Gene Therapy Program, Department of Genetics, Cell Biology and Development, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA.
Gene Ther. 2010 Feb;17(2):238-49. doi: 10.1038/gt.2009.131. Epub 2009 Oct 15.
Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug-resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase the engraftment of gene-modified, hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR-GFP-expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rgammac(null) (NSG) mice after injection of Tyr22-DHFR-hESC-derived cells significantly increases human CD34(+) and CD45(+) cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR cells in vivo, and provides a novel approach for combined human cell and gene therapy.
人胚胎干细胞（hESCs）为基因治疗应用提供了一种新颖的造血和其他细胞群体来源。评估将 hESC 衍生的造血细胞移植到免疫缺陷小鼠中植入的临床前研究仅显示出有限的再定植。表达药物抗性基因，如 Tyr22-二氢叶酸还原酶（Tyr22-DHFR），与甲氨蝶呤（MTX）化疗偶联具有选择性增加基因修饰的 hESC 衍生细胞在小鼠异种移植物中的植入的潜力。在这里，我们描述了 Tyr22-DHFR-GFP 表达 hESC 的产生，这些细胞保持多能性，产生畸胎瘤，并能分化为 MTXr-造血内皮细胞。我们证明，在注射 Tyr22-DHFR-hESC 衍生细胞后，给予非肥胖型糖尿病/严重联合免疫缺陷/IL-2Rgammac（null）（NSG）小鼠 MTX 可显著增加骨髓（BM）和移植 MTX 治疗小鼠外周血中人类 CD34(+)和 CD45(+)细胞的植入。这些结果表明 MTX 治疗支持 Tyr22-DHFR 细胞在体内的选择性、长期植入，并为联合人类细胞和基因治疗提供了一种新方法。
