Radtke Stefan, Kiem Hans-Peter
Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA.
Methods Mol Biol. 2023;2567:87-98. doi: 10.1007/978-1-0716-2679-5_6.
The preclinical development of hematopoietic stem cell (HSC) gene therapy/editing and transplantation protocols is frequently performed in large animal models such as nonhuman primates (NHPs). Similarity in physiology, size, and life expectation as well as cross-reactivity of most reagents and medications allows for the development of treatment strategies with rapid translation to clinical applications. Especially after the adverse events of HSC gene therapy observed in the late 1990s, the ability to perform autologous transplants and follow the animals long-term make the NHP a very attractive model to test the efficiency, feasibility, and safety of new HSC-mediated gene-transfer/editing and transplantation approaches.This protocol describes a method to phenotypically characterize functionally distinct NHP HSPC subsets within specimens or stem cell products from three different NHP species. Procedures are based on the flow-cytometric assessment of cell surface markers that are cross-reactive in between human and NHP to allow for immediate clinical translation. This protocol has been successfully used for the quality control of enriched, cultured, and gene-modified NHP CD34 hematopoietic stem and progenitor cells (HSPCs) as well as sort-purified CD34 subsets for transplantation in the pig-tailed, cynomolgus, and rhesus macaque. It further allows the longitudinal assessment of primary specimens taken during the long-term follow-up post-transplantation in order to monitor homing, engraftment, and reconstitution of the bone marrow stem cell compartment.
造血干细胞（HSC）基因治疗/编辑及移植方案的临床前开发通常在大型动物模型中进行，如非人灵长类动物（NHP）。生理、大小和预期寿命的相似性，以及大多数试剂和药物的交叉反应性，使得能够开发出可快速转化为临床应用的治疗策略。特别是在20世纪90年代末观察到HSC基因治疗的不良事件后，进行自体移植并长期跟踪动物的能力使NHP成为测试新型HSC介导的基因转移/编辑及移植方法的效率、可行性和安全性的极具吸引力的模型。本方案描述了一种对来自三种不同NHP物种的标本或干细胞产品中功能不同的NHP造血干细胞亚群进行表型特征分析的方法。程序基于对细胞表面标志物的流式细胞术评估，这些标志物在人和NHP之间具有交叉反应性，以便立即进行临床转化。本方案已成功用于富集、培养和基因修饰的NHP CD34造血干细胞和祖细胞（HSPC）的质量控制，以及用于猪尾猕猴、食蟹猕猴和恒河猴移植的分选纯化CD34亚群。它还允许对移植后长期随访期间采集的原始标本进行纵向评估，以监测骨髓干细胞区室的归巢、植入和重建情况。
