一种用于定量亚髓细胞消融宿主中经体外操作的小鼠骨髓细胞植入的新型竞争性再增殖策略。

A novel competitive repopulation strategy to quantitate engraftment of ex vivo manipulated murine marrow cells in submyeloablated hosts.

作者信息

Wyss Brandon K, Meyers Justin L, Sinn Anthony L, Cai Shanbao, Pollok Karen E, Goebel W Scott

机构信息

Herman B Wells Center for Pediatric Research.

出版信息

Exp Hematol. 2008 Apr;36(4):513-21. doi: 10.1016/j.exphem.2007.12.002. Epub 2008 Feb 4.

DOI:10.1016/j.exphem.2007.12.002

PMID:18243491

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2373923/

Abstract

OBJECTIVE

Standard competitive repopulation assays have proven valuable in evaluating engraftment potential in ablated hosts, permitting comparisons between various test cell populations. However, no similar method exists to compare engraftment of test cells in submyeloablated hosts, which would be helpful given the applications of reduced-intensity conditioning for hematopoietic gene-replacement therapy and other cellular therapies. Here, we developed a novel assay to quantitate engraftment of hematopoietic stem cells in submyeloablated hosts.

MATERIALS AND METHODS

Engraftment of murine marrow cells transduced with retroviral vectors using two separate protocols was compared to engraftment of fresh untreated competitor cells within low-dose radiation-conditioned hosts using a "three-way" marking system, so that test, competitor, and host cell chimerism could be reliably determined posttransplantation.

RESULTS

We demonstrate that the repopulating ability of marrow cells transduced using two distinct protocols was reduced approximately 10-fold compared to fresh competitor cells in submyeloablated hosts utilizing the novel "three-way" transplant assay.

CONCLUSIONS

Murine marrow cells transduced using a clinically applicable protocol acquire an engraftment defect in submyeloablated hosts, similar to cells transduced using a research protocol. We conclude that the submyeloablative competitive repopulation assay described here will be of benefit to comparatively assess the engraftment ability of manipulated hematopoietic stem cells using various culture protocols, such as to test the impact of modifications in transduction protocols needed to attain therapeutic levels of gene-corrected blood cells, or the effect of ex vivo expansion protocols on engraftment potential.

摘要

目的

标准的竞争性再增殖试验已被证明在评估经消融处理的宿主中的植入潜力方面很有价值，能够对各种测试细胞群体进行比较。然而，目前尚无类似方法可用于比较亚骨髓消融宿主中测试细胞的植入情况，鉴于低强度预处理在造血基因替代疗法和其他细胞疗法中的应用，这一方法将很有帮助。在此，我们开发了一种新型试验，用于定量亚骨髓消融宿主中造血干细胞的植入情况。

材料与方法

使用“三标记”系统，将通过两种不同方案用逆转录病毒载体转导的小鼠骨髓细胞的植入情况，与低剂量辐射预处理宿主中新鲜未处理的竞争细胞的植入情况进行比较，以便在移植后可靠地确定测试、竞争和宿主细胞嵌合情况。

结果

我们证明，在利用新型“三标记”移植试验的亚骨髓消融宿主中，与新鲜竞争细胞相比，通过两种不同方案转导的骨髓细胞的再增殖能力降低了约10倍。

结论

使用临床适用方案转导的小鼠骨髓细胞在亚骨髓消融宿主中出现植入缺陷，类似于使用研究方案转导的细胞。我们得出结论，本文所述的亚骨髓消融竞争性再增殖试验将有助于比较评估使用各种培养方案处理的造血干细胞的植入能力，例如测试为达到基因校正血细胞治疗水平所需的转导方案修改的影响，或体外扩增方案对植入潜力的影响。