The emergence of technology for development of induced pluripotent stem cells (iPSCs) from somatic cells, such as skin and blood cells, has resulted in the ability of researchers to have limitless pool of iPSCs retaining the genetic make-up of the somatic cells from which they were derived.  In conjunction with tools for downstream gene editing, such as clustered regularly interspaced short palindromic-repeat (CRISPR)-Cas9 nuclease systems, iPSCs can be used generate (1) knock-outs to study the impact of genes on cellular processes, or (2) knock-ins to assess the impact of reversal of point mutations on diseased states, or furthermore for generation of reporter cell lines.  Briefly, CRISPR-Cas9 systems provide simple and efficient site-specific targeting that is accomplished by guiding Cas9 nuclease via a variable 20-base guide RNA sequence to the site for formation of a double stranded break. This break can then be repaired via non-homologous end joining (NHEJ) where small insertions or deletions are made in the gene of interest to knock-out its function or via homology-directed repair (HR) in which single nucleotide changes or knock-ins can be accomplished using a donor DNA template for repair.  Together iPSCs and CRISPR-Cas9 systems provide researchers with effective in vitro tools for assessing gene function, disease modeling, and regenerative therapy.  In this webinar, we discuss new technologies available from Thermo Fisher Scientific which support (1) efficient delivery of CRISPR Cas9 to PSCs, (2) improve cell survival following transfection while maintaining normal PSC properties, and (3) improve clonal cell survival following low density seeding of 1, 3, or 5 cells per well of 96-well plate.

Neurons derived from human pluripotent stem cells (hPSCs) and primary rodent neurons both are excellent resources for disease modeling and drug screening.  Human PSCs derived neural stem cells (NSCs) can be expanded in culture and further differentiated into mature neurons for various applications, however, these often contain mixed population of both differentiated neurons and undifferentiated NSCs. Due to the continuing proliferation of undifferentiated NSCs, very high cell densities and cell aggregation are usually observed during the differentiation of hPSC-derived NSCs which increase over time, posing challenges for long-term maintenance and downstream analysis.  Primary rodent neuronal cultures, while highly physiologically relevant, are often challenged by glial cell overgrowth, which may exacerbate assay and analysis issues.  Here we demonstrate the use of CultureOne(tm) - a new supplement which can reduce the proliferation of undifferentiated NSCs without negatively impacting the rate or extent of differentiation for hPSC-derived NSCs.  Further, we will demonstrate the ability to "tune" the glial cell population in primary rodent neuronal cultures.  The overall effect in both instances increases the relevant population of neurons in culture.  Experimental data presented in this webinar will  illustrate the functionality, morphology, and maturity of these neuronal cultures.

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

 

The use of pluripotent stem cell-derived cell types for disease modeling, drug screening and regenerative medicine is an exciting area of activity in health research. Prior to the availability of pluripotent stem cells and differentiation methods, relevant and affected primary cells were difficult to obtain and frequently only accessible post-mortem. Additionally, the recent development of CRISPR/Cas9 based genome editing methods allow the creation of isogenic (matched) disease and control lines that differ only at a specific, disease-relevant locus, or to insert reporter constructs into PSC lines such that the reporter may be used to study disease biology in relevant cell types after differentiation. Recently, CCRM has created a standard protocol for efficient genome engineering in human pluripotent stem cells (hPSC). This talk will describe our protocol, and its application to the creation of a cell line that contains a voltage-sensitive fluorescent reporter that will be used to study cardiac function and disease.

Stem cells play critical roles in the development of organisms, as well as in the maintenance and repair of organs and tissues throughout adulthood.  Advancing our understanding of mechanisms that control stem cell behavior – in particular their two hallmark properties of self-renewal and differentiation into specialized cells – will enable these cells to be increasingly harnessed to repair tissues damaged by disease or injury.  Stem cells reside within specialized microenvironments or niches that present them with a spectrum of regulatory signals to control their behavior. In particular, the niche presents stem cells with a range of molecular cues, and it has also been become increasingly apparent that key biophysical features of the environment modulate the presentation of this biochemical information. For example, spatial and temporal variation in the presentation of cues is important information that can impact fate decisions and tissue structure. In addition, the tissue matrix can have variable bulk mechanical properties and surface topographical properties depending on how its assembled.
 
We have created several technology platforms to investigate these problems, and in particular to understand and control the differentiation of adult neural stem cells and human pluripotent stem cells into neurons. First, we are developing and harnessing optogenetics as a system to investigate how cellular signaling dynamics impact fate decisions. Second, we develop bioactive, synthetic material systems to investigate the effects of cell-matrix and cell-cell interactions on cellular function. Finally, we work towards translating the basic information that emerges from both of these efforts into safe, scaleable, fully defined, robust culture and implantation systems for stem cell based regenerative medicine efforts to treat human disease.

The mission of the California Institute for Regenerative Medicine (CIRM) is to accelerate stem cell treatments to patients with unmet medical needs.  With $3 billion in funding and 300 active programs, it is the world’s largest institution dedicated to helping people by developing cell therapies.  To accomplish its mission, CIRM has funded a breadth of activities spanning from basic research to translation to clinical trials.  In addition to individual grants, CIRM has created resources to help the stem cell community worldwide.  Some resources promote research and drug development using stem cells.  CIRM has established an iPSC Repository maintained by the Coriell Institute that is currently the largest publically accessible pluripotent stem cell bank in the world.  CIRM also has a genomics research initiative that applies cutting edge sequencing and bioinformatics approaches to stem cell research and therapeutic development.  Other resources established at CIRM promote the acceleration of stem cell discoveries into therapeutic development.  CIRM is in the process of setting up a system of translational and clinical centers to help researchers move their discoveries into clinical development.  This system includes 1) a Translating Center to support preclinical IND-enabling activities such as process development, safety testing, and cell manufacturing of stem cell therapy candidates, 2) an Accelerating Center that helps with clinical strategy, regulatory submission, and clinical trial management, and 3) an Alpha Clinics network that conducts clinical trials for stem cell related therapies.  In total these resources are designed to catalyze the flow of stem cell discoveries to the clinic, which can come from both inside and outside of California.  CIRM has supported 16 stem cell based clinical trials and the goal is to have 50.  Beyond an overview of CIRM’s programs, this presentation will touch on perspectives around developing stem cell therapies and regulatory hurdles.

Inherited retinal degenerative disorders such as retinitis pigmentosa are characterized by death of the light sensing photoreceptive neurons of the outer retina. Like the rest of the CNS, the retina has little capacity for endogenous regeneration, and as a result, photoreceptor cell death causes debilitating irreversible blindness. Gene augmentation has the potential to prevent photoreceptor cell death, while cell replacement could actually repopulate the retina with new functioning photoreceptor cells and restore vision. In this talk I will show how we are using patient-specific iPSCs to evaluate disease pathophysiology, test novel gene-based therapeutics and develop autologous photoreceptor cell replacement for the treatment of retinal degenerative blindness.
 

The generation of induced pluripotent stem cells (iPSCs) from adult easily accessible somatic tissues was introduced ten years ago. This technology has revolutionized our opportunities to study human disorders including complex genetics and develop novel therapeutic concepts for regenerative medicine. By differentiating iPSCs to different cell types and stages, we can gain insights into developmental and disease driving molecular networks. Yet, a discussed question of the iPSC model system is the extent to which they retain residual phenotypes from their precursor, which impacts on the study of transcription networks. Moreover, it is open to which degree somatic mutations impact on functional studies of human genetic disorders when patient-specific iPSCs are applied. Here, we will tackle this question in the course of studying healthy and diseased siblings with Tetralogy of Fallot, a frequent congenital heart defect of multigenic origin. Routine genetic screenings are mainly based on genetic material derived from either whole blood or cheek swap sampling. ps-iPSCs are typically derived from dermal fibroblast (skin biopsies) or whole blood derived immortalized lymphoplastoid cell lines (LCLs). We will discuss whole genome frequency and impact of somatic acquired mutations on gene expression and functional studies of complex genetic disorders using ps-iPSCs.
 

• Background: Applications and general workflow of PSCs
• Reprogramming with high efficiency and safety (Summary of current reprogramming methods, Introduction of Cytotune-iPS 2.0 Sendai Reprogramming Kit)
• Optimum PSCs culturing system: (Summary of current culturing system, Introduction of KnockOut Serum Replacement-MultiSpecies and Essential 8)
• Characterization and differentiation of PSCs
• Resources and tools

Culture systems for pluripotent stem cell (PSC) expansion enable generation of a nearly unlimited pool of cells for downstream differentiation, disease modeling, drug discovery, and therapeutic applications.  While a number of PSC feeder-free medium systems exist, there are many challenges encountered by stem cell scientists across the PSC workflow in today’s applications.  Here we sought to improve the robustness and versatility of traditional PSC culture medium systems identify and optimize critical medium components.  Through assessment of over 65 different formulations, an optimum medium composition was identified which provides compatibility across the PSC workflow from somatic cell reprogramming, PSC expansion, downstream differentiation, as well as providing support in stressful applications such as gene editing.  This system additionally provides versatility, allowing for every-other-day or weekend-free feed schedules and compatibility with a broad range of passaging reagents and matrices.  Together this system provides a robust next-generation stem cell medium system for today’s challenging PSC workflow needs.  For Research Use Only

Recent discoveries in stem cell biology have increased the importance of stem cells in therapeutics and have improved researchers’ understanding of normal and disease processes. The inherent diverse nature of differentiating cultures remains a challenge in stem cell research. Multicolor flow cytometry enables researchers to interrogate and quantify heterogeneous cell populations at the single cell level. This webinar will provide numerous examples of how flow cytometry can be used to characterize cells throughout the stem cell workflow. 

The human body is composed of about 200 different cell types. The identity and function of these distinct cell types are precisely programmed by the regulatory networks encoded in the 3 billion base pairs of DNA that constitute the human genome. While 60% of our genome is transcribed, less than 2% of it is translated to proteins. In contrast to previous assumptions, this suggests that a significant majority of the regulatory information from the genome functions as RNAs, termed non-coding RNAs. Emerging evidences suggest that a substantial portion of these non-coding transcripts control myriad biological processes ranging from development to disease, establishing the vital role played by these RNA regulatory elements. In addition, these molecules are regulated by RNA binding proteins at the functional level. We investigate how RNA regulatory elements program cellular identities during cardiac development, aging, and regeneration.
 

In our previous preclinical studies, when neural stem progenitor cells (NS/PCs)-derived from hiPSCs were transplanted into mouse or non-human primate spinal cord injury (SCI) models, long-term restoration of motor function was induced without tumorigenicity, by selecting suitable hiPSCs-lines (Nori et al., 2011; Okano et al., 2013; Okano and Yamanaka, 2014). However, NS/PCs derived from certain iPSC-lines gave rise to late-onset tumorigenicity after transplantation (Tsuji et al., 2010; Nori et al., 2015). Here, to preclude these risks before clinical application, we developed molecular characterization of hiPSCs and hiPSC-derived NS/PCs together with transplantation to injured spinal cord of immune-deficient mice (Nori et al., 2015; Sugai et al., 2016). We investigated global methylation status of tumorigenic hiPSC-NS/PCs and found that aberrant hypermethylation of a tumor suppressor gene was induced along the passage. For addressing the safety issue, remnant immature cells or tumor-initiating cells should be removed or induced into more mature cell types to avoid adverse effects of hiPSC-NS/PC transplantation. Because Notch signaling plays a role in maintaining NS/PCs, we evaluated the effects of γ-secretase inhibitor (GSI) and found that pretreating hiPSC-NS/PCs with GSI promoted neuronal differentiation and maturation in vitro, and GSI pretreatment also reduced the overgrowth of transplanted hiPSC-NS/PCs and inhibited the deterioration of motor function in vivo (Okubo et al., 2016). Based on these findings, we are establishing methods of production, selection and transplantation of clinical grade NS/PCs stocks-derived from human iPSC stocks generated from HLA-homozygous super-donors by CiRA. We aim to commence clinical research (Phase I–IIa) trials for treatments of sub-acute phase SCI using hiPSCs-derived NS/PCs in the near future.
 

Stem cells, specifically induced pluripotent stem cells (iPSCs), offer exciting potential for the future of cell therapy and regenerative medicine. More recently, genetic engineering of stem cells and progenitor lines is enabling in vitro disease modeling for drug development with cell populations that would otherwise be unavailable from live donors.  With the use of TALENs and CRISPRs, this true power is fast approaching.  Having the ability to develop two cell models with isogenic background, except for the site specific edit, gives researchers the potential to study the true effects of a single mutation in a pathway or syndrome.  However, the lack of advanced technologies has been hindering the current pace of research and discovery.  Improved delivery can help accelerate research for emerging new therapies and help the understanding of disease pathways and mechanisms.  We will review multiple delivery solutions for CRISPR based technologies and also highlight protocol details and data outcomes for an iPSC specific CRISPR experiment.

The central nervous system (CNS) is a conglomerate of diverse, interconnected tissues that each contain cell phenotypes specific to their distinct anatomical region. Recent studies have demonstrated that CNS cells derived from human pluripotent stem cells (hPSCs) must be of the appropriate regional phenotype to model tissue-specific disease pathologies in vitro as well as produce a regenerative effect upon transplantation. Yet, only a limited number of regional CNS phenotypes can be derived from hPSCs due to the complex regimen of developmental cues necessary to effectively instruct region-specific neural differentiation.
 
Here, we present a chemically defined protocol for deriving forebrain neural stem cells (NSCs) from hPSCs with greater than 90% efficiency in under 6 days (Lippmann et al. Stem Cells 2014). Also, we have successfully deciphered the regimen of developmental cues that govern differentiation of hPSCs into NSC phenotypes specific to any diverse hindbrain or spinal cord region (Lippmann et al. Stem Cell Reports 2015). These NSC cultures can be differentiated into a spectrum of respective regional hindbrain and spinal cord motor neuron phenotypes, which can be matured to fire action potentials and innervate skeletal muscle fibers in vitro.
 
Hence, our chemically defined protocols vastly expand the diversity of regional CNS phenotypes that can be derived from hPSCs. They enable access to the hundreds of different regional motor neuron phenotypes present in the human hindbrain and spinal cord, which are the sole means of transmitting efferent signals from the CNS to peripheral tissues including skeletal muscles that provide motor function. Our findings have significant implications for modeling degenerative disorders that target hindbrain and spinal cord motor neurons (e.g. Amyotrophic Lateral Sclerosis) and developing regenerative cell therapies for paralysis.    
 

In healthy liver, quiescent hepatic stellate cells (HSCs) participate in the homeostasis of extracellular matrix and store vitamin A. After injury, HSCs activate and participate in the wound-healing response, producing extracellular matrix components and eventually fibrosis.  We have developed a protocol to direct the differentiation of human induced pluripotent stem cells (iPSC) to HSCs. The final HSC–like population was enriched in PDGFRß positive cells and expressed key HSC markers at similar levels than primary HSC. Whole genome transcriptomic analysis revealed that PSC-derived population displayed an intermediate phenotype between activated and quiescent HSCs. Functional analysis showed that PSC-derived HSC-like cells responded to injury mediators and accumulated retinyl esters into lipid droplets. These findings show that we have generated functional HSC-like cells from iPSC, which may have potential for in vitro and biomedical applications

Over the last 15 years, human pluripotent stem cell (hPSC) technologies have progressed from academic curiosities into tools with the promise to underpin commerce, leading to real progress in understanding of disease, improving drug safety and providing novel routes to clinical translation. With an emphasis on the heart, this presentation will discuss our progress in producing models of genetic disease by reprogramming somatic cells into human induced pluripotent stem cell (hiPSC). This includes various conditions such as long QT syndrome, Duchenne muscular dystrophy and CPVT, which affect the function and / or structure of cardiomyocytes. We will show how the Cas9/CRISPR system is being used to produce defined sets of polymorphisms in the ADRB2R and GRK5 loci, which encode proteins that underpin b2-adrenoceptor signaling. These polymorphisms reflect the genotypes in the patient population and we will present early data on how these changes may influence receptor density, internalization and both receptor and heart function. Since these panels of hiPSC and engineered lines can now be created with relative ease, bottlenecks of scaled culture, differentiation and phenotyping are becoming a considerable issue. Thus, we have developed an automation suite that includes a bespoke robotic platform to culture and differentiate hPSCs at scale into cardiomyocytes. Into this suite, we have incorporated high content platforms that allow assessment of structure (confocal plate reader imaging) and function (mitochondrial activity, contractility and electrophysiology). Despite these advances, numerous challenges remain, such as incomplete epigenetic reprogramming of hiPSC relative to hESCs and insufficient levels of expression of key ion channels, which need to be considered for the applicability of these models in biomedical application.
 

PrPC is a conserved lipid-raft associated, GPI-anchored cell membrane glycoprotein. Misfolding of cellular PrPC into the pathogenic PrPSc results in Prion disease, an untreatable and fatal neurodegenerative disorder. Prion induced neurotoxicity is preceded by impairment in metabolism of cholesterol and other lipids which are major component of lipid-rafts in affected neurons. Lipid-rafts deregulation has been implicated in diseases like Prion and AD, the mechanism remains unclear. Understanding the function of cellular PrPC may shed light on such pathological mechanisms. Towards this goal, we utilize a human induced pluripotent stem (iPS) cell model system. We generated isogenic PrP knockout (KO) human primary fibroblasts in order to reprogram them into PrP-KO-iPS cells and human neurons. Metabolomics and RNAseq analysis of these PrP-KO human cells show dysregulation in key CNS pathways like glycerophospholipid and cholesterol metabolism. Here we present a systems biological approach combining RNAseq and metabolomics to understand the functional molecular network of PrPC. The knowledge of key pathways in which PrPC  has important implications could aid in the targeted therapy for prion disorders

Using human pluripotent stem cells as a screening platform, we have identified a novel cell surface maker that identifies foregut endoderm progenitors in pancreas and liver.  We have shown that   cells of the biliary reaction, a regenerative response to liver damage, premalignant and pancreatic ductal adenocarcinoma cells, and the columnar epithelium of Barrett’s Oesophagus, a preneoplastic precursor of oesophageal carcinoma, all express this antigen on their surface.  The antigen is expressed in liver and pancreas during development, and  is also found in the serum of patients with pancreatic or liver cancer.  Cell surface antigens expressed on fetal stem or progenitors cells provide biomarkers of tissue regenerative processes, and can be used to monitor progression neoplasia if repair processes fail to resolve the underlying pathology.
 

Cataract, or vision loss due to clouding of the eye’s lens, is a large and costly international problem. Over 80 million people currently have low vision due to cataract. Cataracts can only be treated surgically, and while this restores vision the ability to focus between near and far objects is lost. Cataract surgery is the most commonly performed ophthalmic procedure and globally costs tens of billions of dollars annually. Due to population aging the incidence of cataract is increasing, and so are the associated costs. It has been estimated that a 10-year delay in cataract formation could halve the number and costs of cataract surgery. To address this issue Dr O’Connor has developed a world-first method to produce large numbers of human lens cells from pluripotent stem cells. The ability to access unlimited numbers of human lens cells, for defining cataract risk factors and performing anti-cataract drug screening, represents a true paradigm shift in international cataract research.
 

Buerger’s disease, commonly known as Thromboangiitis obliterans, is a non-atherosclerotic, segmental inflammatory disease that can affect the small and medium-sized arteries of young people with a history of heavy tobacco use. Its prevalence among all patients with peripheral arterial disease varies widely in different regions of the world.

Critical limb ischemia (CLI) is a severe form of the disease that results in acute rest pain and non-healing ischemic skin ulcers and gangrene of the lower extremity which can results in limb amputation if left untreated. Bone marrow derived MSC (BMMSC) are known to possess strong immunomodulatotory properties, promote angiogenesis and tissue regeneration through paracrine activity. Using a patented pooling technology of BMMSC from different healthy donors, we have developed an allogeneic BMMSC product, Stempeucel. Stempeucel is manufactured in a GMP facility and cryopreserved as an off-the-shelf product [1]. A series of preclinical studies were conducted to establish the safety [2] and efficacy profiles of the pooled BMMSC population. More importantly, results obtained from our phase I/II clinical trial in CLI patients demonstrated the safety of Stempeucel in humans [3]. A large phase II was recently completed with two different doses of Stempeucel. The cells were injected intramuscularly at multiple locations around the calf muscle and also around the ulcer. Analysis of the data six months after Stempeucel administration revealed statistically significant improvements in patients for both the primary clinical end points i.e., relief of rest pain and ulcer healing in the  target limb as  compared to the control group of patients [4]. In addition, ankle brachial pressure index (ABPI) also showed significant improvement in the same cell dose group, suggesting improved blood flow in the limb. As such, Stempeucel has been approved by the regulatory authorities in India for its use in a limited number of “No Option” Buerger’s disease patients.
 

A key aspect of translating great scientific discoveries to commercially viable cell therapies is the establishment of robust manufacturing processes that can produce the right quantity of cells, of the right quality, at the right cost, at the right time. In many instances a significant challenge lies in moving from the generation of millions of cells in the research lab to trillions of cells in compliance with regulatory requirements and Good Manufacturing Practices. There is no single path to achieving this, but establishing your vision early and asking the right questions can build a solid foundation from which to pursue future success. This presentation will explore some of these early considerations and why they are important.

In the last 10 years, PCSK9 emerged as a promising target for the treatment of autosomal dominant hypercholesterolemia (ADH). With the emergence of induced pluripotent stem (hiPS) cells and following differentiation protocols as a model for human pathology studies, we recently demonstrated the modeling of ADH due to mutations in the LDLR gene.  Our strategy to better understand the role of PCSK9 included reprogramming somatic cells from patients carrying the S127R mutation into hiPS cells, differentiate these cells into hepatocytes and characterize the intracellular impact of the mutated form of PCSK9.
To facilitate our strategy, we developed the isolation, culture, amplification and reprogramming of progenitor cells derived from urine samples (UCell). After reprogramming the hiPS cells (UhiPS), showed similar characteristics to hiPS cells classically reprogrammed from skin fibroblast. Through differentiation of the UhiPS cells hepatocytes were obtained that secrete PCSK9, internalized LDL particles and respond positively to statin treatments.To validate our model, in addition to generating hiPS cells carrying the PCSK9-GOF S127R, we reprogrammed cells from a patient carrying the PCSK9-LOF R104C/V114A and showed a spontaneously low level of LDL-cholesterol. Compared to control cells, UhiPS-S127R and –R104C/V114A differentiated in a comparable manner toward hepatocytes. In our hands, while hepatocytes carrying the S127R mutation showed a lower ability to uptake LDL, hepatocytes carrying the R104C/V114A mutations displayed the inverse, when compared to control cells. In addition, cells derived from the hypercholesterolemic patient responded positively to statin treatments, at a level comparable to the clinical response of patients carrying the same S127R mutation. Altogether, we demonstrated that differentiated hiPS cells are a relevant model to decipher PCSK9 functions and patient-derived urine samples represent a convenient source of somatic cells for such study.
 

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/Cas9 gene editing, to elucidate the underlying molecular pathogenesis of XDP. I will also discuss some of the potential problems one might face using hPSC-based disease modeling in combination with gene editing. 
 

The human enteric nervous system (ENS) is derived from the neural crest and represents a complex network of ~500 million neurons with dozens of distinct neurotransmitter and hormone subtypes essential for gastro-intestinal (GI) activities. Despite the significance of the human ENS and its involvement in a wide range of GI disorder, there is very little known about its biology due to the lack of accessible tissue. Directed differentiation of human pluripotent stem cells (hPSCs) offers and alternative approach for the derivation of distinct, functional cell types that can be utilized in a broad range of basic and translational applications. We have recently succeeded in derivation of the ENS lineages from hPSCs and applied them for cell therapy and drug discovery in Hirschsprung disease, which is the most common developmental disorder of the ENS. These studies set the stage for future investigations of the human ENS biology and a better understanding of disease mechanisms and advancement of therapeutic interventions for enteric neuropathies.
 

The human umbilical cord is a promising abundant source of mesenchymal stem cells (MSCs). Compare to other MSCs, the advantages of human umbilical cord MSCs (hUC-MSCs) are easily accessible and they have a short amplification time, high proliferation rate, lower immunogenicity, and abundance. Many studies have shown that hUC-MSCs have the ability to differentiate into three germ layers, to accumulate in damaged tissue or inflamed regions, to promote tissue repair, and to modulate immune response. Thus, they are attractive human cell–based therapies for the treatment of bronchopulmonary dysplasia, chronic obstructive pulmonary disease, ischemic stroke, neuron degenerative diseases, autoimmune diseases, etc.
 

Mesenchymal stromal cell (MSC) therapy is a promising option to support endogenous regeneration and immunomodulation. However, the clinical results are contradictory. We think that the recent studies have to major limitations: poor characterization of MSC(like) cell products which were used and the lack of adequate immune monitoring to better understand  therapy response, mode-of-action, dose-dependency etc.

Because of their high regenerative and immunomodulatory potency shown in various preclinical models and their well-defined manufactoring process in 3-D bioreactors, we focused on the characterization of placental-expanded mesenchymal like adherent cells (PLX) that are aimed to be applied as allogeneic off-the-shelf product.

Interestingly, by minor manipulation in the manufactoring process, Pluristem generated two related products PLX-PAD and PLX-RAD. Both PLX-cell types were comparable regarding their in vitro differentiation capacity and marker profile (CD73+90+105+45-31-34-) that is typical for MSC-like cells. However, the cells showed different properties in some but not all preclinical models. We hypothesized that the protective effects are mediated by the PLX-cells´ secretome which might be different between PLX-RAD and PLX-PAD cells.  In fact, conditioned medium of PLX-RAD expressed a distinct secretome and showed distinct effects in vitro and in vivo. Whole genome DNA methylation analysis and CD screen revealed significant differences between the two products.

PLX-PAD cells are supportive in different tissue regeneration models. In fact, clinical phase I/IIa studies in severe chronic limb ischemia (CLI) and muscle injury demonstrated safety but also clear hints for efficacy. Immune monitoring gave insights into immunogenicity, immune modulation, and dose-related effects which help to design ongoing studies.

In summary, extensive biomarker studies provided mechanistic insights into PLX products and highlighted the need for careful characterization of MSC-like cell products to better understand dosing, indication, mode-of-action etc.

Neurons derived from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are excellent resources for disease modeling and drug screening. Human PSCs derived neural stem cells (NSCs) can be expanded in culture and further differentiated into mature neurons for various applications, however, these often contain mixed population of both differentiated neurons and undifferentiated NSCs. Due to the continuing proliferation of undifferentiated NSCs, very high cell densities and cell aggregation are usually observed during the differentiation of hPSC-derived NSCs which increase over time, posing challenges for long-term maintenance and downstream analysis.  Here we demonstrate the use of a new supplement which can reduce the proliferation of undifferentiated NSCs without negatively impacting the rate or extent of differentiation for hPSC-derived NSCs.  The overall effect increases the relative population of differentiated neurons in culture.  Typically under these conditions by 2-3 weeks, differentiated neurons with extensive neurite networks are seen that are evenly distributed across the culture surface, with very little clumping or aggregation observed.  Further, this more uniform morphology and enriched neuron population greatly facilitates quantitative image analysis, as demonstrated by high content analysis using automated a CellInsight™ CX5 imaging platform which showed differentiated neurons expressing mature neuronal markers MAP2 or HuC&D without the contamination of undifferentiated Nestin positive NSCs, as seen in control samples.  Multielectrode array (MEA) analysis demonstrates that differentiated neurons fired spontaneous action potentials, indicating functional neurons. In addition, use of this new supplement enabled differentiated neurons to be maintained for longer time in culture than untreated control cells.

Human induced pluripotent stem cells (iPSCs) are valuable cells for retinal disease modeling, as these cells are of patient origin and can be differentiated into cell types of interest. This presentation will discuss the differentiation of iPSCs into retinal pigment epithelium cells and retinal ganglion cells and their molecular manipulations for modeling degenerative diseases of the retina and optic nerve.
 

My research program focuses on building the technology to generate diverse human cell-types for application in regenerative medicine and drug toxicology testing. While embryonic stem cells (ESCs) have the potential to generate thousands of distinct human cell-types, this vast array of lineage choices has made it difficult to efficiently differentiate ESCs towards any single desired fate. To more accurately guide ESC differentiation, we have delineated comprehensive roadmaps that describe how ESCs are diversified into a wide variety of endodermal and mesodermal cell-types. These roadmaps have enabled the successful generation of human liver cells and bone progenitors that can engraft in mouse models and respectively regenerate human liver tissue or bones in vivo; as such these ESC-derived tissue progenitors serve as potential sources of human cells for regenerative medicine or drug toxicology testing
 

Autologous cell based interventions, which include mesenchymal stem cells, are mostly unproven therapies increasingly being applied for musculoskeletal and other medical disorders by medical practitioners, with limited regulatory control. A self-regulated Code of Practice written for such Australian practitioners was released in February 2015. It required external review of the medical practices amongst other checks and balances that included (a) practising evidence based medicine; (b) ensuring fully informed consent was obtained from patients; (c) manufacturing the autologous product to be injected into the patient using internationally accepted standards; and (d) following the advertising standards set for medical practitioners by the Australian Health Practitioner Regulation Agency (AHPRA). Whilst the Code of Practice is beginning to be followed by some, with clinical trials approved by a Human Research Ethics Committee, and a Register of Adverse Events created, it may be too little too late. In July 2016, the NSW Coroner released findings of a case whereby an elderly person given autologous stromal cells derived from lipoaspirate to try and assist progressive dementia died within 24 hours of the procedure. This appears to be the first recorded death in Australia of a recipient of an autologous (stem) cell intervention.

In September, the Therapeutic Goods Administration released a Consultation paper inviting feedback on proposed tightening of the regulations. These include no direct marketing to the public, upgrading of manufacturing standards, registering with the TGA, and reporting of serious adverse events. If the regulations are indeed tightened, as now seems likely, the number of unproven autologous cell based interventions should diminish, as will the risk to recipients. However, at the same time innovation is likely to be reduced, as the cost for carrying out the interventions increases to meet the new standards required.
 

Most discussions about pluripotent stem cells center around their promise for regenerative medicine.  The most remarkable quality of these cells is their ability to turn into all of the cell types in the body. This amazing power is driving all of the projects in our lab that range from treatment of human disease to rescue of endangered species. One of our projects is development of an autologous cell replacement therapy for Parkinson’s disease (PD).  We have used non-integrating Sendai virus to reprogram skin cells into induced pluripotent stem cells (iPSCs) from ten people afflicted with PD.  Since death of dopamine neurons in the brain leads to the motor symptoms of PD, we have developed methods for generating dopamine neurons from iPSCs and plan to use these cells for autologous transplants, which should eliminate the need for immunosuppression. On the other end of the spectrum, we are using iPSCs in an effort to save endangered species. We are generating iPSCs from fibroblasts stored in the Frozen Zoo^® at the San Diego Wild Animal Park.  We are focusing on the Northern White Rhino, which will be extinct in our lifetimes.  Only 3 members of this species remain alive.  But there are fibroblasts from 12 individuals stored in the Frozen Zoo, and we have made iPSCs from two so far. We are generating iPSCs from the rest, and plan to differentiate them into gametes- this has been achieved for mice, but we don’t expect it to be easy for rhinos.  If we succeed, and can make fertilized embryos in the lab, there are 6 females from the closely related Southern White Rhino species already at the zoo who will serve as surrogate mothers.  The astounding power of pluripotency has opened doors to new ideas; we’re just getting started.
 

Access this training to learn more about different techniques that can be used for successful colony selection and passage of induced pluripotent stem cells.

Access this training to learn more about the evolution of pluripotent stem cell culture systems and which solution is best for your PSC culture goals.

One of the ultimate goals in Regenerative Medicine is the generation of pluripotent stem cells (PSCs) directly from somatic cells obtained from patients. Although major findings in the definition of in vitro differentiation protocols allowing for the derivation of several somatic cell lineages has been reported progresses on obtaining certain defined populations has faced major obstacles. One of the major problems associated with the in vitro differentiation arises in tissues presenting complex tridimensional (3D) structures, such as the case of cardiac, hepatic or renal lineages. Lately, it has been described that complex differentiation strategies might benefit from the establishment of 3D culture systems.  
The possibility to develop 3D dimensional self-organized tissues, so called organoids, has opened new venues in the generation of protocols dictating human PSCs differentiation. As tissue-derived organoids are compromised by multiple cell types recapitulating part of organ structure and function the possibilities to understand human development are facilitating the development of massive platforms for drug screening and personalized medicine. In this talk, we will summarize part of our recent work in heart and kidney differentiation using hPSCs highlighting potential applications when combined with organ decellularization and 3D bioprinting.
 

Years of significant effort can go into generating key proof of concept data to support moving bench research to clinical development.Yet, much work remains in order to transition from POC to clinical trials for a cell therapy. Raw materials, processes, and cell banks may all require changes to meet regulatory guidances and more stringent specifications for treating a patient. We will discuss the practical aspects of developing a cell therapy to treat heart disease.
 

A major challenge for the clinical application of pluripotent stem cell therapy for neurodegenerative diseases is large-scale manufacturing and cryopreservation of neurons and glia that can be prepared for surgery with minimal manipulation. To address this obstacle, midbrain dopamine (iPSC-mDA) and forebrain (iPSC-FB) lineage neurons were derived from human induced pluripotent stem cells and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved, post-mitotic neurons retained high-viability with gene, protein, and electrophysiological signatures consistent with that of the neuronal lineage. To test therapeutic efficacy, cryopreserved iPSC-mDA neurons were transplanted without sub-culturing into the 6-OHDA-lesioned rat and MPTP-lesioned nonhuman-primate models of PD. Grafted neurons retained midbrain lineage with extensive innervation of both rodent and monkey brain with no aberrant growth. Behavioral assessment in parkinsonian rats demonstrated significant reversal in functional deficits up to 6-months post-transplantation. In addition, cryopreserved iPSC-FB neurons grafted into the striatum of athymic NUDE rats survived and innervated distant anterior and posterior brain structures at 9-months post-grafting. These findings demonstrate a simple and efficacious surgical intervention to deliver cryopreserved iPSC-derived neurons for brain disorders and support translational development of pluripotent cell-based therapies in neurodegenerative disease.
 

Neuropathic pain and bladder dysfunction represent significant quality of life issues for many spinal cord injury patients. Loss of GABAergic tone in the injured spinal cord may contribute to the emergence of these symptoms. Previous studies have shown that transplantation of rodent inhibitory interneuron precursors from the medial ganglionic eminence (MGE) enhance GABAergic signaling in the brain and spinal cord. Here we look at whether transplanted MGE-like cells derived from human embryonic stem cells (hESC-MGEs) can mitigate the pathological effects of spinal cord injury. We find that six months after transplantation into injured mouse spinal cords, hESC-MGEs differentiate into GABAergic neuron subtypes and receive synaptic inputs, suggesting functional integration into host spinal cord. Moreover, the transplanted animals showed improved bladder function and mitigation of pain-related symptoms. Our results therefore suggest that this approach may be a valuable strategy for ameliorating the adverse effects of spinal cord injury.
 

The study of human genetic neurodevelopmental disorders (NDDs) is complicated by the inaccessibility of the relevant tissue for study: it is extremely rare to obtain post-surgical brain samples from patients, and the origins of disease often occur in-utero. The technologies of ex-vivo stem cell culture is a valuable tool to study embryonic development. Derived from the embryo and cultured under appropriate conditions, both embryonic stem and committed neural stem cells display multipotent features of the tissue they are derived from, and are licenced in-vitro with the properties of unlimited self-renewal.  The subsequent differentiation of these cells recapitulate many aspects of in-vivo development and can be used to model embryonic brain development. Because of these attributes, and the ease of genetic and other manipulations afforded in-vitro, we have employed these models to assess the effects of gene mutations that underlie neurodevelopmental disorders. We have been able to assign neurodevelopmental functions to newly discovered novel genes, test the pathogenicity of variants of unknown significance, and identify aspects of brain development likely affected in patients harbouring deleterious mutations.
 

The reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSC) has great potential of applications, not only in basic research, but also in drug screening and cell therapy. With advances in reprogramming technologies, use of diverse media systems and somatic cell sources, there is a compelling need for comprehensive characterizations methods that facilitate the generation of high quality standardized iPSC cell banks

The combined approach described in this presentation utilizes cellular and molecular techniques for qualifying the putative iPSC clones from the master somatic cell reprogramming population and through the subsequent maturation process. The resulting iPSC clones are further vetted for functional pluripotency, via expression of markers of self renewal and the pottential to differentiate into the 3 germ layers.  The resulting validated cell banks can help ensure  high quality of the cells prior to use in downstream applications such as gene editing and directed differentiation to specific lineages. A streamlined method of iPSC qualification and banking is critical for traceability to ensure the identity and quality of cells chosen for drug screening and cell therapy applications.

We work in the framework of the Human Induced Pluripotent Stem Cells Initiative (HipSci) project, funded by the Wellcome Trust and MRC. We provide a dedicated laboratory space for collaborative cell phenotyping to study how intrinsic and extrinsic signals impact on human cells to develop assays for disease modeling and drug discovery and to identify new disease mechanisms.