A Quantitative Hematopoietic Stem Cell Reconstitution Protocol: Accounting for Recipient Variability, Tissue Distribution and Cell Half-Lives

Abstract

Hematopoietic stem and progenitor cell (HSPC) transplantation is the paradigm for stem cell therapies. The protocol described here enables quantitative assessment of the body-wide HSPC reconstitution of different mature hematopoietic cells in mice based on their presence in circulating blood. The method determines donor-derived mature cell populations per mouse, over time, by quantitatively obtaining their absolute numbers in the peripheral blood and utilizing previously assessed tissue-distribution factors. A Markov-based birth/death computational model accounts for the drastic differences in mature cell half-lives. By quantifying the number of cells produced and eliminating host variability, the protocol can be used to directly compare the lineage output of different types of HSPCs on a per cell basis, thereby clarifying the lineage potential and expansion capacity of different cell populations. These protocols were developed for hematopoiesis, but can readily be extended to other contexts by simply replacing the cell types and distributions.HighlightsQuantitative assessment of stem and progenitor cell reconstitution capacityElimination of cell-specific recipient variability for accurate donor cell potentialDirectly comparable lineage output within and between stem and progenitor cellsBlood-based absolute quantification of whole-body repopulation over timeMarkov modelling-based consideration of differential mature cell half-lives

IL7Rα is required for hematopoietic stem cell reconstitution of tissue-resident lymphoid cells

“…S2B). However, a limitation of donor chimerism is that this measure relies on host cell numbers and therefore makes it more difficult to determine the intrinsic requirement of these receptors if the number of host cells change [25,30]. To overcome this limitation, we quantified the absolute cellularity of donor-derived cells, which is purely a measure of donor cells.…”

“…To overcome this limitation, we quantified the absolute cellularity of donor-derived cells, which is purely a measure of donor cells. Quantification of absolute cellularity of donor-derived cells between WT and knockout HSCs [25,30] revealed that reconstitution of B1a cells (Fig. 3C') and MZBs (Fig.…”