CTx-003

Bone marrow (BM)-derived cells (BMDCs) contribute to endometrial regeneration. Our objective was to develop a nongonadotoxic mouse BM transplant (BMT) model using 5-fluorouracil (5-FU) for investigating BMDCs trafficking in reproduction. Female C57BL/6J mice received either single (CTX-1) or paired (CTX-2) 5-FU (150 mg/kg) dose, or single (CTX-1+SCF) or paired-dose (CTX-3+SCF) 5-FU with stem cell factor (SCF). Control mice received BMT only or saline. BM cells (20 × 106) from transgenic green-fluorescent protein (GFP) mice were injected iv. For fertility experiment, mice were mated on day 28 after BMT. Alternatively, mice were killed 1 month after BMT and BMDCs recruitment to the uterus was determined. Mice receiving 5-FU ± SCF showed intact ovarian function and fertility. CTX-3+SCF resulted in greatest BM donor chimerism at 1 month (∼45%). Flow cytometry analysis demonstrated that 6.6% of total uterine cells in CTX-3+SCF mice were GFP+ BMDCs. Remarkably, this was about 40- and 80-fold greater than BMDCs in uterus of CTX-1 or BMT only mice (6.6% vs 0.16% vs 0.08%, respectively, P < .001). Immunohistochemical analysis showed that BMDCs in the uterus were mostly localized to the endometrial stroma (71.8%). The majority of endometrial BMDCs colocalized with the pan-leuokocyte CD45 marker (58.5%), but 41.5% were CD45-negative. Cytokeratin and CD31 staining showed that the GFP+CD45− cells were not epithelial or endothelial, confirming their stromal identity. We demonstrate that paired-dose 5-FU regimen results in efficient BM donor chimerism while maintaining ovarian function and fertility. This model could be used for studying BMDCs trafficking to the uterus in various reproductive physiological and pathological conditions.

To investigate the feasibility, efficacy and safety of high dose immunosuppressive therapy (HDIT) and autologous peripheral blood stem cell transplantation (PBSCT) with CD(34)(+) cell selection in patients with refractory and severe autoimmune diseases.

Twenty-one patients with SLE, RA, pSS, SSc or MCTD were enrolled in the study from 1999. Autologous haemopoietic stem cells were mobilized with CTX 3 approximately 4 g/m(2) and granulocyte colony stimulating factor (G-CSF). CD(34)(+) cells were selected by CliniMACS. After conditioning with CTX (200 mg/kg) and pig antithymocyte globulin (ATG, 90 mg/kg) or CTX (150 mg/kg) and total body irradiation (TBI, 4 approximately 6 Gy), the enriched CD(34)(+) cells were reinfused.

All patients completed the mobilization and leukapheresis procedures successfully, and proceeded to receive conditioning and transplantation. Two patients died of complication related to transplantation, one is CMV infection, the other is severe pneumonia during the course of granulocyte deficiency. A MCTD patient completed the stem cell mobilization and died of severe pulmonary hypertension and heart failure before CD(34)(+) cells reinfusing. Two SLE patients relapsed in 26, 37 months respectively and a RA patient relapsed in 15 months after transplantation. Other patients got improved, with SLE-DAI score decreasing from 17 to 4 score and proteinuria decreasing from 6.7 g to 2.3 g in SLE patients; DAS28 score from 7.9 to 2.1 in RA patient; Symptom improved and lab results recovered in SS.

High dose immunosuppressive therapy followed by autologous peripheral blood stem cell transplantation with CD(34)(+) cell selection is feasible and relative safe. Patients remain free from disease active and improved continuously. Some patients could relapse after transplantation. Long-term effect need to be further observed.