Webber Beau R, Osborn Mark J, McElroy Amber N, Twaroski Kirk, Lonetree Cara-Lin, DeFeo Anthony P, Xia Lily, Eide Cindy, Lees Christopher J, McElmurry Ron T, Riddle Megan J, Kim Chong Jai, Patel Dharmeshkumar D, Blazar Bruce R, Tolar Jakub
Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA.
NPJ Regen Med. 2016;1:16014-. doi: 10.1038/npjregenmed.2016.14. Epub 2016 Dec 8.
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causing mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiation in a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB.
隐性营养不良型大疱性表皮松解症（RDEB）是一种严重的疾病，由基因发生突变导致一种对皮肤完整性至关重要的结构蛋白的生成失活所引起。造血细胞移植可以改善一些症状；然而，同种异体移植过程可能会出现严重的副作用，并且水疱不愈合的区域仍然存在。因此，我们在患者来源的细胞中采用基因组编辑技术，创建了一个用于多种治疗性细胞类型多谱系工程的自体平台。成簇规律间隔短回文重复序列（CRISPR）/Cas9系统有助于纠正原代成纤维细胞中导致RDEB的突变，然后这些细胞被用于诱导多能干细胞（iPSC）的产生。随后，使用特定的分化策略将所得的iPSC重新分化为角质形成细胞、间充质干细胞（MSC）和造血祖细胞。基因校正后的角质形成细胞呈现出典型的上皮形态，并表达角质形成细胞特异性基因和转录因子。iPSC来源的MSC呈现纺锤形形态，并表达CD73、CD90和CD105，能够以与骨髓来源的MSC无法区分的方式进行脂肪生成、软骨生成和成骨分化。最后，我们使用血管诱导策略在基于甲基纤维素的检测中生成了能够进行多谱系分化的强大的确定性造血祖细胞。总体而言，我们已经表明CRISPR/Cas9是一种适应性强的基因编辑策略，可以与iPSC技术相结合，产生多种具有治疗RDEB潜力的基因校正自体细胞类型。
