In vitro maintenance and retroviral transduction of human myeloma cells in long-term marrow cultures

A. Keith Stewart, H. Miles Prince, Darrin Cappe, Peter Chu, Carolyn Lutzko, D. Robert Sutherland, Ian D. Dubé

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplant is attributable to contamination of the autograft with myeloma cells. A requirement in these studies is ex vivo genetic marking of malignant cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of clonogenic myeloma cells in marrow aspirates from 14 patients with MM. To effect gene transfer we utilized a long-term marrow culture (LTMC) system previously shown to facilitate gene transfer into a spectrum of hematopoietic progenitor and stem cells. Transduction of cells in LTMC was performed by multiple supernatant exposure. At LTMC initiation and after 21 days of culture malignant cells were assessed by morphology, flow cytometry, and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoietic progenitors as determined by the number of colonies positive for proviral DNA by PCR, G418 resistance, and X-gal staining was also within the expected range; 65%, 44% and 23%, respectively. Thus, there was no evidence that prior chemotherapy exposure or malignant cell contamination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 21-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 12 informative patients (50%). PCR using allele-specific primers when available confirmed the specificity of IgVH rearrangements for the myeloma clone. In 2 of the 14 patients, expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for transfer of reporter genes (neor and LacZ) into the myeloma clone: morphologically abnormal G418-resistant colonies demonstrated intense staining for β-galactosidase, and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patient, individual colonies positive for β-galactosidase bore a cytogenetic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primers was positive. Our results demonstrate the maintenance of myeloma cells in vitro for up to 21 days in LTMC. They further illustrate that these cells can be genetically marked using transduction protocols currently being tested in clinical trials of hematopoietic cell gene transfer.

Original languageEnglish (US)
Pages (from-to)148-156
Number of pages9
JournalCancer Gene Therapy
Issue number3
StatePublished - Dec 1 1997


  • Gene marking
  • Marrow culture
  • Myeloma
  • Retrovirus

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Cancer Research


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