Hematopoietic stem cells (HSCs) are used extensively in the treatment of both malignant and non-malignant diseases (stem cell transplant) and as the foundation for gene therapy. Thus, an area of intense scientific investigation has been in the development of methods for the ex vivo expansion of hematopoietic stem cells for clinical applications. While there has been significant progress in the ability to expand HSCs ex vivo, this is often accompanied by differentiation of the stem cells to more committed progenitors resulting in a mixed population of HSCs and progenitor cells. A new culture technique developed by the laboratories of Dr. Shaoyi Jiang (Dept. of Chemical Engineering) at the University of Washington and Dr. Colleen Delaney (Clinical Research Division) at Fred Hutch shows promise in the expansion and maintenance of HSCs with minimal differentiation. In order to mimic the natural bone marrow niche where stem cells reside, the labs optimized expansion of stem cells in 3D zwitterionic hydrogels. Zwitterions are hydrophilic compounds with two charged groups – one negative, one positive – that are common in the bone marrow environment. Additionally, they have been shown to interact very little with cellular components and serum proteins, making them a “blank slate” unlike polystyrene flasks commonly used in traditional tissue culture. Their work was recently published in Nature Medicine.
The authors tested whether zwitterionic hydrogels (ZTG) would be suitable for expanding HSC from cord blood compared to conventional and previously optimized culture conditions. Remarkably, after an optimized 24-day culture period, ZTG cultures allowed a 322-fold expansion of cold blood-derived nucleated cells, where 95% of cells expressed CD34, a marker of their “stemness.” Upon further interrogating their cell status, the authors found that the ZTG expansion allowed HSCs to maintain their most primitive state, with ~93% of cells positive for CD34 but negative for markers of differentiation (Lin-). This was in stark contrast to cells expanded with a previously optimized protocol using modified Notch ligand signaling where only 15% of cells were CD34+ Lin-, or traditional culture methods where only 7% of cells maintained their primitive status. Although other culture conditions allowed greater expansion of total cells, the reduction in the number of true primitive HSCs made them less successful at generating clinically relevant HSCs. To that point, the authors tested the ability of cells expanded with various methods to engraft into recipients and found that ZTG-expanded cells reached higher levels of engraftment in immune compromised mice than cells derived from any other method. Using serial transplants and limiting dilution assays, the authors determined that expansion of HSCs using their ZTG method represented a 73-fold increase in long term-HSCs (LT-HSCs) than numbers found in fresh cord blood, an increase that could make a significant difference for patients who receive cord blood transplants.
The authors next sought to determine why their culture conditions were so favorable for HSC expansion. As HSCs normally reside in a low oxygen environment and reactive oxygen species (ROS) have been shown to increase cell differentiation, they measured ROS in each culture. ZTG cultures produced lower ROS levels, consumed less oxygen, and had lower signaling through ROS-related pathways. Gene expression analysis also showed reduction in cell differentiation, activation, and metabolism pathways in ZTG cultures compared to all others, consistent with their ability to maintain their primitive state. The use of this method to expand HSCs without losing stemness has the potential to provide HSCs in clinically relevant numbers for therapeutic applications where stem cell numbers are limited, such as live-saving cord blood transplants or gene therapy.
Bai, T., Li, J., Sinclair, A. l. et al. 2019 Expansion of primitive human hematopoietic stem cells by culture in a zwitterionic hydrogel. Nat Med . 25, 1566–1575.
This work was supported by the National Science Foundation, the Office of Naval Research, the University of Washington, an NIDDK grant, and the NIH/NCI Cancer Center.
Fred Hutch/UW Cancer Consortium member Colleen Delaney contributed to this work.