Hematopoiesis in Regenerated Bone Marrow on the Hydroxyapatite Scaffold.

Background: In bone marrow (BM), osteoblastic cells lining the endosteal surface are a key component of the niche to promote and regulate hematopoietic stem cells (HSCs). Anatomical architecture of BM could be regenerated by culturing BM derived stromal cells on the hydroxyapatite (HA) scaffold. In the present study, we examined whether the regenerated BM has the ability to support HSCs in vivo.

Methods: Our study was designed as follows;

1. BM stromal cells from C57/BL6 mice (Ly5.2) were cultured on the HA scaffold with numerous small pores for 3 days in vitro and the scaffold with attached cells was implanted subcutaneously onto the back of C57/BL6 recipient mice.

2. 4.0x105 Lineage negative (Lin−) Ly5.1 BM cells transduced with a lentiviral vector containing the luciferase (Luc) gene were intravenously administered into the recipient mice after lethal irradiation.

3. Eight weeks after transplantation, the scaffolds were removed from the first recipient mice and subcutaneously transplanted into the lethally irradiated second recipient mice. The mice also received fresh Ly5.2 BM cells for survival.

4. Biodistribution and kinetics of Luc+ Ly5.1 cells were monitored by in vivo bioluminescence imaging and FACS.

Results: In the secondary transplanted mice, Luc+ hematopoitic cells were detected in the scaffolds for at least 6 months after transplantation. Subcutaneous injection of G-CSF resulted in wide distribution of bioluminescence signals from the original scaffolds to whole body including the head, extremities, chest, and abdomen. The presence of Ly5.1 B and T lymphocytes in the circulation was confirmed by FACS analysis 5 months after secondary transplantation.

Conclusions: The regenerated BM on the HA scaffold is capable of supporting HSCs in vivo suggesting that the functional niche is reconstituted. Hematopoiesis in the regenerated BM may have a significant impact for development of new therapeutic strategies for various hematopoietic diseases.