ABSTRACT
The aim of this study was to develop an ex vivo cell culture system for establishing the hematological malignancy model. Mouse bone marrow cells were transfected with GFP-expressed retroviral vectors encoding various leukemia/lymphoma-associated fusion proteins (TEL-PDGFR, Rabaptin5-PDGFR, p210BCR-ABL, AML1-ETO, NPM-ALK). After transfection, the cells were cultured in IMDM containing 10% FCS without growth factors, or with one of the following growth factor combinations: (1) murine c-kit ligand (KL) plus human flt3 ligand (FL); (2) IL-3, thrombopoietin, G-CSF, and hyper-IL-6 (3/T/G/H6); (3) KL/FL plus 3/T/G/H6. The flow cytometry was used to detect the ability of combinations of growth factors to complement the oncogene fusion protein to support self-renewal of the transfected cells. The results showed that the transfected cells could be amplified sustainably in the logarithmic growth way. The indicated combination of c-Kit ligand (KL) with flt-3 ligand (FL) supported the self-renewal of the marrow cells transfected with vectors encoding TEL-PDGFR, Rabaptin5-PDGFR, AML1-ETO and NPM-ALK, in addition to KL/FL, the self-renewal of p210 BCR-ABL transfected-marrow cells also required IL-3. The morphology of cells emerged from culture can be the predictor of the corresponding oncogene-associated malignancy. It is concluded that this study establishes a culture system ex vivo which provides a generalized method for studying hematological malignancies, and may facilitate the screening for therapeutic agents.
Subject(s)
Animals , Humans , Mice , Bone Marrow Cells , Pathology , Cell Culture Techniques , Genetic Vectors , Hematologic Neoplasms , Mice, Inbred C57BL , Neoplasms, Experimental , Oncogene Proteins, Fusion , Genetics , Retroviridae , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To explore the feasibility of regulated expansion and committed differentiation potential of JAK2 gene modified hematopoietic stem/progenitor cells in vitro.</p><p><b>METHODS</b>A murine stem cell virus (MSCV) based retroviral vector MGI-F2Jak2, which encodes a green fluorescent protein (GFP) and a fusion protein containing two copies of modified FK506 binding protein (F36v) linked tyrosine kinase JAK2 was cloned. F36v served as a high-affinity binding site for dimerizer AP20187. GpE + 86 packaging cell was transfected with this vector. Bone marrow cells from C57BL/6 mice were transduced by co-cultured with irradiated (1500 cGy) GpE + 86 producer clone for 48 h. Transduced marrow cells were expanded in X-VIVO 15 and divided into four groups as follows: (1) control group; (2) AP20187 alone group; (3) SCF alone group and (4) AP20187 + SCF group. The phenotypes of the expanded cells were analyzed by directly phycoerythrin-labeled anti-Sca1, c-kit, CD(34), Gr1, CD(11b), TER119, CD(41), B220 and CD(3) monoclonal antibodies for flow cytometry. Committed differentiation, progenitor colony assay and spleen colony forming units (CFU-S) were further evaluated.</p><p><b>RESULTS</b>A significant sustained outgrowth of transduced marrow cells was obtained only in the AP20187 + SCF group. Cells expanded up to 10(14)-fold after 80 days culture. The doubling time was about 30 hs. The phenotypes of the expanded cells were homogeneously strong positive for CD(34), c-kit and Sca1, while were almost negative for Gr1, CD(11b), TER119, CD(41), B220 and CD(3). Functional assay demonstrated that the expanded cells had multipotential to differentiate into granulocyte, macrophage, erythrocyte, or B-cells under different cytokines combinations. A prominent megakaryocytic differentiation was observed when cultured with SCF/Tpo/IL-11 combination. The expanded cells were also capable of forming BFU-E, CFU-GM and CFU-Mix in methylcellulose colony assay. The expanded cells over three months could still form CFU-S.</p><p><b>CONCLUSIONS</b>AP20187 combined SCF mediated activation of JAK2 signaling domain can dramatically expand hematopoietic stem/progenitor cells, and the expanded cells can be regulated and committed to differentiate into multilineage cells. This system may provide important insights into stem cell biology and may be promising for gene and cell therapy.</p>