Abstract
While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25–400 MSCs and 10 umbilical cord blood (CB)-derived CD34+ progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34+ cell expansion outcomes. By contrast, a substantial increase in CD34+CD38− cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34+CD38− cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34+CD38− cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34+ cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.
Highlights
Hematopoietic stem cell (HSC) transplantation is a routine therapy used in the treatment of hematological diseases, including malignancies and immunodeficiencies.[1,2] Umbilical cord blood (CB) is a suitable alternative transplantable cell population when a matched bone marrow (BM) donor is not available
We found that the increase in CD34+CD38- cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice
Our results mirror previous studies,[21,22,35,36] demonstrating that mesenchymal stem/stromal cells (MSCs) trilineage mesodermal differentiation is inducible in 3D spheroids using a microwell platform
Summary
Hematopoietic stem cell (HSC) transplantation is a routine therapy used in the treatment of hematological diseases, including malignancies and immunodeficiencies.[1,2] Umbilical cord blood (CB) is a suitable alternative transplantable cell population when a matched bone marrow (BM) donor is not available. Transplantation of the limited number of HSCs that are present in single CB units is associated with delayed engraftment and increased graft failure and mortality.[1] This has motivated the development of ex vivo expansion technologies designed to increase CB HSC numbers. The previous few years have delivered promising ex vivo expansion systems that incorporated bound signal molecules (Notch ligand: >100-fold CD34+ expansion3), involved coculture with mesenchymal stem/stromal cells (MSCs) (approximately 40-fold CD34+ cell expansion4,5), or utilized pharmaceutical compounds
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