| Background & Objective In adults, hematopoiesis takes place in the bone marrow, where hematopoietic cells grow in close association with a three dimensional microenvironment composed of stromal cells and extracelluar matrix that makes up hematopoietic cell niche. Cell-cell and cell-matrix interactions of hematopoietic cells and hematopoietic niche are facilitated by its three dimensional conformation. Regulation of hematopoietic stem cells by the microenvironment, which induce corresponding biology behavior of hematopoietic stem cells, is mediate by the signal communication of hematopoietic stem cells and microenvironment. Mounting evidence suggests osteoblasts, the bone-forming cell of stromal cell population, takes an essential role in the creation of a hematopoietic stem cell niche and thereby regulate stem cell maintenance, proliferation, and maturation. In vivo study has demonstrated that the number of hematopoietic stem cell is regulated by the size of osteoblast niche, In light of the structure-function relationship of bone marrow topography and reciprocal relationship between hematopoietic stem cells and osteoblasts, we employed the bio-derived bone composited with the marrow mesenchymal stem cells that was induced into osteoblast to increase osteobalst hematopoietic stem cell niche size to replicate the hematopoietic stem cell niche. We have studied the maintenance and expansion character of hematopoietic stem/progenitor cells in this novo biomimetic culture system.Methods Bio-derived bone composited with the marrow mesenchymal stem cells that was induced into osteoblast to construct a three dimensional hematopoietic stem/progenitor cells culture system. Sorted CD 34+ cells or mononuclear cells from umbilical cord blood was cultured for 2~5 weeks in the biomimetic niche (3D culture system) or conventional two dimensional (2D culture system) coculture culture system without additional cytokine supplement. Cultured cells were analyzed for their phenotype and cell function through flow cytometry, colony forming unit (CFU) assay and long term culture initial cell (LTC-IC) assay. Expression of extracelluar matrix and N-cadherin were analyzed in our study.Results In this study, higher extracelluar matrix and N-cadherin were formed in 3D culture system compared to 2D system. After 2 weeks culture, CD34+ cells in 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 predominantly CD34+/CD38-cells. CD34+ cells were decreased in 5 weeks culture; nevertheless CD34+/CD38-cells were maintained at high level. We also observed MNC display a higher frequency of CD34+/CD38-cells after cultured in the 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 dose (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.Conclusion This 3D culture system constructed with the bio-derived bone composited with induced osteoblast expanded the size of hematopoietic stem cell niche, which is capable to allow maintenance and expansion of primitive HPCs in vitro. It may open a new avenue for the study HSCs/HPCs behavior and to achieve sustained primitive progenitor expansion.
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