Targeted expansion and regulation of genetically modified cord blood stem/progenitor cells in vitro

BACKGROUND： Cord blood stem cells are one of ideal target cells for gene therapy, but low gene transferring rate is the main difficulty at recent. Janus kinase tyrosine 2 （JAK2） plays an important role in self-renewing of cord blood stem/progenitor celll2s. Therefore, cord blood CD34~+ cell line modified by target-amplified JAK2 genes has been developed yet by using gene regulating expression technique in order to overcome low transferring rate of cord blood genes. OBJECTIVE： To investigate the feasibility and reliability of a long-term amplified regulation for cord blood stem/progenitor cells mediated by transgene JAK2.SETTING： Department of Hematology, Beijing Hospital, Ministry of Health. MATERIALS： The experiment was carded out in the Laboratory of Hematological Department, Beijing Hospital, Ministry of Health from June 2003 to April 2006. Cord blood was derived from umbilical cord which was immediately cut from healthy, full-term and natural-parturition infants and was provided by Department of Obstetrics ＆ Gynecology, Beijing Hospital. The experiment was approved by the local ethical committee, and informed consent was obtained from expectant mothers and their relatives for the use of cord blood cells. MiniMACS magnetic separation apparatus and immunomagnetic beads adsorbing CD34 single antibody were provided by Miltenyi Biotec Company, Germany; flow cytometer by FACScalibur, USA; recombinant human stem cell factor （rhSCF）, Flt3 ligand （FL）, human interleukin-6 （hIL-6）, granulocyte macrophage colony stimulating factor （GM-CSF）, granulocyte colony-stimulating factor （G-CSF） and thrombopoeitin （TPO） by PeproTec Company; nude mice of the SPF level by Animal Center of Beijing Medical University. METHODS： Retroviral vector MGI-F2JAK2, which was composed of functional catalytic domain of JAK2 genes and two site proteins （2xF36v, F2） combined with synthetic drug （AP20187） of target gene of small molecules, was constructed. AP20187 might specially combine with F36v to cause dimerization of JAK2 so as to activate signal conduction in cells. In addition, the vector included green fluorescence protein reporter gene, which was regarded as a label to detect proliferation. MiniMACS magnetic separation apparatus was used to purify and separate cord blood CD34~+ cells. While, retrovirus supernatant including JAK2 was used to transfer cord blood CD34~+ cells. After transduction, CD34~+ cells were cultured with stem cell factor （SCF）, Flt3 ligand, TPO and IL-6 and divided into control group （not adding AP20187） and experimental group （AP20187）. MAIN OUTCOME MEASURES： ① Flow cytometer was used to detect percentage of green fluorescence protein reporter gene in the CD34~+ cells and to determine gene transferring rate. ② Colony culture results of cord blood stem/progenitor cells after amplification. ③ Nude mice were given subcutaneous injection of ten-week cultured cord blood CD34~+ cells at costa and neoplasia was observed after 30 days. RESULTS： ①Plentiful amplification of CD34~+ cells was observed in both experimental group and control group. With the culture time passing by, positive rate of gel-filtered platelet of amplified CD34~+ cells in the experimental group was gradually increased based on the basic level and more than 95% in the 11^th week; however, positive rate of green fluorescence protein reporter gene in the control group was gradually decreased below the basic level and disappeared finally. ② Transgenic CD34~+ cells in the experimental group still could generate brust forming unit-erythroid （BFU-E）, colony-forming units granulocute/monocyte （cFU-GM） and multipotential hematopoietic progenitors （CFU-Mix）; especially, CFU-GM was the main cell in hemopoietic progenitor cell （HPC）. ③ Nude mice did not have neoplasia. CONCLUSION： Human cord blood CD34~+ cells of transferring JAK2 genes may cooperate with other cytokines to amplify cord blood stem/progenitor cells in vitro for long. Therefore, this is potentially valuable for stem cells to treat some hereditary hematologic disease.