Transdifferentiation-Induced Neural Stem Cells Promote Recovery of Middle Cerebral Artery Stroke Rats

RESEARCH ARTICLE Transdifferentiation-Induced Neural Stem Cells Promote Recovery of Middle Cerebral Artery Stroke Rats Hui Yao1,2☯, Mou Gao1,3☯, Jianhua Ma1,4, Maoying Zhang1,5, Shaowu Li6, Bingshan Wu4, Xiaohu Nie1,5, Jiao Jiao7, Hao Zhao1,2, Shanshan Wang1,2, Yuanyuan Yang7, Yesen Zhang1,4, Yilin Sun6, Max S. Wicha8, Alfred E. Chang8, Shaorong Gao7*, Qiao Li8*, Ruxiang Xu1,2* 1 Affiliated Bayi Brain Hospital, General Hospital of Beijing Military Region, P.L.A, Beijing, 100700, PR China, 2 Neurosurgery Institute of Beijing Military Region, Beijing, 100700, PR China, 3 The Third Military Medical University, Chongqing, 400038, PR China, 4 Anhui Medical University, Hefei, 230032, PR China, 5 Southern Medical University, Guangzhou, 510515, PR China, 6 Beijing Tiantan Hospital, Beijing, 100050, PR China, 7 National Institute of Biological Science, Beijing, 102206, PR China, 8 University of Michigan, Comprehensive Cancer Center, Ann Arbor, Michigan, 48109, United States f of America ☯ These authors contributed equally to this work. * (RX); (QL); (SG) OPEN ACCESS Citation: Yao H, Gao M, Ma J, Zhang M, Li S, Wu B, et al. (2015) Transdifferentiation-Induced Neural Stem Cells Promote Recovery of Middle Cerebral Artery Stroke Rats. PLoS ONE 10(9): e0137211. doi:10.1371/journal.pone.0137211 Editor: Jinglu Ai, St Michael's Hospital, University of Toronto, CANADA Received: March 4, 2015 Accepted: August 14, 2015 Published: September 9, 2015 Copyright: © 2015 Yao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Induced neural stem cells (iNSCs) can be directly transdifferentiated from somatic cells. One potential clinical application of the iNSCs is for nerve regeneration. However, it is unknown whether iNSCs function in disease models. We produced transdifferentiated iNSCs by conditional overexpressing Oct4, Sox2, Klf4, c-Mycin mouse embryonic fibroblasts. They expanded readily in vitro and expressed NSC mRNA profile and protein markers. These iNSCs differentiated into mature astrocytes, neurons and oligodendrocytes in vitro. Importantly, they reduced lesion size, promoted the recovery of motor and sensory function as well as metabolism status in middle cerebral artery stroke rats. These iNSCs secreted nerve growth factors, which was associated with observed protection of neurons from apoptosis. Furthermore, iNSCs migrated to and passed through the lesion in the cerebral cortex, where Tuj1+ neurons were detected. These findings have revealed the function of transdifferentiated iNSCs in vivo, and thus provide experimental evidence to support the development of personalized regenerative therapy for CNS diseases by using genetically engineered autologous somatic cells. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the China Postdoctoral Science Foundation (2013M532138) and Military Twelfth Five-Year Key Sci-Tech Research Projects (Grant Nos. BWS11J002 and BWS12J010). This work was also supported in part by the Gillson Longenbaugh Foundation. Competing Interests: The authors have declared that no competing interests exist. Introduction Neural stem cells (NSCs) promote recovery of neurological disease and show anti-inflammatory, glial scar-inhibitory, and anti-apoptotic effects with neuroprotective functions [1]. These characters make the use of NSCs a promising strategy for the regeneration of damaged brain tissues. However, generation of sufficient numbers of human adult NSCs in vitro has remained to be a major limitation for the application of NSCs. With the implication of transdifferentiation reprogramming, however, large quantities of iNSCs can be produced [2,3]. PLOS ONE | DOI:10.1371/journal.pone.0137211 September 9, 2015 1 / 19 Transdifferentiation-Induced NSCs Recover Stroke Rats Transdifferentiation is defined as direct reprogramming or conversion of one cell type to another by passing the induction of pluripotent stem cells (iPSCs) [4–6]. This process can be used to reprogram adult somatic cells into either adult stem cells or differentiated cells of another germ layer [7–12]. Classic iPSC transcription factors in conjunction with other neural progenitor cell (NPC)-specific factors or microRNA have been used to transdifferentiate human or mouse somatic cells into iNSCs/iNPCs. [13–17] While transdifferentiated iNSCs can now be derived from human somatic cells, which shows significant potential of cell therapy using autologous grafts, it is not known whether iNSCs/iNPCs can function in vivo as NSCs, and few studies have assessed the behavior of iNSCs after transplantation. In this study, we produced transdifferentiation-induced NSC colonies by direct inducing mouse embryonic fibroblasts (MEFs) with conditional expression of OSKM or Oct4, Sox2, and Klf4 (OSK). These cells were readily expanded in vitro. When transplanted into the brains of middle cerebral artery occlusion (MCAO) rats after surgery, the iNSCs significantly improved the motor and sensory functions of the MCAO rats. Importantly, these iNSCs migrated through the lesion region, confirming that they can play an important role in the recovery of MCAO rats. Materials and Methods Method to get MEF cells GFP/rtTA, OG2/rtTA MEFs were isolated from E13.5 embryos. OG2/rtTA embryos were generated by crossing of B6; CBA-Tg (Pou5f1-EGFP) 2Mnn/J mice and B6.Cg-Gt (ROSA) 26 Sortm1 (rtTA M2) Jae/J mice. GFP/rtTA embryos got from crossing of C57BL/6-Tg (CAG-EGFP) 1Osb/J and B6.Cg-Gt (ROSA) 26 Sortm1 (rtTA M2) Jae/J mice. All of the three transgenic mice strain bought from The Jackson Laboratory. To get MEF cells, mice at E13.5 gestation were sacrificed by performing cervical dislocation. Saturate mouse abdomen with alcohol, cut back the skin and peritoneal wall with sterile instruments. Then remove uterine horns and place them into sterile, disposable petri dish. Wash uterine horns 3 times with 10ml PBS. Then cut open embryonic sacs and release embryos and place them in fresh dish and wash 3 times with 10ml PBS. Mince tissue with curved dissecting scissors into grain sized pieces for approximately 5–10 minutes. After that, add 2ml Trypsin and mince for an additional few minutes until pieces are further reduced in size, then pipet the cells vigorously up and down. Then place dish into incubator for 20–30 minutes. After removing the minced tissue from incubator, add about 20ml MEF Derivation Culture Media and transfer contents to a sterile 50ml plastic conical tube. Rinse remaining tissue in the plate with a few milliliters MEF Derivation Culture Media (DMEM, 10% FBS and Penicillin-Streptomycin). Transfer media to the 50ml conical tube. Place 10ml MEF Derivation Culture Media into each T75 Flask. Transfer 5ml minced tissue (...truncated)