To determine the impact of in vitro culture on hematopoietic stem cells (HSCs), we isolated LSK cells (Lin- Sca-1+c-Kit+, a population enriched with HSCs) from mouse bone marrow samples and cultured them under normoxic conditions (20% O2) for two and six days. The chromosome numbers were determined by traditional karyotypic analysis. Assessment of LSK cells after the two day culture noted nearly 50% aneuploid cells (cells with more or less than 40 chromosomes seen in normal cells) ( and ). Following six days of culture, even greater levels of abnormalities were observed (). To further confirm these results, sorted LK cells (Lin- c-Kit+ cells composed primarily of myeloid progenitors) and whole bone marrow cells were also cultured for various periods of time, and similar results were obtained ( and ). These observations suggest that in vitro culture of hematopoietic cells under normoxic conditions causes significant chromosomal instability.
Figure 1 Aneuploidy in hematopoietic cells cultured under normoxic conditions. LSK cells that are enriched with HSCs and LK cells (myeloid progenitors) were sorted from freshly harvested bone marrow cells. A. Representative metaphase spreads with normal (top) (more ...)
Once cells are isolated from the body and cultured in an environment with a higher oxygen concentration (20%), they are exposed to greater levels of oxidative stress and resultantly generate more ROS. Recent evidence has revealed that HSCs in the bone marrow are located in a hypoxic environment known as the stem cell niche [6
]. The niche protects stem cells from oxidative stress and the accumulation of ROS and other free radicals, which are known to cause DNA damage and induce genomic instability [10
]. As a result, only the physiological levels of intracellular ROS can maintain genomic stability in stem cells [5
]. It has also been shown that low oxygen levels can enhance the survival and self-renewal of HSCs during in vitro
]. Thus, these studies now raise the possibility that chromosomal instability of in vitro
cultured LSK cells might be caused by the dramatically different oxygen environment compared to that of the stem cell niche. To test this hypothesis, we cultured LSK cells under hypoxic (3% oxygen) and normoxic (20% oxygen) conditions, and karyotypic analyses were performed after 2 days. The percentage of aneuploid LSK cells was decreased when cells were cultured in the lower oxygen concentration (), indicating that the physiological oxygen level is required for maintaining the chromosomal integrity of hematopoietic cells. The chromosome numbers of LK cells and bone marrow cells were also compared under states of hypoxia and normoxia. Similarly, the frequency of chromosomal instability was reduced after hypoxic culture ( and ), indicating that karyotypic abnormalities in cultured hematopoietic cells are closely related to oxidative stress.
Figure 2 Aneuploidy in cultured hematopoietic cells is associated with oxidative stress. LSK cells that are enriched with HSCs and LK cells (myeloid progenitors) were sorted from freshly harvested bone marrow cells. A. LSK cells and LK cells sorted were cultured (more ...)
To further confirm these results, hematopoietic cells cultured under normoxic conditions were treated with antioxidant N-acetyl-L-cysteine (NAC) and their numbers of chromosomes were also determined. Consistent with the results obtained under hypoxic conditions, the percentage of aneuploid cells was dramatically lower compared to untreated control cells (). However, the rescue effect was only found at a low dosage (0.1 μM) but not at higher dosages (0.5 and 1 μM) (). This may be explained by the recent finding that optimal genomic stability of hematopoietic cells is maintained only in a narrow range of ROS levels. Higher dosages of antioxidants suppress ROS to subphysiological levels which thus may not be sufficient to activate the DNA repair pathway to maintain genomic stability [5
Oxidative stress-induced DNA damage and genomic instability increase with age and are considered as major causal factors in cancer and other age-related diseases [15
]. Consistent with this thought, bone marrow cells from 2-year -old mice showed more aneuploidy than those of 2-month-old mice immediately after isolation from the body (). Interestingly, the difference between young and old mice was even more apparent after in vitro
culture for one day under normoxic conditions, indicating that cells from old mice are more prone to oxidative stress-induced chromosomal instability. Given that ROS are a major source of oxidative stress, the levels of ROS in bone marrow cells from young and old mice were determined by 2’, 7’- dichlorfluorescein-diacetate (DCF-DA) staining and flow cytometric analysis. After one day culture, the ROS levels increased in cells from both age groups of mice (). However, the cells derived from old mice accumulated more ROS, thereby inducing the higher frequency of aneuploidy previously noted (). To further confirm that the chromosomal instability was caused by oxidative stress, bone marrow cells from young and old mice were treated with antioxidant NAC, and the numbers of chromosomes were again counted after karyotypic analyses. Addition of NAC decreased the occurrences of aneuploidy in cells from both young and aged mice, further suggesting that chromosomal instability in cultured hematopoietic cells is related to oxidative stress ().
Figure 3 The cells derived from aged mice are more prone to oxidative stress-induced chromosomal instability. A. Bone marrow cells were freshly harvested from 2-month-old or 24-month-old mice. Bone marrow cells were cultured under normoxic conditions for one day. (more ...)