[Promotion of in vitro long term expansion of primitive multipotential hematopoietic cells by transduced JAK2 gene].
OBJECTIVE
To explore whether activation of the JAK2-signaling pathway can stimulate long-term expansion and regulation of hematopoietic stem cells/multipotential hematopoietic progenitor cells (HSC/MHPC), and evaluate their potential ability of committed differentiation.


METHODS
A retrovirus vector (RV) which contains JAK2 gene and two binding sites for a chemical inducers of dimerization (AP20187) was constructed. JAK2 can be dimerized by adding AP20187. Female C57BL/6 mice were euthanized and marrow cells were harvested. The RV vector was then transduced into murine bone marrow cells. Following transduction, Transduced cells were divided equally into four groups as follow: (1) No drug group, (2) AP2018 alone group, (3) SCF + Flt3-Ligand group, (4) AP20187 + SCF + Flt3-Ligand group. The expanded cells were further analyzed by phenotype, committed differentiation, progenitor colony assay as well as colony forming unite-spleen.


RESULTS
Only the group of AP20187 combined SCF and Flt3-Ligand have a significant sustained outgrowth. The cells reached at 10(19) fold on the day 80. The phenotype of expanded cells were checked by flow cytometry at various time points after two months in vitro culture and we repeated them in five separate experiments. Sca-1 was consistently expressed at the levels in 52% approximately 98%, while 56% approximately 69% of cells expressed c-kit, 40% approximately 85% expressed CD34, About 12% approximately 46% expressed B220, 6% approximately 17% expressed Gr1, 0% approximately 20% expressed TER119, 5% approximately 36% expressed CD41, 35% approximately 46% expressed CD11b and none expressed CD3. The expanded cells could differentiate into granulocytes, macrophages, erythocytes and megakaryocytes under different cytokines combination. They were also capable of forming BFU-E, CFU-GM, CFU-Mix and IL-7 responsive B-lymphoid colonies in methylcellulose colonies assay. Colonies forming unites-Spleen were obtained when the expanded cells injected into lethally irradiated mice.


CONCLUSIONS
The JAK2-mediated transgenic hematopoietic cells could be expanded and regulated in long-term period, and they are capable of maintaining multipotential differentiation. This research is setting up a fundamental basement for cell therapy in the future.
Department of Hematology, Beijing Hospital, Beijing 100730, China.
Sheng-Ming
SM
Zhao
Nai-Bai
NB
Chang
Xi-Chun
XC
Gu
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005260:Female; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D053614:Janus Kinase 2; D008565:Membrane Proteins / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000502:physiology; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D019089:Stem Cell Factor / Q000494:pharmacology; D016559:Tacrolimus / Q000031:analogs & derivatives* / Q000494:pharmacology; D014162:Transfection
C463061:AP20187; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D016559:Tacrolimus