ABSTRACTS

Towards a Regenerative Medicine for Multiple Sclerosis
Robin J. M. Franklin
Cambridge Stem Cell Initiative, University of Cambridge

Remyelination, the process by which new myelin sheaths are restored to demyelinated axons, represents one of the most compelling examples of adult multipotent stem cells contributing to regeneration of the injured CNS. This process can occur with remarkable efficiency in multiple sclerosis (MS), and in experimental models, revealing an impressive ability of the adult CNS to repair itself. However, the inconsistency of remyelination in MS, and the loss of axonal integrity that results from its failure, makes enhancement of remyelination an important therapeutic objective. There is now compelling evidence that ageing is the major contributor to the declining efficiency of remyelination and that this is largely due to a failure of stem cell differentiation. This talk will review recent studies we have undertaken aimed at obtaining a detailed understanding of the mechanisms of regulating differentiation during remyelination and hence identifying novel therapeutic targets.

Modeling Human Retinal Disease with iPS Cells
David M. Gamm
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are both valuable sources of retinal cell types for in vitro and in vivo studies. However, unlike hESCs, hiPSCs can be derived from individual patients, and therefore offer a unique opportunity to model human retinal diseases and perform custom drug testing. In designing a hiPSC modeling study, care should be taken to select retinal disorders that have a reasonable expectation of recapitulating key pathophysiological processes in culture. In addition, a means to enrich for cell type(s) targeted by the disease is necessary; otherwise, the task of assuring a reproducible culture environment becomes very difficult, if not impossible. Taking these and other limitations of hiPSC modeling into consideration, diseases of the retina remain appealing, particularly monogenetic, early-onset diseases that target the RPE. As we improve our understanding of complex disorders and our ability to build more intricate culture environments, the number and types of retinal diseases amenable to hiPSC modeling will broaden. However, even with such improvements, hiPSC technology is envisioned to complement, not supplant, existing laboratory models of disease. In my talk, I will discuss our efforts to characterize and study iPS cell-based models of human retinal disease, with an emphasis on how we intend to use these culture models to better understand disease mechanisms and search for treatments.

iPS Technology and Disease Research: Issues to Be Resolved
Rudolph Jaenisch
Whitehead Institute for Biomedical Research and Department of Biology, MIT, Cambridge, MA 02124, USA

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 as well as the definition of pluripotent cell states need to be understood and will be one focus of the talk.

Human and mouse embryonic stem cells (ESCs) are derived from blastocyst stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse derived epiblast stem cells (EpiSCs). We have rewired the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by exogenous induction of Oct4, Klf4 and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (ERK) pathway. In contrast to conventional human ESCs, these epigenetically converted cells have growth properties, X chromosome activation state (aXa), a gene expression profile, and signaling pathway dependence that are highly similar to that of mouse ESCs. The generation of “naïve” human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans, and may open up new opportunities for patient-specific, disease-relevant research.

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 or TALEN 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.

Self-Organized Formation of Three-Dimensional Tissues in ES Cell Culture
Yoshiki Sasai
Organogenesis and Neurogenesis Group, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan

Over the last several years, much progress has been made for in vitro culture of mouse and human ES cells. Our laboratory focuses on the molecular and cellular mechanisms of neural differentiation from pluripotent cells. Pluripotent cells first become committed to the ectodermal fate and subsequently differentiate into uncommitted neuroectodermal cells. Both previous mammalian and amphibian studies on pluripotent cells have indicated that the neural fate is a sort of the basal direction of the differentiation of these cells while mesoendodermal differentiation requires extrinsic inductive signals. ES cells differentiate into neuroectodermal cells with a rostral-most character (telencephalon and hypothalamus) when they are cultured in the absence of strong patterning signals.

In this talk, I first discuss this issue by referring to our recent data on the mechanism of spontaneous neural differentiation in serum-free culture of mouse ES cells. Then, I focus on self-organization phenomena observed in 3D culture of ES cells, which lead to tissue-autonomous formation of regional structures such as layered cortical tissues. We also discuss our new attempt to monitor these in vitro morphogenetic processes by live imaging, in particular, self-organizing morphogenesis of optic cup and pituitary gland in three-dimensional cultures.

References:
Suga et al, (2011) Nature 480, 57–62; Eiraku et al, (2011) Nature 472, 51-56; Kamiya, D. et al, (2011) Nature 470, 503-509; Muguruma, K. et al, (2010) Nature Neurosci 13, 1171 – 1180; Eiraku, M et al, (2008) Cell Stem Cell 3, 519-532; Sasai, N. et al, (2008) Cell 133, 878–890

Molecular Engineering of Extrinsic and Intrinsic Cues to Control Stem Cell Function
David Schaffer
Stem cell microenvironments present complex repertoires of signals to regulate the processes self-renewal and differentiation. There has been considerable progress in studying soluble signals that regulate stem cell function, but comparatively less work has focused on investigating the “solid phase” of the microenvironment, in large part due to experimental complexities in manipulating a complex mixture of proteins known at the extracelluar matrix (ECM) and other components. Recent work demonstrates that bioactive, synthetic materials can be harnessed to emulate and thereby study the effects of solid phase, biophysical cues on cell function. By using a modular, bioactive material, we have found that that the matrix modulus or stiffness profoundly impacts neural stem cell self-renewal and differentiation, and mechanistic analysis implicates key mechanotransductive pathways in this process that are important in cell culture and in the brain. Furthermore, immobilization of biochemical signals to the solid phase of a natural niche can lead to nanoscale organization of these signals, and nanostructured biological-polymeric conjugates likewise serve as potent effectors of neural stem and human embryonic stem cell function. Finally, the combinatorial presentation of different matrix motifs from a material can generate synthetic systems capable of supporting the self-renewal and differentiation of both neural stem cells and human embryonic stem cells, thereby enabling the dissection or distillation of the ECM into key individual signals necessary to support stem cell function. Biomimetic materials can thus be employed to study mechanisms by which the solid phase of a stem cell microenvironment regulates cell function, as well as offer safe, scaleable, and robust systems to control stem cells for biomedical application.

In addition to engineering the microenvironment, one can control a cell’s behavior by editing its genome. Gene delivery vehicles based on viruses offer a number of advantageous properties, including the potential for safe and efficient gene delivery; however, they face a number of challenges including inefficient delivery to some therapeutically relevant cells such as stem cells. Such shortcomings arguably arise from the fact that viruses did not naturally evolve to be utilized as human therapeutics, and we have thus been developing directed evolution – the iterative generation of large libraries genetic mutants and selection for enhanced properties – as an approach to create new viruses with useful properties. For example, we have evolved adeno-associated virus for enhanced gene delivery to and gene targeting in neural stem cells and human embryonic stem cells. Engineering both extrinsic and intrinsic cues thus provides strong capabilities to study natural mechanisms of stem cell fate regulation, as well as to control these choices for therapeutic applications.

Post-Transcriptional Regulation of Adult Neural Stem Cell Fate
Xinyu Zhao

Neural stem cells in postnatal and adult brains play significant roles in both normal brain functions such as learning and memory, as well as the brain’s responses to injuries and diseases. In addition, neural stem cells provide an excellent model for studying postnatal neurodevelopment and plasticity. My laboratory investigates how noncoding RNA and RNA binding protein-mediated post-transcriptional regulations control the postnatal neurogenesis. Our work unveils novel mechanisms underlying human neurodevelopmental disorders, which may lead to new therapeutic targets for treatment.