The recent demonstration of in vitro reprogramming using transduction of 4 transcription factors by Yamanaka and colleagues represents a major advance in the field. However, major questions regarding the mechanism of in vitro reprogramming need to be understood and will be one focus of the talk.

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved by over-expression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. We have demonstrated that reprogramming is a continuous stochastic process where almost all donor cells eventually give rise to iPSCs upon continued growth and transcription factor expression. Inhibition of the p53/p21 pathway or over expression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPSC formation that was directly proportional to the increase in cell proliferation. These results suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency. In contrast, Nanog over expression accelerated reprogramming in a predominantly cell division rate independent manner.

A major impediment in realizing the potential of ES and iPS cells to study human diseases is the inefficiency of gene targeting. Using Zn finger mediated genome editing we have established efficient protocols to target expressed and silent genes in human ES and iPS cells. Finally, our progress in using iPS cells for therapy and for the study of complex human diseases will be summarized.

Embryonic stem (ES) cells are unique among stem cell populations, set apart by their extensive proliferative capacity and broad differentiative repertoire.  These two properties make ES cells an attractive source for production of cell types useful in therapies for many degenerative diseases.  Through a step-wise differentiation protocol modeled after pancreatic development, we have generated pancreatic endoderm progenitor cells from human embryonic stem cells.  Implantation of the hES cell-derived pancreatic progenitors into rodents results in the efficient generation of glucose-responsive endocrine cells. Moreover, the insulin-expressing cells exhibit many properties that are characteristic of functional beta cells, including expression of critical beta cell transcription factors such as PDX1, NKX6-1, and MAFA, appropriate processing of proinsulin, and the presence of mature endocrine secretory granules. Cells grafted in the epidiymal fat pad, kidney capsule, subcutaneous and omental sites in Scid Beige mice as well as nude mice and rats differentiate into functional glucose responsive beta cells. Finally, as a critical test of their therapeutic potential, we demonstrate that the hES cell-derived pancreatic islet tissue protects against streptozotocin-induced hyperglycemia. Glucose-responsive islet cells are capable of maintaining stable blood glucose levels in mice grafted for more than one year. Development of a replacement cell therapy product will require more than just a renewable pancreatic islet source. Both the scale up and delivery of a safe cell product composition will also be critical requirements.  Incorporation of these additional elements will provide the basis for further clinical evaluation in patients with diabetes.

Benefit Points:
1. Using principles of developmental biology to guide the directed differentiation of human embryonic stem cells.
2. Treating insulin requiring diabetics with a human embryonic stem cell based cell therapy: promises and challenges.
3. Preventing destruction of transplanted cells via encapsulation.
4. Engineering human embryonic stem cells as a platform for drug discovery.

High efficiency and cost-effective directed differentiation of stem cells is technically challenging, but is absolutely required for many applications. Achieving differentiation using small molecule effectors is a longstanding goal in the field, as small molecules are cheaper, more stable and reliable than the recombinant growth factors (or serum) currently used to drive differentiation.

We have developed a high throughput screening system capable of multiplexing extremely large numbers of putative cell differentiation protocols to rapidly pinpoint combinations of variables (such as small molecules) that drive stem cell differentiation into given phenotypes.

In ’Combinatorial Cell Culture’, stem cells grown on microscopic beads are shuffled serially through many different culture conditions, with concomitant labeling of the beads using fluorescent tags. Following phenotypic screening to identify beads bearing differentiated cells, tag analysis allows us to piece together combinations of protocols that result in directed differentiation of stem cells. Algorithms are then used to identify the most effective protocols, which are validated and/or further optimized.

Both extrinsic and intrinsic mechanisms tightly govern HSC decisions of self-renewal and differentiation. However, transcription factors that can selectively regulate HSC self-renewal division after stress remain to be identified. Slug is an evolutionarily-conserved zinc-finger transcription factor that is highly expressed in primitive hematopoietic cells and is critical for the radioprotection of these key cells. We studied the effect of Slug in the regulation of HSCs in Slug-deficient mice under normal and stress conditions by using serial functional assays. We show that Slug deficiency does not disturb hematopoiesis or alter HSC homeostasis and differentiation in bone marrow, but increases the numbers of primitive hematopoietic cells in the extramedullary spleen site. Deletion of Slug enhances HSC repopulating potential but not its homing and differentiation ability. Furthermore, Slug deficiency increases HSC proliferation and repopulating potential in vivo after myelosuppression and accelerates HSC expansion during in vitro culture. Therefore, we propose that Slug is essential for controlling the transition of HSCs from relative quiescence under steady-state condition to rapid proliferation under stress conditions. Our data suggest that inhibition of Slug in HSCs may present a novel strategy for accelerating hematopoietic recovery, thus providing therapeutic benefits for patients after clinical myelosuppressive treatment.

Benefit Points:
• A novel negative regulator of HSC regeneration under stress condition is demonstrated.
• Regulation of c-Kit signaling pathway is dissected.
• The potential application of Slug inhibitors will be explored.

Human ES or iPS-derived cellular therapeutics will likely require novel technologies to achieve purity and document identity. In an effort to generate highly purified, diverse, and scalable embryonic progenitor (EP) cell types for potential use in human cell therapy, we undertook the large-scale combinatorial cloning of EP cell lines from hES cells. We isolated approximately 1,100 diverse candidate human EP cell lines and characterized 242 of the better growing lines by gene expression microarray. Non-negative matrix factorization profiling detected 140 distinct cell types within the 242 cell lines. These lines display a wide array of markers of primitive endodermal, mesodermal, ectodermal, and neural crest types with diverse site-specific homeobox gene expression. A fate space screening protocol yielded three EP lines witha robust chondrogenic phenotype including the expression of COL2A1 LECT1, and EPYC. Immunocytochemical and histological analysis confirmed chondrogenesis and the compatibility of the cells with biodegradable matrices. The three chondrogenic lines showed diverse site-specific homeobox genes with one being LHX8+ BARX1+, one HOXA2, B2+, and one lacking HOX gene expression. Unlike bone marrow-derived mesenchymal stem cells, none of the chondrogenic lines expressed CD74 or the hypertrophic chondrocyte marker IHH. The diversity, scalability, and relative stability of clonal hES-derived embryonic progenitor cell types offers a novel avenue for the study of differentiation in early embryogenesis, the discovery of novel differentiation conditions for hES-derived cells, and when derived from cGMP hES or iPS master cell banks may offer a novel manufacturing protocol increasing the purity of clinical-grade cell-based therapies.

Cancer stem cells (CSC) are a subpopulation of cells within tumors defined by their capacities for self renewal and ability to initiate new tumor growth. As such, CSC may be central to metastasis. Development of successful therapeutic strategies may therefore require the discovery of effective approaches to target this population. We have developed therapeutic monoclonal antibodies that target key stem cell pathways, including the Notch and Wnt pathways, that have been implicated in cancer. OMP-21M18, a monoclonal antibody targeting DLL4, is the first specific inhibitor of Notch pathway to enter clinical trials. In preclinical models OMP-21M18 shows broad anti-tumor activity reducing the growth and tumorigenicity of human tumors. OMP-18R, a first in class inhibitor of the Wnt pathway, shows activity in a range of major human tumor types and inhibits tumor growth by promoting tumor cell differentiation to non-tumorigenic progeny. Both of these agents reduce CSC frequency, in stark contrast to traditional chemotherapeutic agents which fail to eliminate CSC and show evidence of selective sparing of the CSC. The development of therapeutics that disrupt signaling pathways essential to CSC function may therefore provide an attractive therapeutic strategy that is more successful in eradicating residual disease and providing patients with durable clinical benefit.

Although monoclonal in origin, most tumors appear to contain heterogeneous populations of cancer cells. One possible explanation of this tumor heterogeneity is that human tumors are not merely monoclonal expansions of a single transformed cell, but rather caricatures of normal tissues, and their growth is sustained by cancer stem cells (CSCs). These CSCs are thought to be more resistant to apoptosis, to survive therapy and to eventually give rise to second-line tumors, which are harder to eliminate by the first-line therapy. CSCs are not only important in terms of understanding the biology of tumors but also might have an important role in developing novel anti-cancer therapies. With this in mind, we set course to investigate if cells positive for CD133 (an important CSC marker) exist in the culture of primary human MPNST cells.

We have now obtained data showing that while mouse MPNST cells do not contain CD133+ cells, human MPNST cells contain between  up to 28% CD133+ cells. To the best of our knowledge, this is the first report about expression of CD133 in human MPNST cells. Interestingly, non-malignant human Schwann cells (as counterparts to MPNST cells) did not show the expression of CD133. We have also characterized these cells in terms of expression of MPNST-related markers, self-renewal capabilities and activation of Ras signaling pathway (the most important pro-oncogenic signaling pathway highly involved in the pathogenesis of MPNST) and have identified effector pathways which are overactive in CD133+ CSCs. These pathways include Erk, PI3K and Ral which seem to be more active in CD133+ cells as compared to the wild population of human MPNST cells. On such basis we have designed a protocol for targeting MPNST CSCs using pharmacological inhibitors for Ras effector pathways. The results of this study not only enhances our understanding of the biology of MPNST but can also provide is with a novel therapeutic strategy to target this malignancy.

Reliable cell characterization plays a pivotal role in tissue engineering and regenerative medicine. Especially with a perspective on clinical application of a cell product, thorough quality control is essential to ensure product efficacy, patient safety and to fulfil regulatory requirements.

Previously applied techniques for quality control are either based on protein or mRNA markers. To a large extend, these markers describe the current short term status of a cell, whereas the key property of a cellular therapeutic is its cell type identity and long term specialization. In the need of better quality control means, new techniques are sought after.

In several case studies it was shown how epigenetic markers based on differences in cytosine modification of genomic DNA of different cell types facilitate regenerative medicine applications.

For the quality control of Genzyme’s autologous chondrocyte product Carticel®, Genzyme and Epiontis jointly developed a quality control test based on three validated epigenetic markers. Using real time-PCR methods that are specific to epigenetic modifications of certain gene regions, a standard release assay for the therapeutic product has been optimized.

Specific regions of with epigenetic modifications were identified as a superior markers for immune cells (regulatory T cells, overall T cells, NK cells & other subpopulations) compared to formerly known protein markers due to higher specificity and lower sample requirements. Robust epigenetic assays are applied for QC for cellular therapies and immune monitoring applications in regenerative medicine and clinical trials in the cancer, autoimmune and organ transplantation area.

These results demonstrate that analysis of epigenetic DNA modifications qualifies as a suitable technique for cell characterization and routine release quality control tests for products in regenerative medicine and immune monitoring.

Benefit Points:

• A novel technique for cell therapy quality control and its applicability is demonstrated
• Unmet scientific and regulatory needs are addressed
• Proof of industry and initial experiences are presented
• Results were obtained in collaboration with a large biotechnology company
 

Although adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells, its potential for use in regenerative medicine has not been extensively explored. We will report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly, both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF, TGFb, fibronectin and vitronectin, and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative medicine and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance.

Benefit Points:

• Human and mouse adipose-derived stem cells give rise to iPS cells with high efficiencies
• iPS cells are obtained from human adipose cells in feeder-independent and xenobiotic-free conditions
• Adipose-derived stem cells intrinsically express high levels of self-renewal factors and can serve as feeder layers for pluripotent cells
• Adipose-derived cells are abundant and valuable sources for regenerative therapeutics
 

The number of scientists working with human stem cells has exponentially increased creating an urgent need to develop and adopt universal protocols for all stem cell processes. A novel approach for automated isolation and purification of individual stem cell colonies from surrounding cells for the purpose of creating new stem cell lines and genetically modifying existing lines has been developed. Results show that laser-mediated colony purification can be used to efficiently generate iPSC lines from human fibroblasts in a higher throughput manner. In addition, data show that laser-mediated iPSC derivation is more efficient and reproducible than manual colony picking methods. After isolation, iPSC colonies were expanded by laser-mediated sectioning, creating iPSC lines (free of enzymatic disruption) that had normal stem cell morphology, expressed high levels of stem cell-associated genes/proteins and were genetically stable over long term culture. Laser-mediated EB generation was used to reproducibly create size-specific EBs from these cells, which differentiated more efficiently into multi-lineage specialized cell types; ~5-fold increase in the formation of neural progenitor cells and motor neurons, >15-fold increase in cardiomyocyte production, and >35-fold increase in the formation of hepatocyte-like cells, when compared with EBs produced by typical methodology. The use of laser-mediated stem cell technologies provides universal, standardized protocols for efficient derivation and production of GMP-quality stem cell lines as well as reproducible differentiation of human pluripotent stem cells into specialized cell types.

Engineered zinc-finger proteins (ZFPs) extend our therapeutic arsenal to the complete universe of genes implicated in disease by functioning directly at the DNA level.  ZFPs enable two distinct therapeutic modalities (i) gene regulation i.e. turning genes on or off via designed ZFP transcription factors (ZFP TFs) and (ii) genome editing via zinc finger nucleases (ZFNs). SB-509, an investigational ZFP Therapeutic™ targeting the potent angiogenic, neuro-protective and neuro-regenerative factor VEGF-A, exemplifies the clinically most advanced use of the ZFP platform - namely ZFP TF controlled gene expression. SB-509 is in multiple Phase 2 trials in patients with Diabetic Neuropathy and ALS. Building from the clinical success of ZFP TFs the talk will focus on the second therapeutic modality enabled by engineered ZFPs – ZFN induced genome editing of living human cells. ZFNs drive unique outcomes that stand to revolutionize cell-based therapeutics. Indeed, clinical translation of ZFP-driven genome editing technology is underway with two Phase 1 trials of SB-728-T, an investigational ZFP Therapeutic™ targeting the CCR5 gene in T cells for the treatment of HIV/AIDS. These same CCR5-specific ZFNs are also being evaluated in hematopoietic stem cells. Finally ZFNs can be engineered to target and “correct” endogenous genes implicated in monogenic diseases.

Topics that will be discussed are:

• Background information on Spinal Muscular Atrophy.
• Use of genetic modification of Embryonic Stem Cells for disease modeling.
• Pathway of Motor Neuron Differentiation.
• Protein dysregulation in murine SMA ES cells and SMA ESC derived motor neurons.
• Use of iPSC technology for studying disease processes of SMA in affected individuals.

MicroRNAs (miRNAs) bind to mRNA and influence gene expression by destabilizing the target mRNA or repressing translation. miRNAs have a key regulatory role in cellular processes such as apoptosis, oncogenesis, differentiation and development. miRNAs have important functions in neuronal differentiation and recent reports detail essential roles of several miRNA species. The NTERA2 pluripotent human embryonic carcinoma cell line is a simple in vitro model system for characterizing molecular events occurring in early neuronal differentiation. Exposure of NTERA2 cells to retinoic acid induces cellular differentiation into the neuroectodermal lineage. We harvested cell cultures for miRNA and protein expression studies throughout a 14-day differentiation time course study. miRNA signatures of targets were obtained using the MegaPlexTM Preamp Primer Pools and TaqMan® Array MicroRNA cards. In parallel, protein expression profiles were generated using TaqMan® Protein Assays. Our data indicate dynamic regulation of miRNA species throughout the time course – many miRNA species are upregulated during the differentiation process. Several upregulated miRNA species such as miR-21, miR-125b and miR-145 have been reported to interact with specific mRNAs and modulate their respective protein levels. For instance, a major player in the biology of cellular response, the p53 protein, is directly impacted by miRNA cues. We quantified a panel of proteins influenced by miRNA regulation during the temporal differentiation of NTERA2 cells. Our experimental approach and qPCR assay tools have great value for researchers studying the interplay between miRNA dynamics and protein output.

Cytokines play essential roles in expansion and differentiation of human stem cells, which are often the single largest component of the cost for clinical manufacturing. Today, there is no capacity, anywhere in the world, to produce all the essential cytokines at large-scale, which will ideally meet the future human cell therapy requirements. HumanZyme currently offers more than 40 non-GMP, scalable and GMPable human cytokines from HEK293 cells as superior and cost-competitive research reagents and is the leader in providing the cytokines that conventional non-human cell expression sytems either can not produce at all or can not economically produce. HumanZyme manufacturing facility are exclusively dedicated to xeno-free production of human cytokines. The company has implemented a quality system that meets the requirements of major stem cell companies. HumanZyme plans to bolster its leadership position in enabling clinical production of human cell therapy by scaling up major cytokines essential for human stem cell production.

These cytokines provide the following advantages:

• Authentic and higher biological performance
• Xeno- and endotoxin-free
• Cost-effective

We also will discuss how the availability of the current cytokines will improve the speed and quality of both stem cell research and product development.

Congestive Heart Failure (CHF) is a leading cause of morbidity and mortality in the industrialized world. Nearly 5 million individuals in the US alone are estimated to suffer from CHF, with more than 600,000 additional cases diagnosed annually. Numerous small molecule and device therapies have been demonstrated to slow progression of and reduce mortality from CHF; nonetheless 5-year mortality rates remain >50%.

Cell therapies have unique potential for the treatment of heart failure through regeneration of cardiac tissue and restoration of cardiac contractility. Among cell therapies, human embryonic stem cells (hESCs) are unique for their ability to generate bona fide human cardiomyocytes with high efficiency and scalable, cost effective production methods.

I will discuss Geron’s GRNCM1 program, in which we are developing hESC-derived cardiomyocytes for the treatment of heart failure, including methods for high efficiency differentiation of cardiomyocytes from hESCs, characterization of the resulting cell population, preclinical data on cellular function, and next steps for the development of this product towards the clinic.

Although promising, current stem cell therapies for myocardial infarction may exert only a limited benefit due to low retention, survival, and non-cardiogenic nature of implanted cells. Alternatively, the implantation of a pluripotent stem cell-derived cardiac tissue patch may yield improved cell survival and more efficient structural and functional repair of the injured myocardium. However, it remains unknown if stem cell-derived cardiomyocytes can be assembled in vitro into a functional 3-dimensional cardiac muscle tissue capable of physiologically relevant electrical conduction and force generation. In this presentation, I will describe a novel and reproducible cell/hydrogel molding approach that allows in vitro generation of highly functional, 3-dimensional cardiac tissues consisting of highly aligned, cross-striated, and electromechanically coupled mouse embryonic stem cell derived cardiomyocytes. Localized electrical stimulation in these tissues induces a rapid, uniform spread of action potentials with conduction velocities and resulting forces of contraction in excess of 20 cm/s and 1.5 mN, respectively. These unprecedented levels of functional differentiation are similar to those reported for neonatal mouse heart and are achieved after 21 days of culture. Importantly, the use of a multipotent cardiovascular progenitors selected from embryonic stem cells for the expression of cardiac-specific Nkx2.5 enhancer  (but not pure cardiomyocytes selected for expression of α-myosin heavy chain) was sufficient for the formation of 3-dimensional cardiac syncytium. Therefore, this work for the first time demonstrates successful generation of highly functional cardiac tissues derived entirely from an embryonic stem cell source.

Our research group at the Stem Cell Research Center, University of Pittsburgh has shown that a population of muscle derived stem cells (MDSCs), isolated by a modified preplate technique from murine post-natal skeletal muscle, displays a high transplantation capacity in both skeletal and cardiac muscles. The MDSCs’ ability to proliferate in vivo for an extended period of time—combined with their strong capacity for self-renewal, resistance to stress, ability to undergo multilineage differentiation, ability to induce neo-vascularization, and paracrine effects they have on the host—at least partially explain the high regenerative capacity of these cells in vivo. Although the true origin of the MDSCs remains unclear, the similarity to human blood vessel-derived stem cells suggests a putative origin from the vascular wall. I will present a review of the current knowledge and the utility of the MDSCs to improve the healing of various musculoskeletal tissues and injured cardiac muscle and list the potential clinical applications based on this knowledge. I propose that these cells are a successful model for the use of post-natal stem cells in regenerative medicine but emphasize that other factors such as of age and sex of the host and donor cells and paracrine effects should be considered.

Benefit Points:

Beta-cell mass and function are decreased to varying degrees in both type 1 and type 2 diabetes. In the future, islet cell replacement or regeneration therapy may thus offer therapeutic benefit to people with diabetes but there are major challenges to be overcome. Islet cell regenerative therapy could be achieved by in situ regeneration or by implantation of cells previously derived in vitro. Both approaches are being explored and their ultimate success will depend on the ability to recapitulate key events in the normal development of the endocrine pancreas in order to derive fully differentiated islet cells that are functionally normal. There is also debate as to whether beta cells alone will assure adequate metabolic control or whether it will be necessary to regenerate islets with their various cell types and unique integrated function. Any approach must account for the potential dangers of regenerative therapy.  Islet cell regenerative therapy may one day offer an improved treatment of diabetes and potentially a cure. However, the various approaches are at an early stage of preclinical development and should not be offered to patients until shown to be safe as well as more efficacious than existing therapy.

Retinal degenerations, which include hereditary early onset retinal degenerations and age-related macular degeneration (AMD), are major causes of blindness in the western world. Some treatments using repeat intravitreal injections of anti-VEGF antibodies exist for exudative AMD, yet there are no effective treatments for the majority of patients with these diseases. Despite the wide diversity of disease phenotype, in retinal degenerations loss of vision is due to loss of retinal photoreceptors and the retinal pigment epithelium (RPE). In these advanced cases, reversal of vision loss requires replacement of damaged or dysfunctional cells with transplanted cells. Stem cell transplantation is a promising therapeutic approach for the replacement of degenerated retinal cells, since early result suggests that human embryonic stem cells have the potential to differentiate along either a photoreceptor fate, or into RPE. Herein we will review the clinical features of retinal degenerations, the pathological features present at various disease stages, and the status of retinal stem cell transplantation in animal models of retinal degenerations. The potential therapeutic role of these cells, as well as the unique anatomy of the eye that make it a favorable site for cell transplantation, will also be reviewed.

At the end of the talk, the audience should able to understand:

Therapies utilizing cells derived from human embryonic stem cells hold significant promise for treating multiple diseases and conditions. However, this area of biotechnological development is quite new and the traditional regulatory paths should be considered but specific issues and considerations are not well defined.

• What are key regulatory considerations?


• What should be considered in dealing with the FDA?

    - Approaching the Agency as a partner

• How do regulatory professionals deal with various development issues?

    -Pre clinical
    -CMC
    -Clinical Strategies
    -Ethical considerations

We have previously showed that the stem cell marker nestin is expressed in hair follicle stem cells which suggested their pluripotency. We subsequently showed that the nestin-expressing hair-follicle pluripotent stem (hfPS) cells can differentiate in culture to neurons, glial cells, keratinocytes, and other cell types and can promote regeneration of peripheral nerve and spinal cord injuries upon injection to the injured nerve or spinal cord. The location of the hfPS cells has been termed as the hfPS cell area (hfPSCA). Previously, the hfPS cells were cultured for 1–2 months before transplantation to the injured nerve or spinal cord which would not be optimal for clinical application of these cells for nerve or spinal cord repair, since the patient should be treated soon after injury. In the present study, we addressed this issue by directly using the upper part of the hair follicle containing the hfPSCA, without culture, for injection into the severed sciatic nerve in mice. After injection of hfPSCA, the implanted hfPS cells grew and promoted joining of the severed nerve. The transplanted hfPS cells differentiated mostly to glial cells forming myelin sheaths, which promoted axonal growth and functional recovery of the severed nerve. These results suggest that the direct transplantation of the uncultured upper part of the hair follicle containing the hfPSA is an important method to promote the recovery of peripheral nerve injuries and has significant clinical potential.

Benefit points:

• Description of hfPS cells
• Pluripotency of hfPS cells
• Use of uncultured hfPS cells for nerve regeneration
• Comparison of benefits of hfPS with ES and iPS cells
 

The recent availability of human adult and embryonic pluripotent stem cells has the potential to impact drug discovery with new opportunities to develop more predictive, high throughput assays that shorten the timelines for identification of new therapeutics.  Adult stem cells such as mesenchymal stem cells, (MSCs) are isolated from tissues such as bone marrow, adipose and muscle and have a more limited potential but can readily differentiated into chondrocytes, osteocytes, muscle cells, adipocytes and blood cells such as macrophages and mast cells.  These cellular systems may be used in target identification, validation, chemical screening, and in the development of assays for drug efficacy and safety.  In addition, there is the added potential for the identification of clinically relevant biomarkers. Over the past decades most cell-based assay were performed with immortalized or transformed cell lines, which while convenient to grow, harbor abnormal physiology.  In using stem cells based assays we hope to offer the advantage of better (i.e. cellular machinery reflective of the “norm”) cell based tools. The IVD potential of human stem cell cultures isolated from diverse human tissues provides a unique platform for drug discovery and safety for the pharmaceutical and biotechnology industries.

Benefit Points:

• Stem cells provide diverse tools that can overcome the limits of current cell lines
  
• Meet expected functional endpoints of specialized cells produced by in vitro differentiation (IVD) of stem cells
  
• Development of strategies to maximize throughput of cell-based assays established via IVD

Human stem cell-derived neurons (hSCNs) have an enormous potential value as physiologically relevant cells for drug discovery, though there is limited data on their functional properties and even less establishing their validity and reliability for neurotoxicological studies. Our lab has recently begun to determine the neuropharmacological properties of hSCNs and to evaluate them for neurotoxicity and neuroprotection experiments.

Neural cells from the human pluripotent stem cell line, TERA2.cl.SP12, were differentiated and maintained in vitro for up to 35 days. Conventional patch-clamp recording techniques were utilized to evaluate the expression of functional ligand and voltage-gated ion channels. A range of simple cytotoxicity assays and immuno-cytochemical labeling techniques were exploited to determine the sensitivity of stem cells and their neural derivatives to a range of agents, including methyl mercury and the excitotoxin, glutamate.

Our data show that hSCNs express functional voltage-activated sodium, potassium and calcium ion channels consistent with native human neurons. Neurons derived from stem cells also expressed ligand-gated GABAA and glycine receptor anion channels and glutamate and NMDA-receptor cation channels. Our electrophysiological data is therefore consistent with the expression of inhibitory and excitatory receptors described in native neurons.

In the neurotoxicity studies, hSCNs were exquisitely sensitive to the neurotoxin, methyl mercury and to glutamate excitotoxicity. Taken together, our data supports the validity of using stem cells and their neural derivates in neuropharmacological and neurotoxicological studies.

This work was supported by grants from the Johns Hopkins University Center for Alternatives to Animal Testing.