title: Long-term culture of leukemic bone marrow primary cells in biomimetic osteoblast niche.
abstract: We constructed a "biomimetic osteoblast niche" with bio-derived bone as a scaffold, on which we seeded marrow mesenchymal stem cells (MSCs) from CML patients, and induced the MSCs to differentiate into osteoblasts. Bone marrow mononuclear cells from CML patients were cultured in the biomimetic niche (3D culture system) or a 2D culture system with the induced MSCs/osteoblasts as a feeder cell layer for 2 and 5 weeks without adding exogenous cytokines. Cultured cells were analyzed regarding their phenotypes and functions using flow cytometry, colony-forming unit (CFU) assay and long-term culture-initiating cells (LTC-IC) assay. All cultured and colony cells in the LTC-IC assay were collected and analyzed by fluorescent in situ hybridization to identify Ph (bcr/abl)-positive cells. Our results showed that all parameters were higher in the 3D than in the 2D system, either at 2 or 5 weeks, i.e., regarding the number of CD34(+) cells (8,277.00 vs. 4,490.75 or 2,276.75 vs. 786.00 per well on average, respectively), number of CD34(+)/CD38(-) cells (1,207.50 vs. 474.25 or 497.25 vs. 114.25 per well on average, respectively), numbers of CFUs (103.33 vs. 79 or 47.0 vs. 21.67/10(5) MNCs; 189.33 vs. 131.00 or 10.33 vs. 3.67 per well on average, respectively), frequency of LTC-ICs (2.23 x 10(-5) vs. 1.40 x 10(-5) or 1.86 x 10(-5) vs. 0.64 x 10(-5), respectively) and number of remaining LTC-ICs (2.80 vs. 2.03 or 0.46 vs. 0.07 per well on average, respectively). The Ph (bcr/abl)-positive cell fraction was reduced in both systems during culture, but in the 3D system, it was not as rapid as in the 2D system and showed a leukemic predominance. In conclusion, our "biomimetic osteoblast niche" might provide a more adaptive microenvironment for leukemic stem/progenitor cell growth. The biological characteristics of leukemic stem/progenitor cells were partially maintained. It was suggested that the 3D biomimetic niche might be a new tool for studying the behaviors of leukemic hematopoietic stem cells/hematopoietic progenitor cells in vitro.
authors: Department of Hematology, Hematology Research Laboratory, West China Hospital, Sichuan University, 610041, Chengdu, Sichuan, People's Republic of China.
authors: Li
authors: L
authors: Hou
authors: Department of Hematology, Hematology Research Laboratory, West China Hospital, Sichuan University, 610041, Chengdu, Sichuan, People's Republic of China. tingliu@mail.sc.cninfo.net.
authors: Ting
authors: T
authors: Liu
authors: Department of Hematology, Hematology Research Laboratory, West China Hospital, Sichuan University, 610041, Chengdu, Sichuan, People's Republic of China.
authors: Jing
authors: J
authors: Tan
authors: Department of Hematology, Hematology Research Laboratory, West China Hospital, Sichuan University, 610041, Chengdu, Sichuan, People's Republic of China.
authors: Wentong
authors: W
authors: Meng
authors: Laboratory of Stem Cell and Tissue Engineering, West China Hospital, Sichuan University, 610041, Chengdu, Sichuan, People's Republic of China.
authors: Li
authors: L
authors: Deng
authors: Department of Hematology, Hematology Research Laboratory, West China Hospital, Sichuan University, 610041, Chengdu, Sichuan, People's Republic of China.
authors: Hongtao
authors: H
authors: Yu
authors: Department of Hematology, Hematology Research Laboratory, West China Hospital, Sichuan University, 610041, Chengdu, Sichuan, People's Republic of China.
authors: Xingli
authors: X
authors: Zou
authors: Department of Hematology, Hematology Research Laboratory, West China Hospital, Sichuan University, 610041, Chengdu, Sichuan, People's Republic of China.
authors: Yuchun
authors: Y
authors: Wang
mesh_terms: D032701:Biomimetics; D001854:Bone Marrow Cells / Q000166:cytology*; D015820:Cadherins / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D018920:Coculture Techniques / Q000379:methods; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D017404:In Situ Hybridization, Fluorescence; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010006:Osteoblasts / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015820:Cadherins