Microencapsulated Osteoblasts Support Hematopoietic Stem/Progenitor Cell Expansion in Hypoxic Environment*

Abstract

Microencapsulated osteoblasts were cocultured with hematopoietic stem/progenitor cells (HSPCs) under hematopoietic niche oxygen concentration to investigate the promoting effort of hematopoietic microenvironment on the expansion of umbilical cord blood HSPCs. The osteobalsts were isolated from human iliac bone and cultured, the third passage of osteoblasts at a density of 8 ×105 cells/ml were encapsulated in gelatin-alginiate-chitosan (GAC) beads with a diameter of 0.5 mm by the polyelectrolyte-complexation method. Three groups of cells were cultured in 5% oxygen incubator, A′ group with microencapsulated osteoblasts and hematopoietic cells, B′ with only hematopoietic cells and C′ with only microencapsulated osteoblasts. Meanwhile, the similarly grouped cells were cultured under 20% oxygen condition, named as A, B and C groups, respectively. The expansion of HSPCs was evaluated by flow cytometry analysis and colony-forming assays. And the concentrations of two kinds of cytokines, LIF and IL-6, were tested to investigate the mechanism of osteoblast's action. The results showed that human osteoblasts dispersed uniformly and grew well in microbeads. There were amount of micro holes in the beads for nutrients transmission. Lots of hematopoietic cells adhered weakly on the surface of microbeads. After 7 days of culture, the hematopoietic cell expansion folds were (49.0 ± 4.6), (3.3 ± 0.5), (17.7 ± 1.2) and (1.9 ± 0.2) respectively for group A′, B′, A and B. And CD34+ cells in groups A′, B′ and A were expanded (87.6 ± 8.3)-fold, (2.2 ± 0.3)-fold and (14.9 ± 1.0)-fold, but CD34+ cells in group B descended. CFU-Cs expansion folds in group A′, B′, A and B were (9.8 ± 0.8), (3.5 ± 0.4), (6.9 ± 0.7) and (2.6 ± 0.2) respectively. It was indicated that Hypoxic co-culture system could promote HSPCs expansion much more than normoxic co-culture system and somatic cell-free culture system. IL-6 and LIF concentrations in A′ , B′ and C′ were significantly higher than those in groups A, B and C, which were consistent with their expansion results. Moreover, microencapsulated osteoblasts could support the expansion of umbilical cord blood HSPCs, especially in 5% oxygen condition. Osteoblasts lived in low oxygen condition could secrete more cytokines and thus regulate HSPCs expansion.