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的造血系，在过去的几十年里，它们的广泛特征导致了临床应用，包括骨髓移植治疗影响造血室的良性和恶性疾病。基于CD34抗原表达的骨髓细胞的分离丰富了原始的隔室，而分离CD34阳性群体的技术在临床上是常用的。对真正的造血干细胞的分析需要移植和再繁殖，因此很难对人类细胞之间的这种潜力提出质疑。因此，我们开发了一种具有强大的人类造血干细胞植入的异种移植模型，并展示了所有细胞系的植入。这项测试将使我们能够在相关模型中首次在植入的人类细胞中测试珠蛋白载体。此外，造血干细胞沿红系的体外分化可用于检测病毒载体导向的人β珠蛋白基因转移后的表达情况。事实证明，这些系统在开发临床应用的病毒载体系统方面很有用，包括我们团队开发的正向珠蛋白载体，它的载体滴度和长期再繁殖细胞的转导效率分别提高了10倍和10倍。

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.