Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates

Abstract

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.

Figure 1

(a) In vitro cell growth of PB CD34⁺ cells. The CD34⁺ cells were cultured in the presence of MGDF/SCF/FLT/FN for 4 days. On day 4 the cells were split into three equal fractions. One was continued in culture in the presence of SCF/MGDF/FLT/FN (MSF/MSF). The second was transferred to SCF/FN (MSF/SCF). The third was maintained without cytokines on FN (MSF/FN). Shown are the mean ± SD of viable cell numbers obtained in independent experiments using cells from three different animals. (b) Cell cycle analysis. PB CD34⁺ cells were stained with PI and analyzed for DNA content. The percentage of cells in S/G2/M phases of the cell cycle are shown. A significantly lower percentage of cells in active cycle with MSF/SCF treatment than with MSF/MSF at days 6, 8, and 10 (P < 0.05 for each time point). (c) Cell cycle analysis of PB CD34⁺ cells cultured and transduced for 4 days in the presence of MGDF/SCF/FLT/FN with a retroviral vector expressing the eGFP gene, and then either continued in MGDF/SCF/FLT/FN without further transduction (MSF/MSF) or transferred to SCF/FN (MSF/SCF). The percentage of GFP-positive cells in the S/G2/M phases of the cell cycle are shown. (d) Apoptosis analysis. The percentage of apoptotic cells is shown for cells cultured with SCF/MGDF/FLT/FN for 4 days, and then either continued in the three cytokines (MSF/MSF), transferred to SCF/FN (MSF/SCF), or transferred to FN alone (MSF/FN). Transfer to FN alone (MSF/FN) resulted in a significant increase in apoptosis compared with both MSF/MSF and MSF/SCF (P < 0.005 for both comparisons).

Figure 2

Experimental design. In each animal, if G1Na was used to transduce one fraction, LNL6 was used to transduce the other fraction. For each subsequent animal, the vector used to transduce each fraction was alternated.

Figure 3

Analysis of engraftment with transduced cells in vivo. (a) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). DNA from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H₂O: reagent control. (b) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with KpnI, an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.

Figure 4

Summary of in vivo marking levels. DNA marking in PBMCs and PB granulocytes (PB Gr) calculated from PCR analysis, assuming one vector copy per cell, for 96E025, 96E019, and RC706. Animals received cells that were transduced for 4 days in MGDF/SCF/FLT/FN (active), and cells transduced for 4 days and cultured for an additional 2 days in SCF/FN without further vector exposure (rested). The percentage of marked cells was calculated from individual values of neo/β-actin band intensities plotted on the regression curve generated from the G1Na controls. Active: marking level from the vector used to transduce cells cultured for 4 days in MGDF/SCF/FLT/FN; Rested: marking from the vector used to transduce cells cultured in SCF/MGDF/FLT/FN followed by an additional 2 days without vector in SCF/FN.

Figure 5

In vivo analysis of 6-day stimulatory culture versus 4-day stimulatory culture followed by 2 days in SCF/FN. PCR analysis of DNA from an animal receiving cells cultured for 6 days in the presence of the G1Na vector and SCF/MGDF/FLT/FN, versus cells cultured for 4 days in the presence of LNL6 vector and SCF/MGDF/FLT/FN followed by culture without vector for two additional days in the presence of SCF and FN.