Transplant Dose As a Determinant of Proliferative Response and Long-Term Reconstitution of the Hematopoietic Stem Cell Pool

Successful transplantation of hematopoietic stem cells (HSCs) involves both a rapid replenishment of the hematopoietic system through differentiation, as well as a re-establishment of the HSC pool through self-renewal divisions. These processes need to be tightly regulated to balance the acute demands with sustainable hematopoiesis over time.

Here, using transplantation models in mice, we explore how transplant dose affects the proliferative response in HSCs and to what extent the HSC pool regenerates depending on initial transplant dose. We transplanted 10, 50, 200, 1000 or 10 000 purified Lin^-, cKIT⁺, SCA1⁺, CD48^-, CD150⁺ (LSK-SLAM) HSCs, together with 2x10⁵ whole bone marrow cells, into lethally irradiated recipient mice. When analyzing the HSC compartment at 16 weeks, while the total HSC counts were similar for all transplant doses, we observed an increase in numbers of donor HSCs with increasing transplant doses, which plateaued at the 1000 input dose. The 1000 and 10 000 doses showed similar numbers of donor HSCs, indicating a saturation effect. Indeed, while the net HSC expansion from the lower cell doses (10, 50, 200 and 1000) was more than 100-fold, the donor HSCs from the 10 000 dose had expanded significantly less (on average 15-fold). In contrast to homeostatic conditions where the HSC pool can vary in size (Schoedel et al., Blood 2016; Shimoto et al., Blood 2017), our results suggest that the recovery of the HSC pool during regenerative stress is regulated on a systemic level, and that self-renewal and expansion is halted when the size of the HSC pool has reached a certain level.

We next sought to compare the functional features of maximally expanded HSCs from the lower cell doses to those of HSCs from the highest dose with a lower historic fold expansion. We isolated 200 donor HSCs from each primary recipient and transplanted to secondary recipients. Donor chimerism levels were generally low, but to our surprise we observed no significant differences in donor chimerism regardless of the initially transplanted HSC dose. This indicates that, on a per cell basis, the HSCs show similar weakened potential in secondary blood reconstitution capacity despite substantial differences in historic fold expansion. By contrast, when analyzing the HSC compartment of the secondary recipients, we found that donor HSCs from mice initially receiving 10 000 HSCs had expanded 200-fold, compared to a 10 to 20-fold donor HSC expansion for the lower initial doses. These results suggest that the extent of historic fold expansion that is coupled to transplant dose is a strong determinant of sustained HSC self-renewal potential.

We next performed single cell RNA sequencing of HSCs purified from the primary recipients as well as from young (10 weeks) and old (98 weeks) steady state mice, and calculated combined module scores for each sample based on published data sets of divisional history and aging gene signatures (Bernitz et al., Stem Cell Reports 2020; Svendsen et al., Blood 2021). There was a positive correlation between increased transplant dose and genes downregulated with divisional history, in line with the estimated fold expansion. Strikingly, we observed an anticorrelation with extent of HSC expansion and aging indicating distinct molecular features between the “accelerated aging” that is coupled to divisional history, and chronological aging.

In conclusion, transplant dose determines the net expansion and proliferative response of HSCs and strongly influences their long-term self-renewal potential.