Highly efficient retroviral transduction of cord blood hematopoietic stem cells

Retroviral transduction of hematopoietic stem cells(HSC) requires that these cells divide in vitro while retaining their primitive phenotype. We have recently developed the Myeloid-Lymphoid Initiating Cell (ML-IC) assay (Punzel et al, Blood 93(11)), an in vitro culture system that allows for enumeration of single cells capable of generating secondary progenitors that can re-initiate long term myeloid and lymphoid cultures. The ML-IC is therefore believed to be closely related to HSC. CD34+CD38−CD33− cord blood cells were plated in a stroma noncontact culture in CH296 coated transwells above AFT024 stromal feeders in a Flt 3, SCF, IL7 and Tepo containing medium. After 48 hours, and again after 72 hours, they were exposed for 6 hours to GALV pseudotyped MFG-eGFP containing viral supernatant. After a total of 5 days, CD34+CD38−CD33− cells were single cell sorted to assess ML-IC frequency and transduction efficiency. On average there was a 2-fold expansion of total cell number. 14.6±11.3% retained the CD34+CD38−CD33− phenotype, and of these 80.0±14.3% were GFP+, whereas only 39.7±16.1% of the total cell population was GFP+ (p=0.016). Within the CD34+ CD38−CD33− fraction, the ML-IC frequency was 4.3±1.8%, and 73.9±15.7% of these were transduced. In 16% of the transduced ML-IC, GFP expression was silenced in the myeloid arm but present in the NK-IC. 5/69 ML-IC arising from cells in the GFP− population on day 5 proved transduced, i.e. GFP+, upon read-out. The generative potential of the ML-IC, defined as the fraction of ML-IC that generates 2 LTC-IC and 2 NK-IC, rather than just 1 of each, was similar for GFP+ and GFP− ML-IC (39.6±20.5% vs. 51.3±11.8%, p=0.24). In conclusion, we describe a transduction protocol that efficiently targets primitive progenitors, while retaining their generative potential. Selection of GFP+ cells shortly after transduction may lead to underestimation of transduction efficiency. Finally, the ML-IC assay proves to be useful in studying differential silencing in different lineages arising from a single cell.