Hematopoietic stem cells (HSCs) reside within a specialised niche area in the bone marrow (BM). They have tremendous clinical relevance, although HSC expansion and culture ex vivo is not currently possible, reducing BM transplant success. This project expands a novel 3D MSC niche model developed in our lab to include HSCs.

MSCs were loaded with green fluorescent magnetic iron oxide (FeO₃) nanoparticles (200 nm diameter) at a concentration of 0.1 mg ml⁻¹, and incubated for 30 min over a magnet to enhance cellular uptake. The cells were washed, detached and resuspended, then transferred to a plate with magnets above. Spheroids formed within hours and were implanted into 2 mg ml⁻¹ collagen gel. HSCs were loaded with nanoparticles via incubation with suspension, and then introduced to the gel containing the spheroid. Immunostaining, BrdU and Calcein/ ethidium homodimer viability assays were performed to characterise the cells.

Cells in both monolayers and spheroids remain viable up to 7 days in culture. MSCs in monolayers and spheroids were stained with antibodies for: STRO-1, an MSC marker; SDF-1 (CXCL-12), a secreted HSC homing factor; and nestin, a marker for HSC-supportive MSCs in vitro. MSCs in spheroids retain a higher level of expression of all three for 7 days compared to MSCs in monolayers. BrdU assay results show that the MSCs are more quiescent in spheroids compared to monolayers.

Proof of principle studies are promising for the success of the proposed niche model. MSCs express a higher level of MSC markers and retain quiescence when they are in spheroids as compared to monolayers. They also express a higher level of HSC niche factor SDF-1α, which facilitates HSC migration and retention.