Prospective isolation of nonhematopoietic cells of the niche and their differential molecular interactions with HSCs.

A major limitation preventing in vivo modulation of hematopoietic stem cells (HSCs) is the incomplete understanding of the cellular and molecular support of the microenvironment in regulating HSC fate decisions. Consequently, murine HSCs cannot be generated, maintained, or expanded in culture over extended periods of time. A significantly improved understanding of the bone marrow niche environment and its molecular interactions with HSCs is pivotal to overcoming this challenge. We here prospectively isolated all major nonhematopoietic cellular niche components and cross-correlate them in detail with niche cells defined by lineage marking or tracing. Compiling an extensive database of soluble and membrane-bound ligand-receptor interactions, we developed a computational method to infer potential cell-to-cell interactions based on transcriptome data of sorter-purified niche cells and hematopoietic stem and progenitor cell subpopulations. Thus, we establish a compendium of the molecular communication between defined niche components and HSCs. Our analysis suggests an important role for cytokine antagonists in the regulation of HSC functions.

Clonal expansion capacity defines two consecutive developmental stages of long-term hematopoietic stem cells.

Long-term hematopoietic stem cells (HSCs [LT-HSCs]) are well known to display unpredictable differences in their clonal expansion capacities after transplantation. Here, by analyzing the cellular output after transplantation of stem cells differing in surface expression levels of the Kit receptor, we show that LT-HSCs can be systematically subdivided into two subtypes with distinct reconstitution behavior. LT-HSCs expressing intermediate levels of Kit receptor (Kit(int)) are quiescent in situ but proliferate extensively after transplantation and therefore repopulate large parts of the recipient's hematopoietic system. In contrast, metabolically active Kit(hi) LT-HSCs display more limited expansion capacities and show reduced but robust levels of repopulation after transfer. Transplantation into secondary and tertiary recipient mice show maintenance of efficient repopulation capacities of Kit(int) but not of Kit(hi) LT-HSCs. Initiation of differentiation is marked by the transit from Kit(int) to Kit(hi) HSCs, both of which precede any other known stem cell population.

Ultra-high-yield manufacture of red blood cells from hematopoietic stem cells.

Provision of a safe and secure supply of transfusible red blood cells (RBC) is a major global health challenge, and it has been proposed that manufactured RBC could help to alleviate the constraints of the current donor system. Several substantial challenges must be addressed for this approach to be feasible. At the most basic level, this relates to the large quantities of cells that are required: is there sufficient biological capacity, and is it possible to produce RBC using large-scale processes? While it has been demonstrated that, in principle, up to 5 units of RBC could be generated from a single donation of umbilical cord blood (UCB) hematopoietic stem cells, such yields are insufficient to supply demand and existing culture methods are unsuitable for large-scale manufacture. Given the capacity of the hematopoietic system in vivo, we reasoned that an optimized process should give rise to much larger quantities of RBC than previously reported. We successfully developed a robust ultra-high-yield RBC expansion process capable of producing over 500 units of RBC per UCB donation using fully defined culture medium. We obtained near-pure populations of reticulocytes with an enucleation frequency of >90%, mean cell hemoglobin content of 30.8 pg/cell, and mean cell volume of 133 fL. We also show that RBC can be efficiently produced in agitated bioreactor systems, demonstrating that no fundamental barriers exist to the manufacture of RBC using large-scale approaches.