Dao M A, Nolta J A
Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles and University of Southern California School of Medicine, Department of Pediatrics, Los Angeles, CA 90027, USA.
Int J Mol Med. 1998 Jan;1(1):257-64. doi: 10.3892/ijmm.1.1.257.
The potentiality of primitive human hematopoietic cells can be profoundly affected by in vitro culture. Due to the growing number of protocols proposed for stem cell gene therapy and ex vivo expansion, it is crucial to define methods to preserve the generative capacity of human stem cells in culture while promoting self-renewal divisions. Stem cell division, homing, and subsequent lineage development can only be studied definitively by marking of pluripotent cells, followed by tracking and clonal analysis of the progeny in a long-term transplantation system. We have developed a bnx/hu xenograft model, in which transduced human hematopoietic cells can be individually tracked into different lineages over the course of one year post-transplantation. The tracking is accomplished by single cell cloning of individual T lymphoid and myeloid progenitors recovered from the marrow of the mice, and clonal integration analysis by the sensitive technique of single-colony inverse PCR. All cells derived from a stem cell transduced by a retroviral vector will carry the unique restriction fragment length polymorphism (RFLP) created by the random integration event. We have used the bnx/hu xenograft system coupled with single-colony inverse PCR to determine that human stem cells require stromal support, fibronectin support with cytokines, or the presence of Flt3 ligand during a 72-h ex vivo culture to maintain the ability to sustain long-term multilineage hematopoiesis.
原始人类造血细胞的潜能会受到体外培养的深刻影响。由于用于干细胞基因治疗和体外扩增的方案越来越多，因此确定在促进自我更新分裂的同时保持培养中人类干细胞生成能力的方法至关重要。干细胞分裂、归巢以及随后的谱系发育只能通过对多能细胞进行标记，然后在长期移植系统中对后代进行追踪和克隆分析来明确研究。我们开发了一种bnx/hu异种移植模型，在该模型中，转导的人类造血细胞在移植后一年内可以被单独追踪到不同谱系中。通过从小鼠骨髓中回收的单个T淋巴细胞和髓系祖细胞的单细胞克隆，以及通过单菌落反向PCR的敏感技术进行克隆整合分析来完成追踪。所有源自逆转录病毒载体转导的干细胞的细胞都将携带由随机整合事件产生的独特限制性片段长度多态性（RFLP）。我们使用bnx/hu异种移植系统结合单菌落反向PCR来确定人类干细胞在72小时的体外培养过程中需要基质支持、细胞因子的纤连蛋白支持或Flt3配体的存在，以维持长期多谱系造血的能力。
