Abstract
As main hematopoietic organ, bone marrow have three dimensional microenvironment for hematopoietic stem/progenitor cells grow in, so call “hematopoietic cells niche”, which are composed by stromal cells and extracelluar matrix. The interactions of cell to cell and cell to matrix between stem/progenitor cells with hematopoietic niche are facilitated by its three dimensional conformation. The biology behaviors of hematopoietic stem cells are mediate by many signal transductions between stem/progenitor cells with their corresponding microenvironment. Now there are strong evidence from animal model study suggests that osteoblasts might play an essential role in creation of a hematopoietic stem cell niche and thereby regulation of stem cell maintenance, proliferation, and maturation. In light of the structure-function relationship of bone marrow topography, we conceived a biomimetic culture system (3D culture system) with bio-derived bone as framework, composited with human marrow mesenchymal stem cells, and induced the cells into osteoblasts to simulate the effects of hematopoietic osteobalst niche. CD 34+ cells or mononuclear cells separated from umbilical cord blood were cultured for 2∼5 weeks in the 3D culture system and also in a conventional 2D culture system as control without additional cytokine supplement. Based on our results, higher expression of extracelluar matrix and N-cadherin were observed in 3D culture system compared to 2D system. At 2 weeks culture, 3D culture system showed higher number of CD34+ cells and CD34+/CD38- cells when compared with the input (P<0.05), the increased cells were predominant CD34+/CD38-cells. Although CD34+ cells were decreased at 5 weeks culture; nevertheless, CD34+/CD38- cells were still maintained at high level. We also observed that imbedding MNCs with a higher percentage of CD34+/CD38-cells cultured in 3D system (P<0.05), which may represent a down regulation of CD38 phenotype during culture. The function of cultured cells was evaluated in colony forming unit (CFU) assay and long term culture initial cell (LTC-IC) assay. 3D system produced higher expansion of CFU progenitors than 2D system (7.13–8.89 times vs. 1.22–1.31times) after 2 weeks culture. Of note, the colony distribution of 3D system manifested higher percentage of BFU-E and CFU-GEMM, while 2D system showed mainly CFU-GM. LTC-IC represents the primitive progenitor, 3D system showed a 6.2 times increment over input after 2 weeks culture. Furthermore, it was competent to maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. This study demonstrated that our 3D culture system constructed with the bio-derived bone composited with induced osteoblast is capable to allow maintenance and expansion of primitive hematopoietic progenitor cells in vitro. It may open a new avenue to study HSCs/HPCs behaviors and to achieve sustained primitive progenitor cell expansion.
Enhanced maintenance and retroviral transduction of primitive hematopoietic progenitor cells using a novel three-dimensional culture system
