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E. Henry et al.
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Figure 1. Low doses (LD) of ionizing radiations (IR) exposure of human hematopoietic stem progenitor cells (HSPC) leads to deficient serial colony forming unit- cell assay (CFU-C) and primary and extended long-term culture initiating cell (LTC-IC) potentials. CD34+ CD38low CD45RA– CD90+ HSPC were sorted from pools of independent cord blood (CB) samples by cell sorting and exposed to the indicated IR doses prior to in vitro cultures. (A) LTC-IC assay in limiting dilution (pool of 2 experiments, 120 wells/IR dose). Irradiated CD34+ CD38low CD45RA– CD90+ HSPC were seeded on MS5 stromal cells in limiting dilution for five weeks then plated in methylcellulose for 12 days. LTC-IC frequency was calculated using LCALC software. (B) Primary CFU-C assay (cumulative results from 4 independent experiments with HSPC isolated from 4 independent pools of CB samples). HSPC (500 cells/plate) were plated in CFU-C condition for 12-14 days and the number (nb) of CFU-C was quantified. Results are normalized to the non-irradiated conditions. (C) Primary CFU-C were pooled and replated in methylcellulose for 12-14 days. Shown are the nb of secondary CFU-C. Results are normalized to the sham-irradiated conditions (cumulative results from 3 independent experiments). Results are shown as mean±standard error of mean. **P<0.01, ***P<0.001, ****P<0.0001 (Mann-Whitney statistics).
non-parametric statistical analyses were used. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Results
A 20 mGy dose of irradiation decreases serial replating capacity of human hematopoietic stem/progenitor cells
To understand the impact of HSPC exposure to LDIR, we first performed serial long-term culture initiating cell (LTC-IC) and CFU tests as surrogate assays to study human HSPC properties, i.e. self-renewal and differentia- tion capacities.25-27 Human CD34+CD38lowCD45RA–CD90+ HSPC were purified, irradiated and cultured with the MS5 stromal cell line in LTC medium for five weeks or plated directly in semi-solid methylcellulose cultures. In LTC-IC limiting dilution analyses, every single well was harvested independently at the end of the culture and plated in CFU- C assay. The LTC-IC frequency obtained after a 20 mGy irradiation of HSPC was similar to the LTC-IC frequency of sham-irradiated HSPC (1/15) suggesting that LDIR do not affect LTC-IC frequency. In contrast, a high (2.5 Gy) irradiation dose induced a drastic drop in LTC-IC frequen- cy (1/272) (Figure 1A). In addition, 2.5 Gy-irradiated HSPC directly seeded in CFU-C conditions after IR produced
very few colonies compared to control cells whereas 20 mGy-irradiated HSPC generated a similar number of CFU- C to sham-irradiated HSPC (Figure 1B and Online Supplementary Figure S1A). No difference in the quality of CFU-C was observed between sham and 20 mGy condi- tions (Online Supplementary Figure S1B). These primary LTC-IC and primary CFU-C assays show that 20 mGy LDIR do not alter HSPC frequency and clonogenicity in vitro and do not induce any myelo/erythroid differentia- tion bias in primary cultures (Online Supplementary Figure S1B). To characterize whether LDIR have deleterious effects on human HSPC self-renewal potential in vitro, seri- al replatings were performed for both CFU-C and LTC-IC assays.25-27 We observed that 20 mGy and 2.5 Gy-irradiat- ed CD34+CD38lowCD45RA–CD90+ HSPC produced lower numbers of secondary colonies in contrast to sham-irradi- ated HSPC, showing that 20 mGy alters their serial clono- genic potential (Figure 1C and Online Supplementary Figure S1C). This result was also obtained after picking up and replating individual primary CFU-GM colonies (Online Supplementary Figure S1D). In the case of LTC-IC, second- ary/extended cultures were initiated using cells expressing high CD34 surface expression (CD34hi), purified after the initial five weeks of LTC-IC culture (Online Supplementary Figure S1E) and seeded for five additional weeks in LTC- IC conditions at limiting dilution. Of note, we were not
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haematologica | 2020; 105(8)