The Hematopoietic Stem Cell Niche in Whole Bone Marrow Fragments Can Be Maintained In-Vitro.

INTRODUCTIONS: Hematopoietic Stem Cells (HSC) are the most extensively characterized stem cell. Despite our rather extensive knowledge of the HSC, the ability to maintain and expand HSCs in-vitro remains a challenge. The niche, or microenvironment, of the HSC is thought to support and dictate whether HSC will self-renew or differentiate. Many have attempted to create artificial niches, but with limited success. Rather than trying to construct an artificial niche, we sought to examine whether the existent intact bone marrow hematopoietic niche could be maintained and support HSC in-vitro.

METHODS: Femurs, and Humeri were removed from healthy 8–12 week old BA Ly5.1. Bone Marrow (BM) preparations were processed as Whole Bone Fragments (WBF), Flushed, or as intact marrow Plugs. Whole bones were cut into thirds to facilitate perfusion. Flushed BM consisted of completely disassociated cells. Lastly, Plugs were flushed intact from the BM cavity. Preparations were cultured in X-Vivo media with 15% FBS, with or without supplemental cytokines (Flt2, TPO, SCF) and media was exchanged every other day. At serial time points, BM preparations were removed and viability determined. Cells analyzed by flow cytometry to determine presence of c-Kit+/Lin−/Sca-1+/(KLS) populations. 1 million cells were removed from preparations and transplanted into sublethally irradiated (400rads) Ly5.2 recipients which after 6 weeks were analyzed for for donor derived engraftment.

RESULTS: Under all conditions, viability ranged from 13% to 67% at day 9 in culture, with even higher percentages at day 4 (30–94%). On flow cytometry analysis, populations of KLS cells were detectable in flushed, plug and BMF samples. At day 4 of culture, the frequency of c-Kit+, Sca+ cells of Lin- ranged from .43% to 4.00%. Those cultured with supplemental cytokines showed a significantly higher frequency (.99% to 4.00%) than those without cytokines (.36% to 1.43%). The highest mean frequency of KLS was consistently the Flushed BM at 3.52% Cy- and 4.00% Cy+, suggesting the importance of perfusion. A slight increase in frequency was observed in both Plugs (mean of 1.27%) compared WBF (mean of .67%). There was no negligible difference between Cytokine+ and Cytokine- preparations from WBF at both Day 4 and Day 9 time points. Day 9 frequency of KLS cells fell to the range of .29% to 1.23%. However, frequencies decreased proportionally amongst all samples. CFU assay scoring demonstrated the presence of colonies from both Day 4 and 9 samples. In sublethally irradiated mice transplanted with cultured marrow cells, tri-lineage donor derived Ly5.1 cells were detectable at up to3 months post transplant from both flushed marrow plugs and WBF from D+9 culture preparations. Donor granulocyte engraftment from WBF donor cells (without cytokines) was 5.2% to 22.1, as compared to 19% from an equal number of fresh control BM cells, suggesting long-term engraftment was derived from HSC maintained in culture WBF.

CONCLUSIONS: These results suggest that the hematopoietic niche can be maintained in whole bone fragments up to 9 days in culture with minimal manipulation and without cytokine supplementation. Viability however decreases over time, suggesting that in order to maintain a more robust niche, means to increase intact niche perfusion will be necessary.