Ex vivoExpansion Potential of Murine Hematopoietic Stem Cells: A Rare Property Only Partially Predicted by Phenotype

Abstract

Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis, can maintain this function for life, and are the functional units in clinical bone marrow transplantation. The limited HSC numbers not only restricts their clinical use but also several embodiments of experimental research. Recently, a highly defined ex vivo culture system was shown to support several hundredfold expansion of functional in vivo HSC activity. Here, we further explored this system. Over a 3-week culture period, only 0.1% of initially seeded HSCs retained their original phenotype, which contained virtually all functional long-term HSC activity. Despite this low frequency, net HSC expansions were dramatic due to the overall extensive proliferation in cultures. Limited numbers of expanded HSCs allowed for long-term multilineage engraftment of unconditioned hosts, with candidate expanded HSCs rapidly returning to quiescence in this in vivo setting. The ex vivo differentiated progeny from candidate HSCs was rich in progenitor cell activity and allowed for radioprotection from even single cultured HSCs. Finally, clonal barcoding and competitive repopulation experiments demonstrated that successful HSC expansion emanated from rare HSC clones that was only partially predicted by phenotype, demonstrating the large impact of cellular heterogeneity for HSC biology.

Pre-existing stem cell heterogeneity dictates clonal responses to acquisition of cancer driver mutations

“…Next, we leveraged LARRY barcoding to assess the clonal dynamics and cell fate choices of expanded HSCs over time. We found that expansion cultures gradually lose their clonality, despite initiating from highly pure EPCR+ HSCs, in line with recent reports (Figure 1D) (Zhang et al 2024). Sister-cell splitting across independent wells confirmed the preferential expansion of specific clones, which correlated across wells higher than expected based on a null distribution obtained from a sampling simulation (Figure 1D).…”

A Non-Invasive Stem Cell Therapy Boosts Lymphopoiesis and Averts Age-Related Blood Diseases in Mice

“…We and others have previously established the e cacy of a polyvinyl alcohol (PVA)-based culture system in promoting murine HSC expansion. Notably, expanded HSCs enable a degree of HSC-derived reconstitution even in completely unconditioned hosts 11,12 . Given this, we explored the impact of larger quantities of HSCs on in vivo reconstitution outcomes in both non-conditioned and alternatively conditioned hosts.…”

“…Cells from primary recipients were pooled before transplantation. For in vivo proliferation tracking, cultured cells and CD4-enriched splenocytes isolated from C57BL/6-CD45.1 or C57BL/6-CD45.2 mice were labeled with CTV dye as previously described 12 . EE100 HSCs were cotransplanted with 2x10 6 splenocytes into each C57BL/6-CD45.2 or C57BL/6-CD45.1/CD45.2 unconditioned or CD45-SAP treated recipient.…”

“…Additional non-invasive approaches, such as mobilization-based regimens, use agents like granulocyte colonystimulating factor (G-CSF) and AMD3100 to disrupt HSPC-niche interactions, thereby vacating niches for transplanted cells 8 . Another method involves overcoming transplantation barriers by using higher doses of HSPCs [9][10][11][12] .…”