Cycling Marrow Stem Cells Are Lost with Purification.
Authors: Goldberg Laura R , Dooner Mark S , Pereira Mandy , DelTatto Michael , Papa Elaine , Amaral Ashley , Aliotta Jason M , Quesenberry Peter J 
Published: October 2019

Abstract
Abstract 2308
Hematopoietic stem cell biologists have amassed a tremendous depth of knowledge about the biology of the marrow stem cell over the past few decades, facilitating invaluable basic scientific and translational advances in the field. Most of the studies to date have focused on highly purified populations of marrow cells, with emphasis placed on the need to isolate increasingly restricted subsets of marrow cells within the larger population of resident bone marrow cells in order to get an accurate picture of the true stem cell phenotype. Such studies have led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to homogeneity. However, work from our laboratory, focusing on the stem cell potential in un-separated whole bone marrow (WBM), supports an alternate view of marrow stem cell biology in which a large population of marrow stem cells are actively cycling, continually changing phenotype with cell cycle transit, and therefore, cannot be purified to homogeneity. Our studies separating WBM into cell cycle-specific fractions using Hoechst 33342/Pyronin Y or exposing WBM to tritiated thymidine suicide followed by competitive engraftment into lethally irradiated mice revealed that over 50% of the long-term multi-lineage engraftment potential in un-separated marrow was due to cells in S/G2/M. This is in stark contrast to studies showing that highly purified stem cell populations such as LT-HSC (Lineage–c-kit+sca-1+flk2−) engraft predominantly when in G0. Additionally, by performing standard isolation of a highly purified population of stem cells, SLAM cells (Lineage–c-kit+sca-1+flk2−CD150+CD41−CD48−), and testing the engraftment potential of different cellular fractions created and routinely discarded during this purification process, we found that 90% of the potential engraftment capacity in WBM was lost during conventional SLAM cell purification. Incubation of the Lineage-positive and Lineage-negative fractions with tritiated thymidine, a DNA analogue which selectively kills cells traversing S-phase, led to dramatic reductions in long-term multi-lineage engraftment potential found within both cellular fractions (over 95% and 85% reduction, respectively). This indicates that the discarded population of stem cells during antibody-based stem cell purification is composed largely of cycling cells. In sum, these data strongly support that 1) whole bone marrow contains actively cycling stem cells capable of long-term multi-lineage engraftment, 2) these actively cycling marrow stem cells are lost during the standard stem cell purification strategies, and 3) the protean phenotype of actively cycling cells as they transit through cell cycle will render cycling marrow stem cells difficult to purify to homogeneity. Given the loss of a large pool of actively cycling HSC during standard stem cell isolation techniques, these data underscore the need to re-evaluate the total hematopoietic stem cell pool on a population level in addition to a clonal level in order to provide a more comprehensive study of HSC biology.

Disclosures:
No relevant conflicts of interest to declare.