carboline alkaloid harmine, component of the psychoactive plant tea known as “Ayahuasca”, and 5-MeO-DMT (5-methoxy-N,N-dimethyltryptamine), found in the Sonora Desert toad, in cultures of human neural cells and brain organoids derived from induced pluripotent stem cells. Harmine increased the pool of proliferating cells, with DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase) as a target, which suggests a biological activity possibly associated with the antidepressant effects of Ayahuasca in patients with depressive disorder. Analyzing global protein expression of brain organoids exposed to 5-MeO-DMT, we found proteins broadly distributed on functional activities such as cellular protrusion formation, microtubule dynamics and cytoskeletal reorganization, which are correlated to novel dendritic spine formation. These models offer an exciting new range of opportunities to investigate the impact of psychedelics on human neural cells.           

Traditional high throughput screening (HTS) assays for neuronal targets employ non-human primary neuronal cells due to the scale necessary for HTS. Isolation of mouse primary neurons can be unreliable and economically demanding. The discovery of new drugs for neuropsychiatric disorders has further been hampered by lack of access to disease-relevant human primary neurons and appropriate disease models. By using human induced pluripotent stem cell (hiPSC) technology, we can address some of the obstacles which affords the generation of human neurons through (1) embryoid body (EB) formation, (2) cultivation on stromal feeder cells, and, (3) employing lineage specific differentiation factors. Methods exist to reproducibly differentiate hiPSCs into functional cortical induced neurons (iN) in less than two weeks but, they have never been taken to the HTS scale and have been slow and somewhat variable. We have successfully recapitulated the aforementioned technique and leveraged the CRISPR technology to define the path to a plate-compatible format amenable for large-scale HTS implementation. The resulting iN cells exhibit appropriate genetic and fluorescent markers that give confidence of bonafide neuronal differentiation. Imminently, we intent to test the preliminary iN cells for their ability to post-mitotically increase or decrease neurite outgrowth following treatment with LOPAC test compounds via staining and high content analysis. Ultimately, we will determine reliability and reproducibility over time with industrial scale robotics. Furthermore, we also intend to leverage the CRISPR technology to create a library of disease-relevant-phenotypes from hiPSC-derived cellular models that will provide more opportunities for all biologists to study epigenetic mechanisms and scale-up screening initiatives with Scripps Research Institute Molecular Screening Center (SRIMSC).

The enteric nervous system (ENS) plays an essential role in gut motility. Diseases of the ENS result in bowel motility disorders that are some of the most challenging clinical conditions to manage. Cell therapy offers the potential to treat gastrointestinal motility disorders caused by enteric neuropathies. We have previously shown that following transplantation into the colon of recipient mice, enteric neural progenitors proliferate, migrate and differentiate into a variety of neurochemical types of neurons. However, it was unclear whether graft-derived neurons integrate into the circuitry of the recipient and directly regulate gut motility. We have used optogenetic and electrophysiological approaches to examine whether transplanted enteric neural progenitors generate neurons that functionally innervate the colon. Neural progenitors expressing the light-sensitive ion channel, channelrhodopsin, were isolated from fetal or postnatal bowel and transplanted into the colon of postnatal mice. The responses of recipient colonic smooth muscle cells to light stimulation of graft-derived neurons were examined. Light stimulation of graft-derived cells resulted in excitatory and inhibitory junction potentials, the electrical events underlying contraction and relaxation respectively, in colonic circular muscle cells. The pharmacological properties of the junction potentials evoked by stimulation of graft-derived neurons were identical to those of endogenous excitatory and inhibitory motor neurons. Interneurons were also generated from graft-derived cells, but their pharmacological properties varied with the age of the donors from which the progenitors were obtained. Our data demonstrate that transplanted progenitors generate different functional classes of enteric neurons involved in the control of gut motility.

Creating inducible Pluripotent Stem Cell (iPSC) collections of rare patients scattered world-wide can lower the barrier of biological discovery of rare disease, provide a platform for potential drug repurposing and seed new drug development programs where there is a great unmet medical need. Currently approximately 8,000 rare diseases have been identified that affect close to half billion people world-wide of which only 5% have available therapies.   More than half, hundreds of thousands, of children have rare disease of which 30% die before their 5th birthday.  Two main challenges are 1) access to genetic sequencing and 2) if sequencing is available only 30-50% of the time the etiology is identified that leads to clinical intervention. Even with identification of a causal gene, understanding how the gene, gene variants and variants in concert with other genomic mutations change biology and lead to a clinical phenotype is needed.  In the first step, there is a need to find a critical number of families with the same rare disease which necessitates searching for them around the globe.  Secondly, the creation and use of iPSC lines once families have been identified enable validation of variance of significance through techniques such as gene editing in addition to revealing causal biological pathways. This methodology can accelerate identification of therapeutic options in the immediate addressing the urgency needed in addition to seeding longer term new targeted drug development programs.                                                           

The discovery of the CRISPR/Cas9 system has transformed research and now allows straightforward alterations in the genome and can be exploited to introduce changes to mimic diseases. This presentation will highlight the use of CRISPR/Cas9 in the generation of disease models in that are relevant for Parkinson’s disease. A review of our ongoing work on the development of pluripotent stem cell (PSC)-based disease models in the neuronal contexts covers which tools can be used to perform CRISPR/Cas9 based genome editing in PSC, how clonal isolation of edited PSC can be dramatically improved, how edited cells are characterized and how differentiation into the relevant cell type is used to reveal cellular phenotypes caused by the genetic changes.