Inducible motor neuron differentiation of human induced pluripotent stem cells in vivo

Abstract

Objectives: Transplantation of neural progenitor cells (NPCs) derived from humaninduced pluripotent stem cells (hiPSCs) is one of the promising treatment strategies for motor neuron diseases (MNDs). However, the inefficiency in committed differentiation of NPCs in vivo limits its application. Here, we tried to establish a potential therapeutic strategy for MNDs by in vivo directional differentiation of hiPSCs engineered with motor neuron (MN) specific transcription factors and Tet-On system. Materials and Methods:We engineered hiPSCs with three MN-specific transcription factors and Tet-On system. The engineered cells were directly transplanted into immunodeficient mice through subcutaneous, intra-spinal cord and intracerebroventricular injections. Following doxycycline (Dox) induction, teratoma formation, and motor MN differentiation were evaluated.Results: We generated genetically engineered hiPSCs, in which the expression of Ngn2, Isl1, and Lhx3 was controlled by a drug-inducible transgenic system. These cells showed normal pluripotency and proliferative capacity, and were able to directionally differentiate into mature motor neurons (MNs) and NPCs with high efficiency in spinal cords and cerebral lateral ventricles under the induction of Dox. The grafts showed long-term survival in the recipient mice without formation of teratoma. Conclusions:The induced mature MNs and NPCs were expected to replace the damaged endogenous MNs directly, and play a role of de novo stem cell stock for Min Chen, Xia Wang, and Chuan Li contributed as co-first authors.

Improvement of TaC9-ABE mediated correction of human SMN2 gene

“…The nCas9-gSMA expression plasmid (300 ng) and Tada8e-TALE-Tada8e plasmids (200 ng) were co-transfected into cells, and ABE7.10 expression plasmid (500 ng) were transfected into cells as control using lipofectamine 8000 (Beyotime Biotechnology) to find the optimal groups for SMN2 T6>C conversion. NILB-iPSCs [12] were cultured in mTeSR1 medium (STEMCELL Technologies) on matrigel-coated plates (Corning), at 37°C and with 5% CO 2 . Cells were passaged with Accutase (STEMCELL Technologies), and medium supplemented with 10 μM of the rock inhibitor Y27632 (Sigma) when grown to 80% confluence.…”

“…TaC9 is a dual-guide gene editor that includes the expression of transcription activator-like effector (TALE)deaminase and Cas9 nickase (nCas9)/sgRNA protein components [9,10]. The system has the advantages of safety, efficiency, and wide editing window (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), which is suitable for gene therapy through dual AAVs. In this study, we improved the TaC9-ABE for SMN2 gene repair with efficient and accurate base conversion.…”

Bcl-xL Promotes the Survival of Motor Neurons Derived from Neural Stem Cells

“…The PB-Ngn2-Isl1-Lhx3 (PB-NIL) plasmid was a gift from Prof. Alessandro Rosa (Center for Life Nano Science, Rome, Italy) [13]. The PB-Bcl-xL-Luciferase-GFP (PB-BLG) plasmid was generated as previously described [15].…”