Abstract
Current studies involving the clinical application of genetically modified hematopoietic stem cells (HSC) require an ex vivo culture period during which the cells must maintain their ability to self- renew, repopulate the bone marrow, and produce the genetically corrected progeny cells. Recent advances in the formulation of optimized serum-free HSC tissue culture media led us to compare several recently commercially available culture media with the medium used in our current clinical gene therapy protocols for the transduction of HSC. We compared expansion, transduction efficiency, and differentiation of both cord blood (CB) and bone marrow (BM) derived CD34+ cells using X-Vivo15 (BioWhittaker, supplemented with 1% HSA), Cellgro|[reg]| SCGM (Cellgenix), StemSpan|[trade]| H300 (Stemline Technologies), Stemline|[trade]| I (Sigma- Aldrich), and Stemline|[trade]| II (Sigma-Aldrich). We observed comparable cell viability among the various media when using BM and CB derived CD34+ cells after 4 days of culture. Both BM and CB CD34+ cells cultured in X-Vivo 15, Cellgro|[reg]| SCGM, and Stemline|[trade]| II expanded to a greater extent than cells cultured in StemSpan|[trade]| H3000 or Stemline|[trade]|. Higher transduction efficiency using a retrovirus containing the eGFP gene was also observed in X- Vivo 15, Cellgro|[reg]| SCGM, and Stemline|[trade]| II media. A direct comparison between BM and CB CD34+ cells cultured in X-Vivo 15 or Stemline|[trade]| II resulted in 23% (BM) and 27% (CB) more transduced cells in the Stemline|[trade]| II medium. We then examined the clonogenicity of the transduced BM CD34+ cells in a Colony Forming Unit (CFU) assay. BM CD34+ cells cultured in Stemline|[trade]| II medium displayed a two-fold higher (16%) plating efficiency in the CFU assay compared to BM CD34+ cells cultured in X-Vivo 15 (8-9.5%). The number of colonies that were transduced (eGFP+) was higher in the BM CD34+ cells cultured in Stemline|[trade]| II medium compared to those cultured in X-Vivo 15 (29.9% and 10.3% respectively). From these results it is evident that BM CD34+ cells cultured in Stemline|[trade]| II medium have both a higher percentage of transduced cells and a higher percentage of these transduced cells remain capable of producing colonies in a CFU assay when compared with X-Vivo 15. From these data, Stemline|[trade]| II appears to be a more suitable medium for the ex vivo culturing and transduction of HSC for use in clinical therapies.
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