Isolation, characterization, and transplantation of candidate stem cells

造血干细胞在出生后存在于骨髓中，为宿主的生命提供所有造血谱系，并且在过去几十年中它们的广泛表征已经导致临床应用，包括骨髓移植用于影响造血区室的良性和恶性病症。 基于CD34抗原表达的骨髓细胞分离富集原始区室，并且分离CD34阳性群体的技术通常用于临床实践。 真正的造血干细胞的检测需要移植和再增殖，因此在人类细胞中查询这种潜力的研究是困难的。 因此，我们已经开发了具有稳健的人造血干细胞植入的异种移植模型，并且已经证明了所有细胞谱系的植入。 该试验将使我们能够在相关模型中首次在移植的人类细胞中测试珠蛋白载体。 此外，造血干细胞沿着类红细胞谱系的离体分化可用于测定转移人β珠蛋白基因后病毒载体指导的β珠蛋白表达。 这些系统已被证明可用于开发用于临床应用的病毒载体系统，包括由我们的小组开发的正向珠蛋白载体，其显示出10倍更高的载体滴度以及10倍更高的长期再增殖细胞的转导效率。

Hematopoietic stem cells reside within the bone marrow post-natally, providing all hematopoietic lineages for the life of the host, and their extensive characterization over the last several decades has led to clinical application including bone marrow transplantation for benign and malignant disorders affecting the hematopoietic compartment. Isolation of bone marrow cells based upon expression of the CD34 antigen enriches for the primitive compartment, and techniques to isolate the CD34-positive population are commonly used in clinical practice. Assays for true hematopoietic stem cells require transplantation and repopulation, and thus studies querying such potential among human cells are difficult. We have thus developed a xenograft model with robust human hematopoietic stem cell engraftment, and have demonstrated engraftment of all cell lineages. This assay will allow us to test globin vectors for the first time among engrafted human cells in a relevant model. Furthermore, ex vivo differentiation of hematopoietic stem cells along the erythroid lineage can be utilized to assay viral vector directed beta-globin expression after transfer of the human beta globin gene. These systems have proven useful in developing viral vector systems for clinical application including a forward oriented globin vector which has been developed by our group and demonstrates 10 fold higher vector titer as well as 10 fold higher transduction efficiency for long term repopulating cells.