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.

Neural stem cell (NSC) transplantation creates new hope for the treatment of neurodegenerative disorders by direct differentiation into neurons. However, this technique is limited by poor survival and functional neuron deficiency. In this research study, we generated pro−survival murine NSCs (mNSCs) via the ectopic expression of Bcl−xL. A doxycycline (Dox)−inducible Ngn2−Isl1−Lhx3 system was also integrated into the mNSC genome. The four gene−modified mNSCs can rapidly and effectively differentiate into motor neurons after Dox treatments. Ectopic Bcl−xL could resist replating−induced stress, glutamate toxicity, neuronal apoptosis and remarkably promote the survival of motor neurons. Taken together, we established genetically modified mNSCs with improved survival, which may be useful for motor neuron degenerative diseases.