Cell-cycle kinetics and VSV-G pseudotyped retrovirus-mediated gene transfer in blood-derived CD34+ cells

Area of Science:

• Hematology
• Molecular Biology
• Gene Therapy

Background:

• Hematopoietic stem and progenitor cells (CD34+) have a low cell division rate.
• Efficient ex vivo gene transfer requires understanding factors influencing cell cycle entry.

Purpose of the Study:

• To quantify the impact of cytokine combinations on CD34+ cell cycling.
• To correlate cell cycle status with retroviral gene transfer efficiency.

Main Methods:

• Tested nine cytokine combinations for CD34+ cell cycle induction over 72 hours.
• Assessed cell cycle kinetics using the 5-bromodeoxyuridine-Hoechst 33258 (BrdU-Hoechst) assay.
• Correlated gene transfer efficiency using VSV-G pseudotyped retroviral vectors and PCR analysis of reverse-transcribed viral DNA (RT-neoDNA).

Main Results:

• Stem cell factor (SCF) combined with IL-1, IL-3, or G-CSF most effectively induced CD34+ cell cycling, with peak S+G2M phase at 48 hours.
• A significant portion (35+/-5%) of CD34+ cells remained quiescent regardless of cytokine combination.
• The CD34+CD38- subset did not enter the cell cycle and showed no detectable RT-neoDNA post-infection.
• Retroviral integration (RT-neoDNA) was detectable in CD34+ cells immediately after viral exposure, but only persisted in cells cultured with mitogenic cytokines.

Conclusions:

• Specific cytokine combinations can induce CD34+ cell cycling, enhancing retroviral integration.
• The CD34+CD38- subset exhibits resistance to retroviral reverse transcription, independent of cell cycle status.
• Short-term liquid cultures may present additional blocks to retroviral RNA reverse transcription in CD34+CD38- cells.