Gu Angel, Torres-Coronado Monica, Tran Chy-Anh, Vu Hieu, Epps Elizabeth W, Chung Janet, Gonzalez Nancy, Blanchard Suzette, DiGiusto David L
1 Laboratory for Cellular Medicine, Beckman Research Institute , City of Hope, CA 91010.
Hum Gene Ther Methods. 2014 Aug;25(4):221-31. doi: 10.1089/hgtb.2014.043.
Hematopoietic stem cell gene therapy for HIV/AIDS is a promising alternative to lifelong antiretroviral therapy. One of the limitations of this approach is the number and quality of stem cells available for transplant following in vitro manipulations associated with stem cell isolation and genetic modification. The development of methods to increase the number of autologous, gene-modified stem cells available for transplantation would overcome this barrier. Hematopoietic stem and progenitor cells (HSPC) from adult growth factor-mobilized peripheral blood were cultured in the presence of an aryl hydrocarbon receptor antagonist (AhRA) previously shown to expand HSPC from umbilical cord blood. Qualitative and quantitative assessment of the hematopoietic potential of minimally cultured (MC-HSPC) or expanded HSPC (Exp-HSPC) was performed using an immunodeficient mouse model of transplantation. Our results demonstrate robust, multilineage engraftment of both MC-HSPC and Exp-HSPC although estimates of expansion based on stem cell phenotype were not supported by a corresponding increase in in vivo engrafting units. Bone marrow of animals transplanted with either MC-HSPC or Exp-HSPC contained secondary engrafting cells verifying the presence of primitive stem cells in both populations. However, the frequency of in vivo engrafting units among the more primitive CD34+/CD90+ HSPC population was significantly lower in Exp-HSPC compared with MC-HSPC. Exp-HSPC also produced fewer lymphoid progeny and more myeloid progeny than MC-HSPC. These results reveal that in vitro culture of adult HSPC in AhRA maintains but does not increase the number of in vivo engrafting cells and that HSPC expanded in vitro contain defects in lymphopoiesis as assessed in this model system. Further investigation is required before implementation of this approach in the clinical setting.
用于治疗人类免疫缺陷病毒/获得性免疫缺陷综合征（HIV/AIDS）的造血干细胞基因疗法是终身抗逆转录病毒疗法的一种有前景的替代方法。这种方法的局限性之一是，在与干细胞分离和基因改造相关的体外操作后，可用于移植的干细胞数量和质量。开发增加可用于移植的自体基因改造干细胞数量的方法将克服这一障碍。将来自成年生长因子动员外周血的造血干细胞和祖细胞（HSPC）在一种芳烃受体拮抗剂（AhRA）存在的情况下进行培养，该拮抗剂先前已被证明可扩增来自脐带血的HSPC。使用免疫缺陷小鼠移植模型对最低限度培养的（MC-HSPC）或扩增的HSPC（Exp-HSPC）的造血潜力进行定性和定量评估。我们的结果表明，MC-HSPC和Exp-HSPC均能实现强大的多谱系植入，尽管基于干细胞表型的扩增估计并未得到体内植入单位相应增加的支持。移植了MC-HSPC或Exp-HSPC的动物的骨髓中含有二次植入细胞，证实了这两个群体中均存在原始干细胞。然而，与MC-HSPC相比，Exp-HSPC中更原始的CD34+/CD90+ HSPC群体中体内植入单位的频率显著更低。与MC-HSPC相比，Exp-HSPC产生的淋巴系后代也更少，髓系后代更多。这些结果表明，在AhRA中对成年HSPC进行体外培养可维持但不会增加体内植入细胞的数量，并且在该模型系统中评估发现，体外扩增的HSPC在淋巴细胞生成方面存在缺陷。在临床环境中实施这种方法之前，还需要进一步研究。
