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Exposure to 20 mGy radiation decreases HSC functions
A
B
Gated on hCD45+ cells P=0.9548
CD
P=0.9284 Gated on hCD45+ cells
P=0.2845
P=0.7300
Figure 2. Hematopoietic reconstitution capacities of human hematopoietic stem cells (HSC) after in vivo exposure to low doses (LD) of ionizing radiations (IR). NSG mice were transplanted with 5.104 CD34+ CB cells and 13 to 16 weeks post graft mice were irradiated or not with the indicated doses then immediately sacrificed. Bone marrow (BM) cells were recovered and characterized by flow cytometry (Online Supplementary Figure S2A and B). An equivalent of 5.104 CD34+ CD19– BM cells were injected in secondary recipient mice. (A) Dot plots of representative engraftment levels (left: human hCD45+ cells) in secondary recipient mice and human engraftment levels obtained in the BM of 9 secondary NSG mice (right: 2 independent experiments are pooled). Results of a third experiment is shown in Online Supplementary Figure S2D since engraftment levels of control mice were lower. (B) Proportion of engrafted human B cells and myeloid cells based respectively on CD19 and CD14/CD15 expression gated in human CD45+ cells, in secondary recipient mice. (C) Human hematopoietic reconstitution of CD34+ CD38low Linneg BM cells purified by cell sorting from primary mice and transplanted in secondary recipient mice. Shown are human cell reconstitution in the BM of secondary recipient mice 13 weeks later. (D) Proportion of engrafted human B cells and myeloid cells gated within human CD45+ cell compartment in secondary mice after human CD34+ CD38low Linneg BM cell transplants. Results are shown as mean±standard error of mean. *P<0.05; **P<0.01, ***P<0.001 (Mann-Whitney statistics).
able to perform such extended LTC-IC with the 2.5 Gy condition due to very low cell quantities in the cultures. Interestingly, there was a 2-fold decrease in the extended LTC-IC frequency of 20 mGy-irradiated HSPC compared to sham-irradiated HSPC (1/275 vs. 1/128) (Online Supplementary Figure S1F) showing a defect in long-term HSPC maintenance. This decrease in secondary LTC-IC frequency induced by 20 mGy LDIR was also found in bulk culture conditions with 20 mGy-irradiated HSPC generating fewer CFU-C than sham-irradiated cells after secondary LTC-IC cultures (Online Supplementary Figure S1G). Taken together, these results suggest that a single exposure to 20 mGy LDIR impairs the in vitro self-renewal potential of human CD34+CD38lowCD45RA–CD90+ HSPC.
A 20 mGy dose of irradiation decreases human hematopoietic stem/progenitor cell hematopoietic reconstitution potential
We then studied the effect of 20 mGy LDIR on in vivo HSC functions. To do so, NSG mice were first engrafted with human CD34+ cells. Sixteen weeks later, once human hematopoiesis was stabilized, engrafted mice were exposed to 0, 20 mGy and 2.5 Gy IR doses and sacrificed immediately after irradiation. Bone marrow (BM) cells were harvested and phenotype analysis was performed. The levels of human CD45+ cells and the percentage of LinnegCD34+ cells recovered from NSG BM were similar in the irradiated and non-irradiated groups (Online Supplementary Figure S2A and B). Furthermore, no increase of apoptosis of human cells engrafted in NSG mouse BM
was observed in mice irradiated at 20 mGy compared to non-irradiated mice (Online Supplementary Figure S2C). To study the consequences of irradiation on human HSC functions (i.e. reconstitution and differentiation potential), BM cells containing 5.104 human CD45+CD34+CD19–cells were transplanted in secondary NSG mice. Human hematopoietic development in the secondary recipient mice was analyzed 13-16 weeks later. Human engraft- ment levels in mice receiving 20 mGy and 2.5 Gy-irradiat- ed BM cells were decreased compared to sham-irradiated BM cells (Figure 2A and Online Supplementary Figure S2D) showing that 20 mGy-irradiated BM cells are less efficient than non-irradiated BM cells to reconstitute human hematopoiesis in secondary recipient mice. However, in the 20 mGy condition, some human cells were still detect- ed in the BM of secondary recipient mice. Among them, human B CD19+ lymphocytes and CD14+/CD15+ myeloid cells were produced at the same proportion than in non- irradiated conditions, indicating that the few HSC that survived LDIR had maintained their differentiation capac- ities (Figure 2B). To exclude a non-cell autonomous effect of LDIR on HSPC mediated by irradiated hematopoietic or non-hematopoietic BM cells during the transplantation, we purified human CD34+CD38low cells from the BM of control and 20 mGy-irradiated mice before transplanta- tion in secondary NSG mice. As in the previous experi- ment, human cell engraftment in secondary recipients showed a reduced hematopoietic reconstitution capacity when 20 mGy-irradiated CD34+CD38low cells were used (Figure 2C) and again no difference in the differentiation
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