Richard Josephson

GlobalStem, Maryland, United States

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Publications (7)26.92 Total impact

  • Richard Josephson, Jonathan Auerbach
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    ABSTRACT: Pluripotent human stem cell lines from embryos or reprogrammed adult cells are not all alike. Cell lines differ widely in their propensity for differentiation, their chromosomal integrity and epigenetic state, immunological profiles, and their availability for research. It is important that all pluripotent cell lines be protected from loss by being properly banked and authenticated, which will also protect current experimental data by enabling its future reproducibility. This unit considers basic guidelines for banking and authentication of pluripotent stem cells that should be easily implementable within any laboratory. Cell Banking is the disciplined preservation of a cell stock in the originally obtained state, as well as stocks representing the baseline state for experimental efforts. Each of these stocks must be authenticated appropriately. Authentication of pluripotent lines verifies five properties: the unique identity of the line, its sterility or freedom from contaminating microorganisms and pathogens, the integrity and stability of its genome, its expression of typical markers of the stem cell phenotype, and its pluripotency upon differentiation. This unit lists and compares several assays to verify each of these stem cell line properties. Thanks to recent advances in molecular biology and the availability of state-of-the-art assays from service providers, the time and material costs of banking and authentication are not excessive for the typical research laboratory.
    Current protocols in stem cell biology 12/2009; Chapter 1:Unit 1C.9.
  • Richard Josephson, Jonathan Auerbach
    04/2009: pages 169 - 202; , ISBN: 9780470454923
  • Richard Josephson
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    ABSTRACT: Regenerative therapies based on transplantation of cells derived from human embryonic stem cells (hESC) are currently being prepared for clinical trials. Unfortunately, recent evidence indicates that many kinds of changes can occur to hESC during expansion in culture, and alterations to the growth control mechanisms may be required to establish hESC lines at all. Changes in the genome and epigenome can affect the validity of in vitro and animal studies, and put transplant recipients at increased risk of cancer. New molecular cytogenetic technologies enable us to examine the whole human genome with ever-finer resolution. This review describes several techniques for whole-genome analysis and the information they can provide about hESC lines. Adoption of high-resolution genotyping into routine characterization may prevent highly discouraging clinical outcomes.
    Expert Review of Molecular Diagnostics 08/2007; 7(4):395-406. · 4.09 Impact Factor
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    ABSTRACT: As the number of human embryonic stem cell (hESC) lines increases, so does the need for systematic evaluation of each line's characteristics and potential. Comparisons between lines are complicated by variations in culture conditions, feeders, spontaneous differentiation, and the absence of standardized assays. These difficulties, combined with the inability of most labs to maintain more than a few lines simultaneously, compel the development of reference standards to which hESC lines can be compared. The use of a stable cell line as a reference standard offers many advantages. A line with a relatively unchanging hESC-like gene and protein expression pattern could be a positive control for developing assays. It can be used as a reference for genomics or proteomics studies, especially for normalizing results obtained in separate laboratories. Such a cell line should be widely available without intellectual property restraints, easily cultured without feeders, and resistant to spontaneous changes in phenotype. We propose that the embryonal carcinoma (EC) line 2102Ep meets these requirements. We compared the protein, gene, and microRNA expression of this cell line with those of hESC lines and alternative reference lines such as the EC line NTERA-2 and the karyotypically abnormal hESC line BG01V. The overall expression profiles of all these lines were similar, with exceptions reflecting the germ cell origins of EC. On the basis of global gene and microRNA expression, 2102Ep is somewhat less similar to hESC than the alternatives; however, 2102Ep expresses more hESC-associated microRNAs than NTERA-2 does, and fewer markers of differentiated fates.
    Stem Cells 03/2007; 25(2):437-46. · 7.70 Impact Factor
  • Richard Josephson, Jonathan M. Auerbach
    Cell Stem Cell. 01/2007; 1(3):251-252.
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    ABSTRACT: Human embryonic stem cells (hESCs) offer a renewable source of a wide range of cell types for use in research and cell-based therapies. Characterizing these cells provides important information about their current state and affords relevant details for subsequent manipulations. For example, identifying genes expressed during culture, as well as their temporal expression order after passaging and conditions influencing the formation of all three germ layers may be helpful for the production of functional beta islet cells used in treating type I diabetes. Although several hESC lines have demonstrated karyotypic instability during extended time in culture, select variant lines exhibit characteristics similar to their normal parental lines. Such variant lines may be excellent tools and abundant sources of cells for pilot studies and in vitro differentiation research in which chromosome number is not a concern, similar to the role currently played by embryonal carcinoma cell lines. It is crucial that the cells be surveyed at a genetic and proteomic level during extensive propagation, expansion, and manipulation in vitro. Here we describe a comprehensive characterization of the variant hESC line BG01V, which was derived from the karyotypically normal, parental hESC line BG01. Our characterization process employs cytogenetic analysis, short tandem repeat and HLA typing, mitochondrial DNA sequencing, gene expression analysis using quantitative reverse transcription-polymerase chain reaction and microarray, assessment of telomerase activity, methylation analysis, and immunophenotyping and teratoma formation, in addition to screening for bacterial, fungal, mycoplasma, and human pathogen contamination.
    Stem Cells 02/2006; 24(3):531 - 546. · 7.70 Impact Factor
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    ABSTRACT: Human embryonic stem cells (hESC) offer a renewable source of a wide range of cell types for use in research and cell-based therapies to treat disease. Inspection of protein markers provides important information about the current state of the cells and data for subsequent manipulations. However, hESC must be routinely analyzed at the genomic level to guard against deleterious changes during extensive propagation, expansion, and manipulation in vitro. We found that short tandem repeat (STR) analysis, human leukocyte antigen (HLA) typing, single nucleotide polymorphism (SNP) genomic analysis, mitochondrial DNA sequencing, and gene expression analysis by microarray can be used to fully describe any hESC culture in terms of its identity, stability, and undifferentiated state. Here we describe, using molecular biology alone, a comprehensive characterization of 17 different hESC lines. The use of amplified nucleic acids means that for the first time full characterization of hESC lines can be performed with little time investment and a minimum of material. The information thus gained will facilitate comparison of lines and replication of results between laboratories.
    BMC Biology 02/2006; 4:28. · 7.43 Impact Factor