Multiparameter-fluorescence activated cell sorting analysis of retroviral vector gene transfer into primitive umbilical cord blood cells

G. B. McCowage, K. L. Phillips, T. L. Gentry, S. Hull, J. Kurtzberg, E. Gilboa, C. Smith

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

Retroviral vector gene transfer strategies are currently being developed to treat a variety of hematopoietic disorders. To date, genetic modification of human pluripotent hematopoietic stem cells has been inefficient. In the present study we developed reagents and procedures for rapidly screening retroviral vector gene transfer conditions using a multiparameter fluorescence-activated cell sorting (FACS) assay. To identify transduced cells using FACS analysis, we developed a retroviral vector, termed MN, which stably expressed high levels of a truncated version of the low-affinity nerve growth factor receptor (LNGFR). In addition, procedures were developed for enriching CD34+ cells from cryopreserved umbilical cord blood. These cells were transduced with MN and evaluated using multiparameter FACS analysis for expression of CD34, CD38, and LNGFR. Stem cell maintenance was determined by measuring the CD34(hi) and CD34(hi)CD38(lo/-) cells remaining after ex vivo gene transfer. Gene transfer into these cells was measured by evaluating cells expressing high levels of LNGFR. Initial studies with this assay and with in vitro functional assays indicated that retroviral gene transfer following pre-incubation with a variety of cytokines in serum containing conditions resulted in 1) poor maintenance of hematopoietic stem cells and 2) gene transfer predominantly in relatively mature cells. When gene transfer in serum-free conditions was performed, some improvement was observed in the maintenance of cells retaining primitive immunophenotypes with no reduction in the gene transfer efficiency. The MN vector and multiparameter FACS analysis will be useful in efficiently screening ongoing efforts designed to improve stem cell gene transfer.

Original languageEnglish (US)
Pages (from-to)288-298
Number of pages11
JournalExperimental Hematology
Volume26
Issue number4
StatePublished - 1998
Externally publishedYes

Keywords

  • Gene therapy
  • Retroviral vector
  • Stem cell
  • Umbilical cord blood

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

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