Stem Cell Workshop 2007


BACKGROUND

Sixteen NIH Institutes and Centers (ICs) that support research on the nervous system formed the NIH Blueprint for Neuroscience Research to develop research tools and resources that can catalyze science advances across the neuroscience community.  Human embryonic stem cells (hESC) have many potentially exciting applications in neuroscience research, from understanding fundamental questions about nervous system development and functions to possibly even treating neurological conditions.  Accordingly, the NIH is exploring whether there are opportunities for Blueprint (BP) to stimulate and facilitate hESC research.  Any aspect of hESC biology as applied to the neuronal, glial, and ependymal elements of the central or peripheral nervous system is of interest to the Blueprint.  The BP encourages visionary ideas to generate novel research tools and resources that could rapidly advance the capacity to differentiate hESC into specific neuronal and glial cell types.

Based on a recommendation at the June 2006 BP IC Directors’ retreat, the NIH BP decided to hold a workshop in the spring of 2007 to address the feasibility and usefulness of promoting research on the differentiation of hESCs in the nervous system.  The workshop focused on ways to facilitate research related to targeted differentiation of hESCs along specific neuronal and glial lineages, and identified tools and resources that are needed to advance the field.  The creation of additional tools and resources will be critical for elucidating the fundamental genetic, molecular, and cellular processes associated with hESC biology in the nervous system, as well as fulfilling the potential that stem cell research offers to the area of regenerative medicine.

WORKSHOP SUMMARY

The workshop was held June 28-29, 2007 at the Hyatt Regency Bethesda.  The workshop co‑chairs were Jeanne Loring, PhD (The Burnham Institute) and Lorenz Studer, MD (Sloan-Kettering Institute).  There were an additional 12 invited participants and one keynote speaker.  The workshop began with a casual evening orientation to present an overview of the BP and the workshop charge to the group.  In the keynote presentation, Arlene Chiu, PhD (California Institute of Regenerative Medicine) emphasized the important role for NIH in setting standards for stem cell research and possibly coordinating stem cell research funded by individual States.

Plenary Sessions

The second day of the workshop featured three plenary presentations in the morning.  The three topics focused on various aspects of hESC differentiation into cells of the nervous system.  These were: 1) hESC Differentiation:Basic Biology, 2) hESC Differentiation:Characterization and Genetics and 3) hESC Differentiation:Scaling, Purification, Quality Control, and Source Materials.  These three plenary talks were given by Drs. Lorenz Studer, Jeanne Loring, and Martin Pera (University of Southern California), respectively.

Session 1: Basic Biology: Identify stages of neural/glial differentiation of hESC

There are several common strategies for directed neural differentiation of hESCs.  They range from 1) Hypothesis-driven basic   biological approaches using   key developmental signaling pathways or by high-throughput screens (HTS) and 2) Genetic identification and isolation of hESC lineages using a variety of transgenic techniques, homologous recombination, and BAC transgenesis with various marking techniques.  The identification and characterization of neural stem cells or progenitors derived from hESCs still needs some ‘defined intermediates’ when starting with a heterogeneous population.  Various starting lines will be critical for scale-up, purification, and assays.  Plus, recreating complex systems will be a real challenge.

With this in mind, the participants agreed that the key questions for this topic are to:

1. Determine cell ‘types’ for defining lineages.

  • Determine the multi-step process of directed neural differentiation of hESCs
  • Each stage has different potential for differentiation, for behavior in vivo, and for modulation by signaling molecules.

2. Evaluate/Standardize the factors for neural induction, and hESC maintenance

  • Critical to understand the cellular responses to these factors to identify lineages
  • Differentiation, survival, and aging raise further issues in each category, including aspects such as phenotype stability, difference between in vivo/in vitro, disease vs. healthy. 

3. Improve efficiencies and outcomes, which can vary 1000-fold in cell numbers.

  • Use of engineered matrices can provide shaped or textured surfaces, such as arrayed biomaterials, nanotopography, and 3-D scaffolding, which can encourage particular growth patterns.

Session 2: Characterization and Genetics: Develop methods and reagents to monitor neural/glial differentiation

To address the development of methods to monitor differentiation of hESCs, the Systems Biology approach has developed into a significant method for studying these cells.  Systems Biology examines the interaction of molecular elements, including genetic and epigenetic components, messenger RNAs and microRNAs, and the integration of these elements into biological networks.  Following precedents set in cancer research, several components are necessary to establish a “systems biology of stem cells,” including 1) an appropriate, large cell-type collection, 2) methods for global analysis of systems elements, and 3) methods for data integration.  Taken together, these global molecular analyses are defining the systems elements of hESC.  Although molecular expression studies might be considered descriptive, they are truly important: unique gene and miRNA expression combined with specific DNA methylation patterns will lead to a clear definition of cells as hESC, as well as their progeny.

The overarching goal of these studies is to use the information gained to establish testable hypotheses, determine clinical relevance and to generate clear outcome measures for the identification and therapeutic application of hESC.  Systems biology might move hESC biology toward cell therapy for human disorders.  The ability to reprogram differentiated cells to a pluripotent state is one major goal.  Recent research has shown that differentiated mouse cells can be reprogrammed to pluripotent (-like) states.  However, the ultimate test of pluripotence and embryonic stemness in mice is germline transmission, studies that are not possible in humans.  However, stem cell systems biology is poised to provide the appropriate diagnostic tests.  These tests may include unique molecular interactions that can be identified by expression of genes, miRNA, and unique DNA methylation patterns.  This unique interaction network will allow researchers to explore the factors that are underlying the pluripotency of hESC.

Session 3: Scale-up, Purification, and Quality Control: Develop quality control and scale-up methods consistent with future translational studies

In order to implement robust, reliable, and safe therapies using stem cells, standards and quality control will be paramount.  Current issues that must be addressed include:

  • hESC quality: identification, history, and purity
  • Genetic/epigenetic stability
  • Scale-up and GMP manufacturing (highest proportion of the cells, and readily scalable culture conditions)
  • Assays (markers, gene expression, systems biology solutions, or in vitro functional assays) to predict function and safety of the various stages proposed for clinical use.
  • Characterization of cellular maturation stages and right stage for transplantation (i.e. relating in vitro phenotype to safe, functional engraftment)

Issues of heterogeneity will have a profound effect on determining phenotype at the population level and scale up protocols.  Controlling (or even fully defining) the stages of development are difficult even in small cultures, and will require more markers than we currently have.  We need to understand the differences between a hESC culture at one hour post passage and an established culture.  The microenvironment of the culture undoubtedly plays a big role in standardizing and controlling cellular and population responses.  Scaling this process up will be a real challenge.  There are some reports of high density cell cultures yielding up to 1010 or 1011 cells, but achieving this across many different cell types and “on demand” is not yet possible.  There seem to be various recipes circulating for optimizing growth and/or differentiation within large culture formats, but there is little standardization—it’s very much still an art.

There are some very practical “non-scientific” issues that need to be clearly defined in order for the community to fully share their lines and information.  Intellectual property limitations (e.g. reach through rights and responsibilities) must be clearly defined if investigators intend to share resources.  It will be particularly important to establish unambiguous guidelines on how “derivatives” and information from non-approved cells lines might be used in federally funded research.  Such considerations will be crucial for neuroscientists exploring how hESCs can be differentiated into neurons and glia, etc.

Recommendations

The workshop participants then discussed the above topics in more depth in three afternoon roundtable discussions.  The participants recommended initiatives to address three identified needs:

  1. Validated methods for defining the stages of neural/glial cell lineages
  2. Robust gene targeting methods and reporters for hESCs
  3. The production of large quantities of well-characterized differentiated neural and glial cells from hESCs

Identified Need #1: Validated methods for defining the developmental stages of neural/glial cell lineage

  • Needed Tools and Resources
  1. Develop consistent criteria for defining stages, both undifferentiated and differentiated
  2. Develop assorted profiling assays
  3. Generate antibody markers
  4. Develop high throughput in vitro functional assays that predict in vivo activity
  5. Develop the ability to finely control targeted differentiation on demand

Identified Need #2:  Robust gene targeting methods and reporters for hESCs

  • Needed Tools and Resources
  1. Develop gene targeting methods for hESCs
  2. Develop a distribution system to share gene-targeted cell lines
  3. Develop a toolbox of gene targeting methods and reagents specific to hESCs

Identified Need #3: The production of large quantities of well-characterized differentiated neural and glial cells from hESCs

  • Needed Tools and Resources:
  1. Develop  a database of standardized “culture systems”
  2. Develop measures and standards of safety
  3. Develop high throughput genetic screens
  4. Extend conditions to 3D cultures and scaffolds

Conclusion

The BP IC Directors commended the team for a successful workshop.  The workshop report and recommendations will now be reviewed by the BP IC Directors and the BP Coordinating Committee to consider whether opportunities exist for Blueprint for Neuroscience involvement in supporting the development of shared tools and resources in this important area.