Vitalant Research Institute San Francisco, California, United States
Background/Case Studies:
The oldest form of stem cell therapy, the bone marrow transplant, involves transplanting a small number of hematopoietic stem cells (HSCs) and progenitors to reconstitute hematopoiesis. Ex vivo expansion of the number of progenitors and HSCs in a graft can accelerate hematopoietic reconstitution. Mesenchymal stromal cells (MSCs) can support the growth of HSCs. This study aimed to better understand and improve culture conditions using a feeder layer of MSCs of fetal bone marrow origin to support ex vivo HSC expansion. How different conditions affected the cytokine production profile of MSCs was a major focus of the study.
Study
Design/Methods:
Human fetal bone marrow derived MSCs were immortalized using SV40-LTA creating FBM8 and FBM1 cell lines. Optimal seeding density and mitomycin C (MMC) treatment concentration required to halt cell proliferation were defined using a titration assay. Gelatin substrate was evaluated for its ability to improve cell retention. To confirm mitotic inactivation, MMC treated FBM8 cells were transplanted into immunodeficient mice and monitored for 24 weeks. Luminex assay was performed to compare the effects of the optimized culture conditions on the cytokine profiles between FBM8, FBM1 and fresh fetal bone marrow cells.
Results/Findings:
After 8 days in culture, there was a statistically significant reduction in cell proliferation in FBM8 cells seeded at 12500 cells/ml and treated 10 ug/ml MMC compared to untreated cells (p< 0.05). Mice that received MMC-treated FBM8 cells displayed a 100% survival rate and did not develop any tumors. PCA analysis of the Luminex data suggested that cytokine production of MSCs ex vivo was affected by cell origin and culture media; however, neither MMC treatment nor gelatin coating modified their cytokine profile significantly.
Conclusions:
Ex vivo culture of FBM8 cells was optimized at 12500 cells/ml on 2% gelatin coating while treated with 10 ug/ml MMC. Since transplantation of FBM8 cells into immunodeficient mice did not show any adverse effect, the next step involves setting up a FBM8-HSC co-culture to test their long-term engraftment in vivo. Interestingly, gelatin coating and MMC treatment did not significantly alter the cytokine profiles of MSCs, suggesting that their cytokine interactions with HSCs may remain less affected by external treatments required to create an effective feeder layer. Confirming that in imitation of the bone marrow niche ex vivo, crucial cell-to-cell interactions were conserved.
