Study On Multidrug Resistance Gene Transfer And Expansion In Vitro Of Hematopoietic Stem Cells To Support High-Dose Chemotherapy

Background:Ovarian cancer is the leading cause of death from gyneocologic cancer. It is hard to be detected at early stage and 2/3 of patients with ovarian cancer have advanced ovarian cancer at the time of the primary diagnosis. Chemotherapy plays an important role in cancer therapy. To some degree, the chemotherapy effect is intensified with the increasing doses of chemotherapeutic drugs. However, myelosuppression, which is the major side effect of most drugs, restricts the application of high-dose chemotherapy. The human multidrug resistance 1 (MDR1) gene is a candidate gene encoding the membrane-located drug-efflux pump P-glycoprotein (P-gp). P-gp confers resistance to a wide array of cytostatic agents by pumping these drugs out of the cells. The transfers of MDR1 genes into hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are expected to confer drug-resistance to the bone marrow (BM) of patients so as to protect them from myelotoxicity which is the main adverse effect of high-dose chemotherapy. However, low gene transfer efficiency in vitro and a low number of reconstituting MDR1 gene-made cells and/or inefficient expression of the transgene in vivo has hampered its wide application in clinical trials.Retroviral vector is the safest and the most extensively used gene transfer system. The prerequisite for higher gene transfer efficiency is that the target cells must be in division. However, human HSCs are mostly quiescent in nature. That's the main reason for low efficiency of gene transfer into human HSCs. Based on the farther study of biological characteristics of human HSCs, investigators are exploring more desirable gene vector system and optimizing the transduction conditions in order to find a perfect and feasible clinical protocol with high gene transfer efficiency. Published optimized protocols demonstrated the improvement of the level of gene transfer into human HSCs. These optimizations include: the use of alternate vectors, such as pseudotyped vectors (GALV envelope, VSV-G envelope, RD114 envelope, and so on), adeno-associated virus (AAV) vector, lentivirus vectors (HIV vectors) and foamy virus (FV) vector; the use of early acting cytokines and/or their combinations inducing HSCs into cell cycles, such as stem cell factor (SCF), Flt-3 ligand (FL) and thrombopoietin (TPO); the use of stromal cells mimicking hematopoietic microenvironment and the use of recombinant fibronectin colocalizating retroviral particles and target cells. Prestimulation of HSCs with cytokines almost becomes a routine procedure in HSCs transduction with retroviral vectors. However, these stimulatory signals forcing HSCs into the cell cycles result in impaired long-term engraftment in vivo. So how to induce HSCs into cell cycles without impairing their engraftment ability will be an appealing attempt.The culture system with supporting stromal cells can mimick the hematopoietic microenvironment in vivo and counteract the HSCs differentiating action induced by cytokines so that HSCs can self-renew in vitro. Stromal cells can be isolated from umbilical cord, bone marrow and fetal liver. These stromal cells were transformed into cell lines with SV40, such as yolk sac CD34⁺ endothelial cells, human umbilical vein endothelial cell line (HUVEC), MS-5, M2-10B4, HESS-5, HES-1, AFT024, 2018, 2012 cell lines. Every cell line can support HSCs in vitro independently and has its unique mechanism and effect. Therein, AFT024 cell line was proved to be able to maintain the long-term hematopoietic reconstitution and multileage differentiation capabilities of HSCs and be superior to other stromal cell lines. AFT024 cells can recruit significant numbers of HSCs into cell cycles and increase asymmetric divisions of HSCs which result in a balance between self-renewal and differentiation. Thus, it would be possible to introduce retroviral markers into HSCs efficiently when HSCs were co-cultured with the AFT024 cells. In this paper, we isolated CD34⁺ cells from umbilical cord blood and co-cultured them in the presence and absence of the AFT024 feeder cells for 7 days. Then in the subsequent 14 days, the MDR1 retrovirus was added to infect cultured CD34⁺ cells in the presence or absence of the AFT024 feeder cells. MDR1 gene transfer efficiency and expansion of CD34⁺ cells was detected in vitro. Then we transplanted MDR1-transduced cells or freshly isolated CD34⁺ cells into NOD/SCID mice. 28 days after transplantation, survived mice received paclitaxel chemotherapy. We investigated whether MDR1-transduced CD34⁺ cells can complete hematopoietic reconstitution and ameliorate hematopoietic toxicity induced by paclitaxel chemotherapy.Part I: Effect of AFT024 Cells on the Multidrug Resistant Gene1 (MDR1) Transfer Efficiency and Expansion in Vitro of CD34⁺Cells Derived From Umbilical Cord BloodObjective: To investigate the effect of AFT024 cells on the transfer efficiency of multidrug resistant gene 1 (MDR1) and the expansion in vitro of CD34⁺cells derived from umbilical cord blood.Methods: Retroviral vector pHaMDR1/A containing the full-length human MDRl cDNA was transfected into PA317 packaging cell line. Clones of transfected PA317 cells were selected in colchicine and retrovirus-containing supernatant was harvested. CD34⁺ cells were isolated from human umbilical cord blood (UCB) by MACS CD34 Progenitor Cell Isolation Kit and co-cultured with AFT024 cells (AFT024 group) or cultured alone (control group) for 7 days. In the subsequent 14 days, the retrovirus carrying MDR1 gene was supplemented twice a week to transfect CD34⁺ cells with or without the support of AFT024 cells. RT-PCR method was used to assay the level of MDR1 mRNA in the transfected cells. The expression of P-glycoprotein (P-gp) was evaluated by flow cytometry (FCM) assay and and the function of P-gp was evaluated by rhodamine-123 (Rh-123) efflux assay. The gene transfer efficiency was calculated by drug-resistant colony-forming cells assay. The expansion folds of total nucleated cells (TNCs), CD34⁺ cells and colony-forming cells (CFCs) were counted on the seventh, fourteenth and twenty-first days after culture.Results: (1) A higher level of MDR1 mRNA transcription was detected in the transfected cells in AFT024 group by RT-PCR than in control group. The gene transfer efficiency was significantly higher in the transfected cells in AFT024 group than in control group (46.0% vs 15.2%) (P<0.01). The expression of P-gp in AFT024 group was (31.7%±10.2%), significantly higher than that in control group (12.6%±3.9%) (P<0.01). Rh-123 efflux assay detected (35.5%±11.4%) of cells with P-gp efflux function in AFT024 group, significantly higher than in control group (16.6%±3.2%) (P<0.01). (2) On the seventh day, the expansion folds of TNCs cells, CD34⁺ cells and CFCs in control group were (12.1±2.6), (2.5±1.0) and (4.5±1.4) respectively, slightly more than in AFT024 group [(8.2±2.4), (2.3±1.1) and (3.5±1.0) respectively], but the differences were not significant (P>0.05). On the fourteenth day, the expansion fold of TNCs in control group (102.9±26.6) was significantly more than in AFT024 group (78.9±16.6) (P<0.05). However, the expansion folds of CD34⁺ cells and CFCs in control group were (6.6±2.8) and (6.7±2.1), less than in the AFT024 group [(16.2±7.6) and (9.4±4.0)]. Therein, there was significant difference in the expansion fold of CD34⁺ cells between the two group (P<0.05). On the twenty-first day, the expansion folds of TNCs, CD34⁺ cells and CFCs were (145.0±26.8), (37.9±13.9) and (27.1±13.3) in AFT024 group and (132.0±26.7), (9.1±2.3) and (7.7±3.6) in control group respectively. The expansion folds of CD34⁺ cells and CFCs in the AFT024 group were significantly more than in control group (P<0.01).Conclusions: AFT024 cells can facilitate the transfer of MDR1 gene into CD34⁺ cells and improved the expansion in vitro of primitive hematopoietic cells. Part II: Hematopoietic Reconstitution and Chemoprotectionin NOD/SCID Mice Transplanted with MDR1-transducedHematopoietic Stem CellsObjective: To investigate whether MDR1-tranduced hematopoietic stem cells transplantation can reconstitute hematopoiesis in sublethally irradiated NOD/SCID mice and protect them from myelotoxicity induced by paclitaxel (PAC) chemotherapy.Methods: We transduced MDRl gene into UCB-derived CD34⁺ cells with the support of AFT024 cells for 3 weeks and transplanted them into sublethally irradiated NOD/SCID mice (MDR1 group). Mice in control group were transplanted with freshly isolated CD34⁺ cells. Peripheral blood was analyzed once every week. 28 days after transplantation, survived mice received PAC injection intraperitonially (12mg/kg/d×5d) or normal saline (NS). Peripheral blood was analyzed once every week. 21 days after chemotherapy, mice were sacrificed and bone marrow was dectected for the percentage of human CD45⁺ cells (NOD/SCID repopulating cells, SRCs) and Rh-123^dull CD45⁺ cells (SRCs expressing MDR1 gene) by flow cytometry. Results: (1) MDR1 mice showed an accelerated return of WBC counts in comparison to control mice and the number of WBC in MDRl mice arrived at (4.5±0.6)×10⁹/L at day 21, which is significantly higher than that in control mice[(2.8±0.8)×10⁹/L] (P<0.001). At day 28 when PAC began to be administrated, WBC counts reached (4.4±0.9)×10⁹/L in MDRl mice and (4.7±0.9)×10⁹/L in control mice and there was on difference between the two group (P>0.05). (2) MDR1-PAC group showed significantly improved survival rate (90%, 9/10) than control-PAC group (50%, 5/10) after PAC chemotherapy. The time for WBC counts dropping to the nadir in MDR1-PAC group was shorter than that in control-PAC group. WBC counts in MDR1-PAC group dropped to a nadir of (2.3±1.4)×10⁹/L at day 7 after chemotherapy. Beyond day 7, WBC counts in MDR1-PAC group increased to (3.1±1.4)×10⁹/L at day 14 while that in control-PAC group continued to drop and reached a significantly lower nadir of (1.5±1.2)×10⁹/L at day 14. Because the nadir of WBC counts in MDR1-PAC group is significantly higher than that in control-PAC group (P<0.05) and WBC counts in MDR1-PAC group began to increase earlier than in control-PAC group, WBC counts in MDR1-PAC group increased to (5.4±1.2×10⁹/L) by day 21, significantly higher than (3.8±1.0)×10⁹/L in control-PAC group (P<0.05). (3) We detected equivalent CD45⁺ cells in the BM of mice receiving NS treatment in MDR1 group (48.8%±17.0%) and control group (50.2%±12.7%). A higher level of human CD45⁺ cells in MDR1-PAC group (31.0%±12.5%) was detected than in control-PAC group (21.8%±8.5%). (4) The percentage of Rh-123^dull CD45⁺ cells in the MDR1 mice treated with PAC was 38.8%±12.9%, significantly higher than a proportion of (18.7%±7.5%) in the MDR1 mice receiving NS only (P< 0.01). A much lower level of human Rh-123^dull CD45⁺ cells were detected in BM of control mice (4.1%±1.8% in control-PAC group and 3.8%±1.1% in control-NS group).Conclusions: MDR1-transduced human hematopoietic cells could facilitate hematopoietic recovery and completely reconsistitute hematopoiesis in sublethally irradiated NOD/SCID mice as well as freshly isolated CD34⁺ cells. Mice transplanted with MDR1-transduced human hematopoietic cells were protected from PAC chemotherapy with higher survival rate and higher level of WBC counts compared with mice transplanted with untransduced hematopoietic stem cells.