Chromosomal Instability in Cultured Mouse Hematopoietic Cells Associated with Oxidative Stress

Abstract 4819

Hematopoietic stem cells (HSCs) that give rise to all blood cell types are important vehicles for cell-based and gene therapies. After isolation from the bone marrow, HSCs are often cultured in laboratory settings for purposes of ex vivo expansion, gene transduction, and bone marrow transplantation for the treatment of various disorders of the blood and immune systems. While undergoing proliferation and differentiation in vitro, test tube and dish culturing can potentially induce genomic instability in HSCs due to prolonged culturing periods or the exposure to increased levels of oxygen. Here we demonstrate that in vitro culturing outside their bone marrow niches, HSCs may change even under very short durations of time. Lineage⁻ Scal-1⁺ c-Kit⁺ (LSK) cells that are enriched with HSCs revealed significant levels of genomic instability in culture, as evidenced by the emergence of aneuploidy cells. To further determine the effects of in vitro culturing conditions, whole bone marrow cells were cultured in a hypoxic environment of 2–3% oxygen, mimicking conditions inside the body's bone marrow. In this case, cells proved to undergo less genetic alterations. Proper dosages of the antioxidant N-Acetyl-Cysteine (NAC) similarly decreased occurrences of chromosomal changes. Furthermore, in vitro normoxic culture-induced chromosomal instability was enhanced in aged hematopoietic cells compared to that in young hematopoietic cells due to noted increased oxidative stress in aged cells. These results reveal that in vitro cell culturing does indeed cause genomic instability in hematopoietic cells. Reduced oxygen levels and additions of antioxidants can be employed as a possible agent to lower oxidative stress and decrease chances of transformation. Additionally, since hematopoietic cells are commonly developed in laboratory settings before transplantation for patient treatment, our findings raise a concern for using cultured hematopoietic cells for therapeutic purposes.

Note: Alice Liu and William Qu contributed equally to this work.