Abstract
We have constructed a biomimetic hematopoietic niche (3D culture system) with bio-derived bone as framework, composited with human marrow mesenchymal stem cells, and induced the cells into osteoblasts. Our primary results showed that the biomimetic 3D culture system is capable to allow maintenance and expansion of primitive hematopoietic progenitor cells in vitro. But so far, leukemia primary cells long-term culture from patients marrow are still difficult because it is not clear how does the regulation of leukemic cells grow ex vivo, and lack of adequate investigation between leukemic stem cells with stromal cells. Based on our previous research, we cultured bone marrow mesenchymal stem cells from chronic myelogenous leukemia (CML) patients, and conceived a “pathologic biomimetic osteoblast niche”, to explore the growth of leukemia bone marrow primary cells from CML patients. Bio-derived bone was composited with marrow mesenchymal stem cells from CML patients and constructed a 3D biomimetic osteoblast niche. The mononuclear cells (MNCs) were collected with standard Ficoll-Paque separation from newly diagnosed CML patients. The MNCs were cultured for 2∼5 weeks in the 3D culture system and compared with 2D culture system. The results showed that the proportion of CD34+ cells are increased either in 3D or 2D culture systems. Compared to input, the proportion of CD34+ cells were increased 6.52(1.87∼9)vs. 3.18(1.07∼6.8)times at 2 weeks culture, and 13.6(3.59∼26.31)vs. 7.86(0.78∼18.0)times at 5 weeks culture. The proportion of CD34+/CD38- was higher in 3D culture system than 2D system. It was 5.55(2.1∼11.7)% vs. 2.4(0.9∼3.4)%, and 13.5(3.4∼34.2)% vs. 4.83(2.1∼8.9)% at 2 weeks and 5 weeks respectively. 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 more colonies than 2D system {103.33(82∼144)vs. 79(53∼122)} at 2 week culture and 47(33∼66)vs. 21.67(16∼27)at 5 week culture. LTC-IC are widely used as a surrogate in vitro culture for pluripotent stem cells, and those primitive progenitor cells responsible for leukemia in mice are named SL-IC or leukemia stem cells (LSCs). 3D system showed higher frequency of LTC-IC than that of 2D system after 2-week culture(2.23E-05(1.73∼2.56)vs.1.40E-05(1.21∼1.73)). FISH showed the proportion of Ph+ cells declined in both system during the culture, but not as rapidly as it did in 2D system{65%(3D)vs.63%(2D)at 2 week, 55%(3D)vs.35%(2D)at 5 week}, and the Ph+ cells were predominant derived from 3D culture. Our 3D culture system constructed with induced osteoblasts from mesnchymal stem cells in CML patients might provide a more suitable microenvironment for leukemic cells growing in vitro. The leukemic stem cells seemed to be regulated by the molecular signals mediated by osteoblast, and the biological characteristics of leukemia stem cells at least partially is maintained. It may be become a new method for studying leukemic HSCs/HPCs behavior in vitro.
Cell Phenotype and Matrix Regeneration of Human Nucleus Pulposus Cells of Different Ages in a Laminin-Rich Pseudo-3D Culture System in Vitro
