| BackgroundHematopoietic stem cells (HSCs) are defined by their ability to self-renew and to contribute differentiation into all lineages of mature blood cells. Expansion of stem cells has long been a hot spot for worldwide scientists. A large number of studies have shown that a modest expansion of HSC numbers can be achieved in both adherent and nonadherent culture systems using several different cytokines combination. It is important to note that in most of these studies, HSC expansion occurred in a relatively transient manner and existed many undesired problems.As we know, many of the critical cell function of cells, such as cell division, development into specialized cell types, response to stimuli, and even cell death, take place through the process of cell signaling and gene activation. Research over the decades has revealed that dimerization is a heart of many signaling pathway in activation of intra-and extracellular proteins. When two proteins were dimerized, their signaling activities would be activated. So, we have exploited this finding to develop gene regulated expression technology, a powerful technology for controlling cellular events using small molecules, and makes possible the activation of specific cell signaling, gene transcription in cultured cells. Very recent studies have identified an essential role for the JAKs-STATs pathway in specifying stem cell self-renew, and a large amount of experimental data has accumulated to indicate that most cytokines transmit their signal via a family of JAK kinase. JAK2 is one of the members play a pivotal role in the self-renewal of hematopoietic stem/multipotential hematopoietic progenitor cells (HSC/MHPCs). Upon above the principle of biology, we constructed a retrovirus vector that contains a JAK2 functional domain linked a protein that provides a high-affinity binding site for a drug called AP20187. AP20187 is a novel gene-targeted drug that is being developed a small molecule on the basis of the structure-based drug design and protein engineering. This dimerizer drug can make JAK2 dimerization and activates its signaling pathway in combination with either SCF or FL cytokine. The application of our gene regulated expression technology has overcome several of the major limitations to the development of stem cell therapy--the inability to efficiently transfer genes into these rare cells, to control their growth, and direct their differentiation towards the desired cell type and tissue.Objective To explore the feasibility of regulated expansion and committed differentiation potential ofJAK2 gene modified hematopoietic stem/progenitor cells in vitro.Methods A murine stem cell virus (MSCV) based retroviral vector MGI-F2Jak2, which encodes a green fluorescent protein (GFP) and a fusion protein contains two copies 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 line was generated by transfection with this vector. This vector was first transfected into BaF3 cells and the engineered BaF3 cells expressing JAK2 were treated with AP20817 for MTT assay. Then the murine primary bone marrow cells from C57BL/6 were isolated and transduced by co-cultivated with irradiated (1500cGy) GpE＋86 producer clone for 48h. Transduced marrow cells were expanded in X-Vivo 15 and divided into eight groups as follows: 1) No drug, 2) AP20187 alone, 3) SCF alone, 4) FL alone, 5) SCF＋FL, 6) AP20187＋SCF, 7) AP20187＋FL, 8)AP20187＋SCF＋FL. The phenotyping of the expanded cells were analyzed by flow cytometry; committed differentiation, colony assay, CFU-S and introthymic injection were evaluated under specific conditions; signal inhibitor study performed by western blotting; IgH rearrangement performed by PCR; cells reversibility and mice tumorigenesis were also evaluated in the experiments.Results MTT assay showed AP20187 could support and stimulate cell growth of BaF3 cell in the absencene of IL-3. We then set up 100nmol/L concentration of AP20187 for our bone marrow study. Our data shown a significant sustained outgrowth of transduced marrow cells were accomplished in AS, AF and ASF groups, on the contrary, sustained growth failed to occur using SCF alone, FL alone or SCF plus FL in combination. Cells cultured for 80days expanded up to 1012-19-fold, the doubling time was about 30h. Cells cultured in ASF displayed the fastest rate of growth, while cell growth was least rapid with AF combination. Distinctive phenotypic and functionally characteristics populations of HSC/MHPCs were obtained, depending on c-kit or FL was activated. Cell expanded in AP20187+SCF (AS) displayed a phenotypically homogeneous population of blast-like cells that were strong positive for CD34, c-kit and Seal; while Gr1, CD11b, TER119, CD41, B220, CD3 were almost negative. Cells expanded in AP20187 plus FL (AF) exhibited two subfractions of both Pro-B and CLPs markers. Seal, c-kit, IL-7, CD43 and B220 were positive expression, while CD3, Gr1, TER119, CD41, Thy1.1 and IgM expression were all negative. But Cells expanded in AP20187+SCF+FL (ASF) demonstrated substantial heterogeneity. Seal expressed at 52～98%, while c-kit expressed 56～69%, 40～85% expressed of CD34, 0～20% positive for TER119, 5～36% positive for CD41, 12～46% positive for B220, 35～46% positive for CD11b and negative expresse for CD3. Functional assay demonstrated that AS and ASF cells had multipotential differentiation into granulocyte, macrophage and erythocyte under different cytokine culture conditions. A prominent megakaryocytic differentiation was observed under cultured in SCF/TPO/IL11 combination. BFU-E, CFU-GM and CFU-Mix were also capable formed in methycellulose colonies assay. Both ASF and AF cells could form IL-7 responsive colonies. PCR for IgH demonstrates DJ rearrangement but no VDJ rearrangement in AF cells. Intrathymic injection showed AF cells could form functional CD4, CD8, CD3 positively subset T cells and CD25, Seal positive Pro-T cells in the thymus of in vivo sulethally irradiated mice. CFU-S was observed in the lethal radiated mice from all three expanded cell groups. Experiments have also demonstrated that selfrenewal was abrogated in the presence of inhibitors targeting either the MAPK or P13Kpathway. The expanded cells had reversibility, and also no tumorigenesis was observed in nude mice from those cells.Conclusion AP20187 combine either SCF or FL mediated activation of the jak2 signaling domain can dramatically expand hematopoietic stem/progenitor cells, and the expanded cells can be regulated and committed differentiate into multileages. This system may provide important insights into stem cell biology and may have applications for gene and cell therapy. Background Cord blood (CB) is an established source of HSCs for allogeneic or autologous transplantation. To date, UCB has being increasingly used as an alternatively hematopoietic stem cell source for stem cell transplantation, marrow failure syndromes and hereditary immunodeficiency. Recent studies indicate that human CB have lots of advantages such as self-renewal, multipotential differentiation, long-term repopulation ability and high immunological tolerence, which are one of the most ideal targets for gene therapy. But the inefficiency of gene transfer into human CB cells poses a major obstacle for gene therapy of inherited and acquired blood cell disorders. The development of therapeutic applications for stem cell gene transfer depends on markedly increasing the proportion of genetically corrected stem cells. Recently, Several lines of evidence suggest that the tyrosine kinase JAK2 might play a significant role in HSC/MHPC self-renewal. In order to overcome the obstacle of inefficiency gene transfer of CB, we take advantage of the principle of gene regulated expression technology, developed a system which can be used to expansion targeted genetically modified CD34+ purified cells. This approach allows for the specific delivery and activation of intracellular JAK2 signals to CD34+ population and greatly expansion of transduced CB CD34+ cells.Objective To determine the feasibility of long-term regulated expansion of genetically modified human CD34+ purified cord blood cells in vitro and evaluate their biological characteristics and safety. Methods A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducers of dimerization (AP20187) was cloned. JAK2 can be dimerized through administration of AP20187. The RV vector encoding a green fluorescent protein (GFP) served as a marker. CD34+cells were isolated from cord blood cells by MiniMACS. The purified CD34+cells were tranduced with supernatant from the RV packaging cell line. Following transduction, cells were cultured in suspension with in a combination of growth factors (SCF, Flt3, TPO and IL-6), either in the presence or absence of AP20187 (100nM). We then analyzed their GFP expression efficiency, immunophenotype and multipotent committed progenitor colony assay for JAK2 transduced CD34+cells. Meanwhile cells were evaluated the safety, karyotype and nude mice model tumorigenesis. Results The purity of selected CD34+ cells was over 91% and gene transfer rate was 49.3%a±6.2%. We obtained CD34+cells proliferation both in the presence of ASFTI and SFTI groups, but the GFP expression efficiency significantly difference between two groups. The percentage of GFP+ cells over time in culture was strongly influenced by the presence of the AP20187. The absence of AP20187 group was associated with a steady decline in the percentage of GFP+ cells from baseline immediately following transduction, gradually to disappear in culture. In contrast, the addition of AP20187 group consistently produced a rise to the 95% peak level in the percentage of GFP+ cells by the end of 11th week of culture. Thereafter, cultures performed in the presence of AP20187 maintained a higher frequency of GFP+ cells than cultures performed in the absence of AP20187. ASFTI group appears to continue express CD34+CD38-/CD34+CD38+ subpopulation that their augmented folds have significantly difference compared with SFTI control group at the time culture around 6 weeks. Flow cytometry analysis also showed the phenotype of the expanded cells were CD33+, CD61+ and Gly-A+ partial positive; CD38+ and HLA-DR+ strong positive, while CD2,CD7 and CD19 were almost negative by the end of 8th week of culture. The expanded CD34+ cells could still form CFU-GM, BFU-E and CFU-Mix colonies over 12 weeks culture. Neither abnormality in karyotype analysis nor tumorigenesis was observed in nude mice from the expanded cells. Conclusions Our results demonstrate that human CB cells can be expanded for prolonged periods in culture by transduced JAK2 in response to AP20187 and cytokines combination. These findings have potential useful application of cell therapy in treatment of genetically manipulated hematopoietic stem cell disorder.
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