RG Reprogramming and Gene Therapy, Hannover Medical School, Hannover 30625, Germany.
Institute of Experimental Hematology, REBIRTH, Hannover Medical School, Hannover 30625, Germany.
Int J Mol Sci. 2020 Apr 3;21(7):2481. doi: 10.3390/ijms21072481.
Induced pluripotent stem cells (iPSCs) offer great promise for the field of regenerative medicine, and iPSC-derived cells have already been applied in clinical practice. However, potential contamination of effector cells with residual pluripotent cells (e.g., teratoma-initiating cells) or effector cell-associated side effects may limit this approach. This also holds true for iPSC-derived hematopoietic cells. Given the therapeutic benefit of macrophages in different disease entities and the feasibility to derive macrophages from human iPSCs, we established human iPSCs harboring the inducible Caspase-9 (iCasp9) suicide safety switch utilizing transcription activator-like effector nuclease (TALEN)-based designer nuclease technology. Mono- or bi-allelic integration of the iCasp9 gene cassette into the AAVS1 locus showed no effect on the pluripotency of human iPSCs and did not interfere with their differentiation towards macrophages. In both, iCasp9-mono and iCasp9-bi-allelic clones, concentrations of 0.1 nM AP20187 were sufficient to induce apoptosis in more than 98% of iPSCs and their progeny-macrophages. Thus, here we provide evidence that the introduction of the iCasp9 suicide gene into the AAVS1 locus enables the effective clearance of human iPSCs and thereof derived macrophages.
诱导多能干细胞（iPSCs）为再生医学领域带来了巨大的希望，并且 iPSC 衍生细胞已经在临床实践中得到应用。然而，效应细胞中残留的多能细胞（例如，致瘤起始细胞）或效应细胞相关的副作用可能会限制这种方法。这对于 iPSC 衍生的造血细胞也是如此。鉴于巨噬细胞在不同疾病实体中的治疗益处，以及从人 iPSCs 中衍生巨噬细胞的可行性，我们利用转录激活因子样效应物核酸酶（TALEN）基于的设计核酸酶技术，建立了携带诱导型 Caspase-9（iCasp9）自杀安全开关的人 iPSCs。iCasp9 基因盒的单等位基因或双等位基因整合到 AAVS1 基因座对人 iPSCs 的多能性没有影响，也不干扰它们向巨噬细胞的分化。在 iCasp9-单等位基因和 iCasp9-双等位基因克隆中，0.1 nM AP20187 的浓度足以诱导超过 98%的 iPSCs 及其祖细胞-巨噬细胞凋亡。因此，在这里我们提供的证据表明，将 iCasp9 自杀基因引入 AAVS1 基因座可有效清除人 iPSCs 及其衍生的巨噬细胞。
