Day 1 - Wednesday, October 20, 2010

Plenary Keynote Session

1:30

Therapeutic Modalities – Moving Discovery to the Next Level

Hans-Joachim Boehm, Ph.D.
Global Head of Chemistry and Center Manager
Pharma Research and Early Development
Roche, Basel Switzerland
 

Small Molecules and biologics are the backbone of modern medicinal therapy. Both therapeutic modalities have intrinsic advantages but there are also challenges that currently limit the application range for them In my talk, I will outline the strategy we have implemented at Roche to address the challenges and to develop safe and efficacious next generation therapeutics.

For small molecule discovery, new technologies such as Genetic Chemistry, In Silico prediction tools or Encoded Library technologies play an increasing role and I will describe the Roche approach in this field.

I will also describe how we at Roche have created a new chemistry strategy focusing on innovation [1].

[1] Drug Discovery Today, 2010, p.260-264

2:15

A High Throughput Quantitative Stem Cell Screening Platform to Find New Therapeutics

Tom Novak, Ph.D.
Senior Vice President
Research & Development
Fate Therapeutics
 

A significant need exists in the pharmaceutical industry for the development of cell-based assays that are more predictive of human physiology and clinical response and that provide better models of human disease. There is great promise for improving the drug discovery and development process by deriving relevant differentiated cell types from human stem cells, especially induced pluripotent stem cells (iPSCs), for more effective primary and secondary screening. To fully leverage the potential of stem cells in screening, careful and accurate quantification of relevant cell states and a well-developed knowledge base of stem cell biology are essential. Quantitative biology approaches, such as gene and protein expression and epigenetic analysis, provide the characterization of normal and diseased states necessary to create new screening paradigms. This talk will discuss recent advances in the industrialization of iPSC technology as well as provide examples of how a high-throughput quantitative platform for stem cell screening is being used to aid our drug discovery and development activities.

3:00

Networking and Refreshment Break

3:30

Development of a New Apoptosis-Targeting Agent, HGS1029, a Small Molecule IAP (Inhibitor of Apoptosis Protein) Inhibitor, as Potential Cancer Therapeutic

Gilles Gallant, B.Pharm. Ph.D.
Vice President
Clinical Research — Oncology
Human Genome Sciences
 

Apoptosis is a fundamental process responsible for maintaining homeostatic cell populations in tissues and its dysregulation has been identified as an important factor in the development and resistance of many types of cancer. Overexpression of IAPs confers protection against a number of proapoptotic stimuli in a variety of tumors. XIAP, the best characterized IAP family member, binds to and inhibits the activation of caspase 9, so targeting caspase activation through the inhibition of IAP function may be a promising innovative anti-cancer strategy. HGS1029 is a novel IAP inhibitor designed to directly kill tumor cells by actively inducing apoptosis in selected tumor types. The in vitro apoptosis inducing activity of HGS1029 can be enhanced by pretreatment with a diverse range of chemotherapeutic agents and, in particular with mapatumumab, an agonistic human monoclonal antibody that targets specifically the TRAIL-R1 receptor on the surface of cancer cells. In vivo, HGS1029 administered as a single-agent or in combination, can significantly inhibit tumor growth in xenograft models of human tumors. HGS1029 is currently undergoing Phase I clinical development.

4:15

Multivalent Design in Drug Discovery

Mathai Mammen, M.D., Ph.D.
Senior Vice President
Research and Early Clinical Development
Theravance
 

Multivalency is a natural phenomenon that describes co-operative binding to two or more binding sites to provide favorable thermodynamics of binding and thus obtain high potency at a particular biological target. In the context of drug discovery, Theravance leverages Multivalent Design to install useful molecular features, such as high potency, high selectivity, duration of action, and activity at a second biological target to create novel and highly differentiated medicines. Examples of drugs and candidates in Development created by the use of Multivalent Design include the following: VIBATIV^TM (telavancin) an antibiotic with two complementary pharmacologies approved in 2009 for the treatment of adult patients with complicated skin and skin structure infections; TD-1792, a second antibiotic with two different complementary pharmacologies that has completed a Phase 2 study in complicated skin and skin structure infections; and TD-5959, which as a single molecule acts to both agonize the b₂ adrenoreceptor and antagonize the M₃ muscarinic receptor, has completed Phase 2a studies in Chronic Obstructive Pulmonary Disease. The discovery and characterization of these compounds will be described to illustrate the utility, depth and breadth of Multivalent Design in Drug Discovery.

5:00

Networking Reception and Poster Presentation

             Top of the page                                                      Day 3
 

Day 2 - Thursday, October 21, 2010

7:30

Registration and Continental Breakfast

Session I: Pre-Clinical & Clinical Trials of Stem Cell Therapy

Moderator: Edward Rebar, Sangamo

8:00

Addressing a Chronic Ischemic Disease Market of $11B with Novel Cell Therapies

Ed Field, M.B.A., Executive Vice President and Chief Business Officer, Aldagen

Cardiovascular disease is the class of disease which involves the heart or blood vessels. Inadequate blood flow, or ischemia, can cause a shortage of oxygen and resulting damage to tissue. Common examples of clinical effects of ischemic include heart attacks, strokes, heart failure and leg ulcers. The presentation will focus on novel cell therapies that are aimed at improving perfusion in ischemic tissues and improving clinical status of patients.  In particular, the presentation will focus on clinical and preclinical data in the area of critical limb ischemia, ischemic heart failure and ischemic stroke.

8:30

Zinc Finger Nuclease – Edited Stem Cells: New Possibilities for Research and Therapy

Edward Rebar, Ph.D., Vice President - Technology, Sangamo

The ability to engineer precise genetic modifications into human stem cells would both accelerate research and extend the range of potential therapeutic applications. This possibility is now being realized via the use of zinc finger nucleases (ZFNs). ZFNs are customizable, sequence-specific endonucleases that can be designed to introduce a discrete cleavage event at any user-chosen location within the stem cell genome. By adjusting conditions under which the cleavage event is subsequently repaired, one may efficiently and precisely disrupt or edit the targeted locus, or integrate a larger, gene-sized DNA fragment. This technology – which is portable to any eukaryote – has been used for diverse applications, including therapeutic gene modification in primary cells, trait stacking of producer cell lines for improved manufacture of biologics, and gene targeting in previously refractory species such as nematodes, rabbits, zebrafish and rats.

This talk will describe recent applications of this technology in human stem cells. Examples include gene tagging in embryonic stem cells, gene targeting in induced pluripotent stem cells, and gene addition at safe harbors in a variety of stem cell types. Preclinical proof-of-concept studies towards the development of autologous, CCR5-disrupted CD34 stem cells as a treatment for HIV will also be presented.

9:00

Tissue Repair Cells (TRCs) for Cardiovascular Repair - Identification of Potency in a Mixed Cell Product

Michelle Kreke , Ph.D., Senior Scientist , Aastrom

Aastrom Biosciences has developed an ex vivo expanded autologous bone marrow-derived product, referred to as tissue repair cells (TRCs), for the repair and regeneration of ischemic tissue. Recent clinical trials examining TRC therapy in the treatment of critical limb ischemia and dilated cardiomyopathy have shown clinical promise. Despite positive clinical outcomes, the precise mechanism by which TRCs induce benefit remain unclear. Elucidation of mechanism is complicated by the fact that TRCs are a mixed cell product containing multiple bioactive stem and progenitor cells. In vitro and in vivo data is presented that seeks to characterize the TRC product and its subcomponents. The goal of these studies are to develop a potency assay that is based upon a well characterized mechanism of action and accurately reflects the biological activity of the TRC product.

9:30

Neural Stem Cell-Mediated Cancer Therapy: From Bench to Bedside

Karen Aboody M.D., Associate Professor, Department of Neurosciences and Division of Neurosurgery, City of Hope National Medical Center and Beckman Research Institute

Despite aggressive multimodal therapy, high-grade gliomas remain incurable and lethal. Neural stem/progenitor cells (NSCs) offer an unprecedented advantage over conventional approaches because of their exceptional ability to cross the BBB, target invasive tumor cells throughout the brain, and provide a platform for the production of localized chemotherapy. Used as a delivery vehicle, NSCs have been engineered to express a variety of anti-cancer agents, demonstrating >70% therapeutic efficacy in pre-clinical models of glioma, medulloblastoma, melanoma brain metastases and disseminated neuroblastoma.
We now report FDA approval to initiate a first-in-human clinical trial using a clonal human NSC line, HB1.F3, modified to express cytosine deaminase (CD), an enzyme that converts the prodrug 5-Fluorocytosine (5-FC) to the active chemotherapeutic 5-Fluorouracil (5-FU) in patients with recurrent high-grade glioma. Injected into the tumor cavity wall at the time of surgical resection, we postulate that NSCs will localize to residual and invasive brain tumor foci, and convert orally administered 5-FC to 5-FU at the tumor sites. This strategy also minimizes toxicity to normal tissues, potentially reducing undesirable side effects of current chemotherapies.
The HB1.F3.CD clonal NSC line was generated from15 wk fetal telencephalon by retroviral transducion with v-myc. In vitro cytogenetics, migration and activity assays demonstrate that HB1.F3.CD NSCs are chromosomally and functionally stable. Identification of a single copy and insertion site for both CD and v-myc genes was determined by LAM-PCR. In vivo preclinical biodistribution, safety/toxicology and therapeutic efficacy studies conducted in normal and glioma-bearing immunocompromised and immunocompetent adult mice indicate this NSC line is non-toxic, minimally immunogenic (HLA class II negative), and non-tumorigenic. Clinical use of this expandable, well-characterized, allogeneic NSC line circumvents problems associated with primary stem cell pools, and the need for continued sources of new cells. We postulate that our HB1.F3.CD NSCs will localize to invasive glioma foci and convert 5-FC to 5-FU, causing preferential killing of dividing tumor cells and improve clinical outcome in recurrent glioma patients. Demonstration of safety/feasibility in a phase I study will provide the foundation for further therapeutic development and applications to other invasive cancers.

10:00

 Networking and Refreshment Break

Session II: Regulatory Aspects and Commercialization

Moderator: Jim Wang, Sanofi-Aventis

10:30

Commercialization of Regenerative Medicine: Translating Great Science into Successful Business

Gregory Bonfiglio, J.D., Founder and Managing Partner, Proteus Venture Partners

The presentation will address the commercialization of Regenerative Medicine technologies – moving Regenerative Medicine from bench to bedside. We will review market for Regenerative Medicine Companies, and will try to place that environment in context of historical market trends. We will discuss the current funding environment, and the new funding strategies driven by the current economic realities.  We will examine the venture process, the criteria used by investors for making funding decisions, and typical deal structures. Finally, we will outline a new capital efficient model for the development of Regenerative Medicine technologies.   

A synopsis of the presentation is as follows:
 “Commercialization of Regenerative Medicine: Translating Great Science into Successful Business"

In my presentation, I will address a number of key issues involved in the commercialization of stem cells and regenerative medicine, including the following:

• A Brief Review of the RM Market

  • Where are we and how did we get here?

  • RM Commercialization Challenges

• The RM Funding Environment: New Economic Realties

  • Public Markets: A World Without Biotech IPOs

  • Venture Capital: Only “Pristine” Deals Need Apply

  • Alternate Funding Sources

• Crossing the Valley Of Death With Friends

  • The Current Model Is Broken

  • Capital Efficiency Is Critical

  • A New Collaborative Model

11:00

Regulatory Considerations: Getting to the Clinic

Edmund Mickunas, M.D., Vice President of Regulatory, Advanced Cell Technology

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?
  What does it take to get to the clinic?

11:30

Stem Cell Based Products - FDA and EMA Regulatory Considerations

Jim Wang, M.B.A., Ph.D., Associate Director, Global Regulatory Affairs, Sanofi-Aventis

  12:00

[FEATURED PRESENTATION]
 

Stem Cell-Derived Therapies—Challenges and Opportunities in Product Characterization

Malcolm Moos, M.D., Ph.D., Medical Officer, Cell and Tissue Therapies Branch, CBER, OCTGT, FDA

Therapeutic interventions with cells or engineered tissues promise to provide effective treatments for conditions refractory to conventional approaches. However, development of such products poses new obstacles. Because living cells and tissues cannot be sterilized, microbiological safety is an even greater concern than for other regulated products. The difficulty is compounded by product shelf lives that are often so short that conventional safety testing using compendial methods is not practical. Though rapid methods are under development, there is limited experience with these techniques, and their use may require careful qualification studies. Another challenge is identification of analytical procedures that can predict the clinical performance of these products reliably. A crucial technological issue is that these products are highly complex with respect to the analytical methods available for their characterization and analysis. A formidable problem for products consisting of single cell types, it is even more challenging for products containing mixed cell populations. Furthermore, analytical characteristics associated with both desirable and undesirable in vivo characteristics of these products may be unknown, necessitating a well-planned and comprehensive set of development studies to characterize them analytically and ultimately select tests suitable for monitoring product manufacture and ultimately as product release specifications. During the presentation, we will explore these questions and discuss possible strategies for addressing them in a practical manner, first to meet regulatory requirements for initiation of human trials, and subsequently for marketing approval.

Benefits:
Attendees will:

• Understand the basic regulatory requirements for initiation of human trials

• Understand the basic regulatory requirements for marketing approval

• Understand the direct connection between scientific questions concerning cell fate specification, competence, signal pathway status, etc. and regulatory requirements

• Appreciate how the interface between product biology and analytical biochemistry fits into the overall strategy of product development

12:30

Lunch On Your Own

  2:00

[FEATURED PRESENTATION]
 

GRNCM1: Human Embryonic Stem Cell-derived Cardiomyocytes for the Treatment of Heart Failure

Katharine Spink, Ph.D., Vice President, Program Operations, Geron

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.

Session III: Regenerative Medicine & Tissue Engineering

Moderator: Robert Halliwell, University of the Pacific

2:30

Nestin-Expressing Cells from the Dermal Papilla and Bulge Area in the Mouse Vibrissa Can Equally Repair Spinal Cord Injury

Robert M. Hoffman, Ph.D, President, AntiCancer; Professor of Surgery, University of California, San Diego

Nestin-expressing cells were found in the dermal papilla (DP) and bulge area (BA) in the mouse vibrissa. We have previously shown that nestin-expressing bulge area cells, termed hair follicle pluripotent (hfPS) cells, are pluripotent. hfPS cells from the mouse and human can differentiate into many cell types, including neurons and glial cells, and effect nerve and spinal cord repair in mouse models. Nestin-expressing cells in the hair follicle DP also appear to be multipotent. These cells are termed skin precursors or SKP. The objective of this study was to compare the ability of the nestin-expressing cells from both regions to repair spinal cord injury. Matrices of cells from both areas were made by culturing them on Gelform®, where they grew very well. Spinal cord hemisection was performed on nude mice. Upon spinal cord transplantation, the BA cells + Gelform-and DP cells + Gelfoam®-groups had similar locomotor recovery, both better than the Gelfoam®-only group or negative controls. Immunofluorescent staining of BA or DP implanted cells in the injured spinal cord identified Tuj1-expressing neurons and GFAP-positive glial cells differentiating from cells from both areas. These results suggest BA and DP cells cultured on Gelfoam have clinical potential for spinal cord injuries.

Benefit points:
Introduction of classes of stem cells of the hair follicle
Comparison of hfPS and SKP cells for spinal cord regeneration
Advantages of hfPS, SKP, ES and iPS cells

3:00

Multi-organ Engraftment by Human Embryonic Stem Cell-Derived CD34+ Cells, with Implications for use as Cellular Therapy for Diabetes

A. Daisy Goodrich, Ph.D., On-campus Affiliate, Research Assistant Professor, Department of Animal Biotechnology, University of Nevada, Reno

Hematopoietic stem cells, commonly isolated via their cell surface expression of the CD34 molecule, have previously been shown to possess multi-organ engraftment potential. While cord blood and bone marrow are common sources for these cells, human embryonic stem cells (hESC) also produce CD34+ cells upon differentiation in culture. Our objective was to determine if hESC-derived CD34+ cells had a potential for multi-organ engraftment as observed for CD34+ cells from hematopoietic sources. To this end we transplanted pre-immune fetal sheep recipients in-utero via intra-peritoneal injections. Tolerance towards foreign antigens, acquired when cells are introduced prior to the development of the immune system in gestation, persists in the adult animal. We transplanted sorted hESC-derived CD34+ cells populations as well as non-sorted whole differentiated populations.

The hESC-derived CD34+ cell population demonstrated engraftment and long-term presence in multi-organs. This activity was not augmented by transplanting the entire differentiated cell population from which the CD34+ cells were isolated. We further tested cellular activity and function of these grafts in adult sheep via assaying for human hematopoiesis, and human albumin and C-peptide production.

 Benefit points:

• Long-term data (5 year) on the potential of hESC-derived CD34+ cells for multi-organ engraftment and function, without any teratomas.

• Long-term data (5year) demonstrating the persistence of in-vivo beta-cell-like activity which could prove useful for cellular therapy in regenerative medicine.

• The same cell population could be used for the induction of immunological tolerance with bone marrow chimerism as well as cellular therapy for diabetes.

• A feel for cell numbers transplanted and the level of C-peptide measured in a large animal model.

3:30

Networking and Refreshment Break

Session IV: Technology in Stem Cell Research

Moderator: Robert Halliwell, University of the Pacific

  4:00

[FEATURED PRESENTATION]
 

Molecular Elucidation and Engineering of Stem Cell Niches 

David Schaffer, Ph.D., Co-Director, Berkeley Stem Cell Center; Professor, Chemical Engineering, Bioengineering, and Neuroscience, University of California, Berkeley

Stem cell self-renewal and differentiation are regulated by complex cues within the stem cell microenvironment, or niche, which include soluble small molecules and proteins, extracellular matrix (ECM) signals, and mechanical cues. While there has been considerable progress in investigating soluble cues that regulate stem cell function, far less is known about the “solid phase” of the microenvironment, in large part due to experimental complexities in studying large matrix and other proteins. Recent work demonstrates that bioactive, synthetic materials can be harnessed to emulate and thereby study the effects of solid phase, or biophysical, signals on stem cell function. We have harnessed biomaterials to investigate principles by which the solid phase regulates human embryonic stem cell (hESC) and adult neural stem cell (NSC) function. Extracellular mechanical properties can profoundly impact NSC cell self-renewal and lineage commitment. Furthermore, nanoscale organization of biochemical factors can modulate their signaling properties, and we have utilized biomaterials to oligomerize signaling factors and thereby greatly enhance their bioactivity and potency on NSCs and hESCs. Finally, the combinatorial presentation of ECM motifs from a material substrate can generate biomimetic substrates that support the self-renewal and differentiation of neural stem cells and hESCs, thereby enabling the dissection of the ECM into key signals necessary to support cell function. Biomimetic materials can therefore be used to study principles by which the solid phase of a stem cell microenvironment regulates cell function, as well as offer safe and scaleable systems to precisely control stem cell function for biotechnological and biomedical application.

4:30

Development Perspectives on Adherent Stem Cell Therapy in CNS Injury and Disease

Anthony Ting, Director of Regenerative Medicine, Athersys, Inc.

Clinical development support for cell therapy in the CNS has been limited due to a lack of insight into mechanistic benefit pathways, as well as the poor performance history of small molecules in treatment of stroke. Cell therapies represent a multi-modality approach in treatment of CNS disease, and elaboration of mechanistic pathways can provide a rationale for dose design and delivery route to optimize success and encourage broader investment in this therapeutic area.
MultiStem® is an adult adherent stem cell derived from bone marrow with an established manufacturing platform and safety base in clinical studies including GVHD prophylaxis and acute MI, and an open IND for use in treatment of stroke. In addition to understanding the cytoprotective and angiogenic potency of these cells, MultiStem exerts strong influence on activated inflammatory cells. Through action on activated endothelium, MultiStem restricts inflammatory cell extravasation into ischemic tissue. In CNS injury such as TBI, MultiStem dramatically influences the migration of activated macrophages from spleen into brain associated with recovery benefit. In models of spinal cord injury, MultiStem is capable of reversing macrophage induced neuronal die-back from the injury site and facilitates regrowth through proteoglycan scars.
Development parameters necessary for CNS cell therapy clinical entry will be described.

• Pre-clinical safety requirements for CNS cell therapy
• Preserving neuronal function following stroke and spinal cord injury
• Brain/immune cell cross-talk in CNS injury

5:00

Investigating the Interdependencies of OCT3/4, NANOG, SOX2 and LIN28 Protein Expression in Embryonic Germ Cells and Tumors of the Testis

Mark Shannon, Ph.D., Senior Staff Scientist, Molecular Biology Systems, Life Technologies

Human testicular germ cell tumors (TGCTs) arise during embryonal development as a component of the primordial germ cell (PGC) lineage and first appear as carcinoma in situ (CIS) later in life. The resulting invasive tumors mimic embryonal cells to a great extent as demonstrated by their capacity to form all differentiated lineages, including the germ line, and by the expression of pluripotency factors OCT3/4, NANOG, SOX2, and LIN28.
In this study, the expression patterns of these proteins during normal human male germ cell development and in primary TGCTs (fresh-frozen and formalin-fixed, paraffin-embedded) were investigated using immunohistochemistry and novel TaqMan® Protein Assays. These highly sensitive and quantitative assays are based on the Proximity Ligation Assay (PLATM) technology. The effect of siRNA-induced down-regulation of LIN28 and OCT3/4 on expression of the pluripotency proteins in tumor-derived cell lines was also determined using the same approaches. LIN28, OCT3/4, and NANOG were expressed in highly similar patterns during germ cell development and in TGCTs; however, a broader range of expression was observed for LIN28. In addition, down-regulation of LIN28 in the tumor cell lines resulted in the loss or substantial reduction in the levels of LIN28, OCT3/4, and NANOG (and SOX2 in embryonal carcinoma), while down-regulation of OCT3/4 produced more independent outcomes for LIN28 and SOX2 compared to OCT3/4 and NANOG. In conclusion, this study clearly highlights the similarities between embryonic stem cells, PGCs, and their malignant counterparts with respect to the retention and interdependencies of the key pluripotency proteins.

5:30

End of Day 2

             Top of the page                                                      Day 2
 

Day 3 - Friday, October 22, 2010

7:30

Continental Breakfast

Session V: Induced Pluripotent Cells, Reprogramming & Epigenomics

Moderator: Enal Razvi

8:00

From iPS to RPE and Back: Memory in iPS Cells

Dennis Clegg, Ph.D., Professor and Chair, Molecular, Cellular, and Developmental Biology (MCDB), University of California, Santa Barbara

Induced pluripotent stem (iPS) cells have the potential to differentiate into ocular cells, which may prove useful in the treatment of retinal diseases, particularly retinitis pigmentosa and age-related macular degeneration (AMD). AMD and related diseases are the leading cause of blindness in elderly people of the western world and retinal pigment epithelial (RPE) cell dysfunction and death are thought to be major causative events. Here we show that human iPS cells derived from embryonic lung cells using Oct4, Sox2, Lin28 and Nanog (Thomson et al.) can spontaneously give rise to RPE cells that are similar but not identical to native RPE with respect to structural, functional and molecular properties. iPS RPE were shown to carry out phagocytosis of photoreceptor outer segments in vitro and rescue vision in the RCS rat model of retinal dysfunction. Furthermore, some but not all human iPS cells derived from embryonic RPE show a tendency to redifferentiate into RPE. RPE derived from iPS or hESC have potential as cellular therapies for AMD.

8:30

Epigenetic Markers for Cell Therapy Quality Control and Clinical Monitoring

Ulrich Hoffmueller, MBA., Ph.D., Founder and Chief Business Officer, Epiontis

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 fulfill 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 B-cells, granulocytes) 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.

• 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

9:00

Feeder-Free Reprogramming of Adipose-Derived Stem Cells As a System to Study Metabolism: Potential Roles of Nuclear Receptors

Shigeki Sugii, Ph.D., Senior Researcher, The Salk Institute for Biological Studies  

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 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 self-renewal factors such as basic FGF, 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. Using these cells as a model system, we studied expression changes of nuclear hormone receptors, a family of ligand-activated transcription factors that regulate a number of metabolic and developmental processes. Potential involvement of a subset of nuclear receptors in reprogramming will be discussed.

• 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

• Reprogramming of adipose-derived cells offers a valuable system for studying nuclear receptors

9:30

Stem Cell Transcriptomics: A Systems Approach to the Pluripotent State

Kitchener D. Wilson, M.D., Ph.D., Senior Postdoc, Stanford

The discovery and isolation of human embryonic stem cells (hESCs), and the more recent generation of induced pluripotent stem cells (iPSCs) from adult cells, has given medical science the tantalizing prospect of one day regenerating organs and tissues in human patients, as well as a revolutionary method for investigating heritable human diseases in a petri dish. However, forcing these cells to change their phenotype is an imperfect science, and is often time-consuming, resource-intensive, and plagued by poor yields. Fundamentally, what is needed is better control over the factors that induce, maintain, and repress pluripotency. My approach is taken in large measure from systems biology, in which understanding biology at the systemic level, rather than its individual parts, is the central dogma. My work has therefore attempted to characterize the sets of molecules, both messenger RNAs and microRNAs, that constitute the “transcriptome” of the stem cell. This has included understanding how the stem cell transcriptome changes in the face of external insult (e.g. ionizing radiation), how it changes during differentiation to adult phenotypes such as cardiomyocytes and endothelial cells, and how knowledge from it may be used to induce pluripotency. Taken together, interrogating and ultimately controlling the stem cell transcriptome will be an essential step before we can realize the promise of regenerative therapy.

10:00

MicroRNA Regulation of Induced Pluripotency

Robert Blelloch, M.D.,Ph.D., Associate Professor - Urology; Obstetrics, Gynecology and Reproductive Sciences; Pathology, University of California, San Francisco

Induced pluripotent stem (iPS) cells hold great promise for the study and treatment of disease. However, current roadblocks include the low efficiency of iPS production, genetic modifications associated with most current methods, and the challenge of differentiating iPS into tissues of interest. One promising means of addressing these roadblocks is the use of small non-coding small RNAs called microRNAs (miRNAs) to manipulate cell fates. Our lab has been pursuing this approach. In particular, we have been using miRNAs to both promote the differentiation of embryonic cells into somatic cells as well as the dedifferentiation of somatic cells to iPS cells. We have discovered how different miRNA families interact to control these cell fate decisions. We have also been uncovering the downstream pathways regulated by the miRNAs providing us insights into additional nodes of control. This work is leading to novel means of producing and expanding cells of interest and possibly to more direct uses in patients suffering from degenerative diseases or cancer.

10:30

Networking and Refreshment Break

 11:00

[FEATURED PRESENTATION]
 

CIRM Grant Applications - Fostering Innovation and Translation in Stem Cell Research in California

Patricia Olson, Ph.D., Executive Director, Scientific Activities, California Institute for Regenerative Medicine (CIRM)

Session VI: Differentiation of Stem Cells

Moderator: Robert M. Hoffman, University of California, San Diego

11:30

Combinational Cell Culture™ Experiment Analysis Facilitated By Bioinformatics

Yen Choo, Ph.D., CEO and Chief Scientist, Plasticell Limited

Differentiation of pluripotent (ES and iPS) cells will improve our understanding of mammalian development and provide a source of progenitor and differentiated cell types for use in basic research, drug discovery and cell replacement therapy. However, stem cell differentiation is technically challenging and is considered a bottleneck in the field.

We have developed a bead-based high throughput screen capable of multiplexing extremely large numbers of cell culture protocols to discover those which result in high efficiency and cost-effective directed differentiation into any given phenotype.

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 labels. Following phenotypic screening to identify beads bearing differentiated cells, deconvolution of labels allows us to piece together combinations of protocols that result in differentiation.

We will present screens in which up to 10,000 combinations of protocols (equivalent to millions of combinations of growth factors), typically result in the elucidation of hundreds of putative differentiation protocols. We have developed bioinformatics software that allows us to collate data from the various steps of the experimental workflow, and to perform statistical analysis of the results in order to predict those protocols which are likely most effective. In this way a shortlist of protocols can be subjected to secondary screening to validate and quantitate their efficacy.

12:00

Pluripotent Stem Cells, a Potential Source of Beta Cells for Diabetes Therapy

Mattias Hansson, Ph.D., Project Manager, Novo Nordisk

Beta cell failure cause diabetes and is currently treated by exogenous insulin administration. Absolute or relative beta cell deficiency characterizes type 1 diabetes (T1D) and type 2 diabetes (T2D), respectively. Diabetes-associated late complications are devastating to the diabetic patients and are caused by inadequate blood glucose control. The reconstitution of a functional beta cell mass by transplantation of isolated islets from organ donors can restore normoglycemia in T1D patients - and protect against the development/escalation of late complications as long as sufficient numbers of functional beta cells survive the grafting. However, a shortage of donor material is one of the factors preventing the general use of cell replacement therapy for diabetes treatment. Advances in the directed differentiation of pluripotent stem cells toward beta cells by the stepwise recapitulation of embryonic development have generated proof of concept demonstrating that stem cells may be an appropriate source of cells for the generation of therapeutic beta cells. However, the progress toward a clinical application of this technology is slow and challenging. Robust cell differentiation protocols for the generation of fully functional beta cells from pluripotent stem cells are yet to be defined and the development of large scale production methods of clinical grade pluripotent stem cells, pancreas progenitors as well as mature beta cells are still in its infancy. Furthermore, novel approaches to avoid the immune-mediated destruction of the transplanted beta cells are needed as the existing methods deliver poor long-term results. Some of these critical issues that impede the translation of stem cell-based diabetes therapies to the clinic will be discussed.

Benefits:
• Diabetes, beta cells and cell replacement therapy
• Directed differentiation of beta cells from pluripotent stem cells
• Progression towards clinical application – challenges and pitfalls
• Strategies to avoid the immune-mediated destruction of transplanted beta cells
• In vitro modeling of T1D using iPS cells – a novel tool to study beta cell killing and its prevention?

12:30

Fate Space Screening of Clonal Human ES-Derived Embryonic Progenitor Cell Lines for Chondrogenesis

Michael D. West, Ph.D., CEO, BioTime, Embryome Sciences, and OncoCyte Corporation

1:00

Lunch

Session VII:  Stem Cell Applications in Drug Discovery

Moderator: Petter Bjorquist, Cellartis AB

2:00

Functional Properties of Neurons derived from Human Stem Cells

Robert F. Halliwell, PhD, Professor, School of Pharmacy, University of the Pacific

Human pluripotent stem cells have enormous potential value in both neuropharmacology and neurotoxicology yet there is little data on the receptors and ion channels expressed by neurons derived from these stem cells. Moreover, there is currently little data on the sensitivity of stem cells and their neural derivatives to neurotoxins rendering their validity as models in neurotoxicology uncertain. Recent studies in this lab have therefore begun to determine the expression profile of ligand and voltage-gated ion channels in neurons derived from the human stem cell line, TERA2.cl.SP12, using single cell patch-clamp electrophysiology. We have also utilized immuno-cytochemistry and flow cytometry to determine the impact of a range of drugs and environmental agents on the viability of stem cells and their neural derivatives. This presentation will describe the pharmacological and electrophysiological properties of neurons derived from human stem cells and the impact of several neuro-active agents on these cells and their ability to differentiate into neurons.

Co-authors: Leanne Coyne, Mu Shan, Ryoko Hirakawa, William Cao & John Livesey.

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

2:30

[Oral Presentation from Exemplary Submitted Abstracts]

To be considered for Oral Presentation, please submit an abstract here.

3:00

Human Pluripotent Stem Cells: Current Technologies and Emerging Industrial Interest

Petter Bjorquist, Ph.D., Senior Principal Scientist, Project Manager, Cellartis AB

The drug discovery process is extremely time consuming and expensive. Consequently, novel approaches for improving these processes and for reducing late-stage attrition are of great value for the pharmaceutical industry. In pre-clinical drug development, the lack of functional human cell models leads to the extensive use of either cell systems with low clinical relevance, or complex and costly animal models. However, it is anticipated that the access to specialized cells derived from human stem cells will improve the quality of targets, hits, and leads, reduce attrition rate, and thus shorten the time and cost of drug development. The utilization of stem cells in drug discovery spans from early target finding and evaluation studies, via the use of functional human cells in screening and pharmacokinetic studies, to the use of various stem cells technologies in toxicological testing.
Here will be illustrated some of the opportunities presented by human pluripotent stem cells, and the different level of interest the industry express for these possibilities will be discussed. Some of the advantages these cells provide for the development of novel and improved tools for drug discovery and toxicity testing will also be illustrated. Finally, the role of the growing Biotech industry in projects tackling regenerative medicine challenges, including LMW compound screening programs and cell therapy, will be outlined.

3:30

Pharmaceutical Applications of Stem Cells

Huseyin Mehmet, Ph.D., Director, Exploratory Biomarkers, Diabetes, Merck

 4:00

Conference Concludes

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