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Retroviral gene transfer into cord blood stem/progenitor cells using purified vector stocks
Author(s) -
Asch Julie,
Weinberg Rona S.,
Mueller Lisa,
Galperin Yelena,
Kiang Lily,
Jolly Douglas,
Isola Luis M.
Publication year - 1998
Publication title -
american journal of hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.456
H-Index - 105
eISSN - 1096-8652
pISSN - 0361-8609
DOI - 10.1002/(sici)1096-8652(199801)57:1<16::aid-ajh3>3.0.co;2-4
Subject(s) - transduction (biophysics) , multiplicity of infection , biology , microbiology and biotechnology , progenitor cell , ficoll , cord blood , viral vector , stem cell , cd34 , genetic enhancement , peripheral blood mononuclear cell , fetal bovine serum , flow cytometry , transfection , cell culture , immunology , gene , in vitro , biochemistry , genetics , recombinant dna
Cord blood (CB) progenitor/stem cells (P/SC) are ideal targets for early gene therapy in individuals prenatally diagnosed with genetic disorders. Most retroviral transduction protocols were developed using adult peripheral blood stem cells (PBSC) and bone marrow (BM). Less is known about retroviral transduction of CB P/SC. We examined how timing, multiplicity of infection (MOI), and polycations in the transduction media affect transduction efficiency. Rates of transduction were determined in recently isolated CD34 + enriched CB cells and in colonies derived after various times in liquid cultures (LC). CB mononuclear cells (MNC) were separated by ficoll‐hypaque centrifugation and enriched for CD34 + cells. Purity was assessed by flow cytometry. Transduction were performed with clinical‐grade retroviral stocks at MOIs of 1–20. Transduction was performed with fetal bovine serum (FBS) or autologous plasma, IL‐3, GM‐CSF, IL‐6, and SCF. The retroviral vector contained LacZ and neomycin resistance ( neo ) reporter genes. Transduction was determined by X‐gal stain and by PCR amplification of the reporter genes. No drug selection was used. Twenty‐five experiments were done. CB volumes ranged from 35–150 ml. MNC and CD34 + cell counts ranges were: 0.14–840 × 10 6 and 0.1–4.2 × 10 6 , respectively. Transduction efficiency in liquid cultures ranged from 4–63%. Higher rates were seen using MOI ≥ 10, 2 μg/ml polybrene, and 10% autologous CB plasma. In colonies, transduction rates were 63 to 72% by PCR and 32% by X‐gal staining. In LTC‐IC derived colonies, transduction was 7% by PCR. Short incubations of CD34 + CB cells with purified retroviral stocks, polybrene, and autologous sera result in high transduction rates of committed progenitors and moderately low efficiencies of transduction of LTC‐IC in the absence of drug selection. Am. J. Hematol. 57:16–23, 1998. © 1998 Wiley‐Liss, Inc.