Proliferation and functional characterization of human CD34+ bone marrow cells cultured in simulated microgravity

Abstract

The ability of the rotating wall vessel (RWV) bioreactor, developed by NASA for benchtop simulation of microgravity (μ-G), to support expansion of primitive human hematopoietic progenitor cells (HPC) was assessed during short-term cultures. Human bone marrow (BM) enriched for CD34+ cells (mean purity = 85%, n = 3) was seeded at 1.1 to 2.0 × 10e5 cells/mL in 10 mL in RWV, and incubated in the presence of SCF, IL-3, IL-6, GM-CSF, Flt-3L, and MGDF in either simulated μ-G or earth gravity (1-G). Proliferation of these cells after 5 days of culture averaged 11± 10-fold in μ-G, compared to 4.1 ± 3-fold in 1-G control cultures. Expansion of CD34+ cells was also greater in μ-G cultures compared to 1-G (2.6 ± 2.6 vs. 1.0 ± 0.8, respectively), as well as expansion of more primitive CD34+ CD38−/lo cells (1.9 ± 1.4 vs. 0.8 ± 0.6, respectively). To further examine the hematopoietic potential of CD34+ cells propagated in μ-G, long-term repopulating potential was assessed in NOD/SCID recipients. Conditioned NOD/SCID mice were transplanted with either fresh day 0 CD34-enriched BM cells (570K to 990K/mouse), or progeny of equivalent cell numbers expanded in μ-G (2.5 to 11.4 × 10e6/mouse) or 1-G (1.5 to 3.9 × 10e6/mouse). Fresh CD34+ cells provided 39 ± 14% chimerism at 8 weeks of post-transplantation. Despite possessing in μ-G were deficient in NOD/SCID-repopulating potential compared to 1-G controls (2.1 ± 1.4, n = 4 vs. 14.8 ± 13, n = 4, respectively). To examine whether diminished homing may contribute to the reduced engraftment of μ-G cells, adhesion molecule expression was analyzed. While no differences were found between μ-G and 1-G CD34+ cells in their adhesion molecule repertoire, these cells expressed considerably different levels of CD11a, CD43, CD44, CD49d, CD49e, CD62L, and CXCR4 in comparison to day 0 CD34+ cells, possibly contributing to their reduced engraftment potential. These results in simulated μ-G shed further light on stem cell biology, and may begin to explain some of the hematologic changes occurring in humans exposed to true microgravity.