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Stem Cell Information

Summary of NIH Stem Cell Unit 08/21/2003 Meeting

NIH Stem Cell Unit
Steering Committee Conference Call
Meeting Summary
Thursday, August 21, 2003
12:30 p.m. EST

Participants: James F. Battey, Jr., M.D., Ph.D. (chair); Ronald D.G. McKay, Ph.D. (Unit Director); J. Carl Barrett, Ph.D.; David M. Bodine, Ph.D.; Marvin Gershengorn, M.D.; Robert G. Hawley, Ph.D.; Story C. Landis, Ph.D.; Mahendra Rao, M.D., Ph.D.; Janet Rossant, Ph.D.; Anne White-Olson; Baldwin Wong; and Lisa Montney, Ph.D.

Unable to Participate: Curt Civin, M.D., and Brigid L.M. Hogan, Ph.D.

  1. Welcome—Dr. Battey and Dr. McKay

    Dr. Battey introduced the meeting by thanking the committee for helping to manage the new NIH Stem Cell (SC) Unit. The purpose of the Unit is to have a side-by-side comparison of the available Human Embryonic Stem Cell (hESC) lines on the NIH Human Embryonic Stem Cell Registry. The Unit's main goal is to identify—and share with the research community—the similarities and differences between the available hESC lines when subjected to a standardized paradigm.

    Dr. Battey discussed the status about the International SC effort. There are SC Units in Canada and the UK (Dr. Peter Andrews). The UK will be able to compare hESC lines within and outside of the NIH Registry. Dr. Rossant announced that the Canadian SC Network is a federally funded effort to bring together Canadian scientists that are studying hESC and adult stem cell lines. There is funding for deriving, scaling-up and characterizing hESC lines. The Network is interested helping with the International effort toward standardizing the characterization of cell lines. Dr. Battey expressed a need for continued coordination with the UK and Canada as the NIH SC Unit develops. He also expressed a need for standardization in 1) assay conditions and 2) monoclonal antibodies for cell surface characterization, and sources for those antibodies.

  2. Stem Cell Unit Status Report—Dr. McKay and Dr. Battey

    Dr. McKay and Dr. Battey outlined the status of the NIH Stem Cell Unit. They announced that the Unit is staffed and work is underway, with some cell lines growing. Dr. McKay estimates that six of the available 12 hESC lines will be analyzed and characterized within a year. The basic project plan of the SC Unit is to: 1) develop standardized methods to grow the cell lines or develop simplified growth protocols, 2) test for pathogens, 3) test for normal karotype, 4) develop an assay to monitor the cell pluripotent state (collaboration needed here to establish a standardized assay to serve as a marker of the pluripotency of the cell; Dr. McKay has spoken with Peter Andrews and Martin Pera about collaborations), and 5) establish a genomic fingerprint for each line and develop gene expression technology.

    Mouse fibroblast feeder cells are also being grown in the SC Unit. Stem cell growth under feeder and feeder-free conditions will be tested.

  3. Discussion of stem cell characterization assays—all participants

    Dr. Battey asked about using genomic "fingerprint" technology to identify each cell line and how to perform the protocols. Dr. McKay described the following techniques available: 1) DNA tests similar to those used by FBI labs or 2) Comparative Genome Hybridization (used by Thomas Reid at NCI, and UCSF labs). The Committee highly advised the Unit in conducting both assays. The cells currently growing in the SC Unit have a normal karotype. HLA typing (Immune typing) was also suggested. Dr. Landis suggested that the SC Unit could utilize the resources at the NIH Clinical Center for HLA typing. Dr. Battey suggested that the Unit should use NIH-wide resources to get the work done and the Steering Committee was in favor of the Unit exploring those collaborations.

    Gene Expression Techniques

    Dr. Rao said NIH is working on characterizing the cell line genomes (collaboration with FDA). The following techniques were also discussed as possible assays for the Unit:

    1. Lynx Massively Parallel Signature Sequencing (MPSS)™
      Dr. Rao and a Singapore group are currently doing MPSS on hESCs. The NCI may start soon. MPSS is highly quantitative (gives a Digital database to X per million). MPSS analyses the level of gene expression in a sample by counting the absolute number of individual mRNA molecules produced from each gene. MPSS involves a set of new technologies for cloning and sequencing a 16-20 base signature sequence from more than a million molecules simultaneously.

      Protocol Features

      • 2 million sequences
      • can get standard deviation on the data
      • great for seeing rare mRNAs
      • medium level expression at decent quantitative accuracy
      • get a taste for rare transcripts
      • possible Issues with MPSS:
        1. extracting enough RNA
        2. costly ($30K per assay)

       

    2. Affymetrix™ GeneChip®
      Affymetrix is widely used worldwide. This would allow for easier implementation.

    3. Serial Analysis of Gene Expression (SAGE)
      It was noted that Dr. Civin should be consulted about SAGE technology for hESC. SAGE focuses attention on the differences between the cell lines.

      Protocol Features

      • couple hundred thousand sequences; i.e., 50,000 transcripts are quantified per analysis
      • highly expressed items at high quantitative accuracy
      • medium level expression at decent quantitative accuracy
      • get a taste for rare transcripts

       

    4. cDNA libraries

      Dr. Rao noted that the FDA has just published guidelines for researchers wishing to use cells for therapy. Participants agreed that it would be good for the Unit to consult these guidelines when determining which assays to perform.

      Dr. Battey noted that approximately half of the mRNA transcripts in a cell are rare. The NIH SC Unit does not want to limit itself to a less stringent assay that will not be able to provide the amount of information needed. Lynx™ and Affymetrix™ are receptive to putting the data into the public domain. The FDA is receptive to the idea of developing a gene chip if funding is available.

  4. Final suggestions of the Steering Committee—all participants

    Dr. McKay expressed his goal of establishing a reputation for the Stem Cell Unit as a top worldwide resource for tools and techniques to determine the characteristics of hESCs.

    It was suggested that the data from the NIH SC Unit be posted on the Internet as soon as it was verified. It was also suggested that the NIH SC Unit post this information under the existing NIH stem cell Web site. If needed, a separate Web site for the Unit may be appropriate in the future.

    FDA/CBER has recently issued draft guidance for public comment on Human Somatic Cell Therapy Investigational New Drug Applications. It was suggested that the NIH SC Unit should consider testing the hESC lines for the pathogens listed in the FDA guidance. It was noted that c-type particles are not mentioned in the guidance, but nonetheless, the hESC lines should be screened for c-type particles. The Unit proposes to maintain contact with UK SC Bank, which also has a strong pathogen analysis program for stem cells (UK-Dr. Andrews, Australia-Martin Pera, and Canada-Peter Zandstra).

Dr. Battey thanked everyone for their participation on the conference call. The meeting adjourned at 1:10 p.m.