Trivedi H L, Mishra V V, Vanikar A V, Modi P R, Shah V R, Shah P R, Firoz A
Institute of Transplantation Sciences, Department of Nephrology and Transplantation Medicine, Civil Hospital, Gujarat, India.
Transplant Proc. 2006 Nov;38(9):3103-8. doi: 10.1016/j.transproceed.2006.08.173.
We generated an human embryonic stem cell (hESC) line to augment chimerism-associated tolerance. A 40-year-old African with chronic glomerulonephritis-chronic renal failure with 100% G6PD enzyme deficiency presented for renal transplantation with a 27-year-old, 6/6 HLA-matched sister as a willing donor.
We generated an hESC line from the donor's oocytes using long ovarian stimulation protocol simultaneously with tolerance induction protocol. A nuclear transfer (NT)-hESC line was derived by transferring a donor cumulus cell into an enucleated oocyte, subjected to electrical fusion, and cultured for 5 days. ESCs hatched from the blastocyst on day 6 were cocultured with her unmodified bone marrow for 2 days and suspended in Ringer's lactate. Five milliliters of suspension were collected for cell counting, viability, pluripotency, flow cytometry, and karyotyping. The remaining suspension was infused into the periphery of the recipient. Transplantation was performed 1 week later following a negative lymphocytotoxicity cross-match test using no immunosuppression. Peripheral blood chimerism (PBC) was studied using fluorescent in situ hybridization technique. Allograft biopsy was performed on day 7.
NT-hESC CD34+ count was 7.6%, viability 100%, karyotyping normal, pluripotency markers: SSEA-1, SSEA-4, OCT-3/4, TRA-1/60:positive; 12% PBC was noted at 1 week after transplantation. Serum creatinine was 1.2 mg%, graft biopsy was unremarkable, and G6PD enzyme deficiency was corrected to 0% at 100 days posttransplant. Liver function tests and hematology profile were unremarkable for graft-versus-host disease.
This is the first report of tolerance induction using NT-hESC-induced hematopoietic chimerism with synergistic use of adult bone marrow. It was safe and effective.
我们生成了一条人类胚胎干细胞（hESC）系以增强嵌合相关的耐受性。一名40岁患有慢性肾小球肾炎-慢性肾衰竭且100%葡萄糖-6-磷酸脱氢酶（G6PD）酶缺乏的非洲人，因肾移植前来就医，其27岁、HLA配型6/6相合的妹妹作为志愿供体。
我们使用长期卵巢刺激方案并同时采用耐受性诱导方案，从供体卵母细胞中生成了一条hESC系。通过将供体卵丘细胞转移至去核卵母细胞中，进行电融合，并培养5天，获得了一条核移植（NT）-hESC系。第6天从囊胚孵化出的胚胎干细胞与她未修饰的骨髓共培养2天，然后悬浮于乳酸林格氏液中。收集5毫升悬浮液用于细胞计数、活力检测、多能性检测、流式细胞术分析和核型分析。其余悬浮液注入受体外周。1周后，在淋巴细胞毒性交叉配型试验呈阴性且未使用免疫抑制的情况下进行移植。使用荧光原位杂交技术研究外周血嵌合现象（PBC）。在第7天进行移植肾活检。
NT-hESC的CD34+细胞计数为7.6%，活力为100%，核型正常，多能性标志物：阶段特异性胚胎抗原-1（SSEA-1）、阶段特异性胚胎抗原-4（SSEA-4）、八聚体结合转录因子3/4（OCT-3/4）、肿瘤相关抗原-1/60（TRA-1/60）均为阳性；移植后1周时检测到12%的外周血嵌合现象。血清肌酐为1.2毫克%，移植肾活检无异常，移植后100天时G6PD酶缺乏纠正至0%。肝功能检查和血液学指标在移植物抗宿主病方面无异常。
这是关于使用NT-hESC诱导造血嵌合并协同使用成人骨髓进行耐受性诱导的首份报告。该方法安全有效。
