The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.

Department of Anesthesiology School of Medicine University of California San Diego La Jolla CA USA

Department of Biomaterials and Bioanalogous System Institute of Macromolecular Chemistry Czech Academy of Sciences Prague Czech Republic

Department of Neurosurgery School of Medicine University of California San Diego La Jolla CA USA

Division of Stem Cell Medicine Department of Advanced Medicine Medical Research Institute Kanazawa Medical University Uchinada Japan

Institute of Animal Physiology and Genetics AS CR v v i Liběchov Czech Republic

Murayama Medical Center Department of Orthopaedic Surgery Tokyo Japan

Regenerative and Cellular Medicine Kobe Center Sumitomo Dainippon Pharma Co Ltd Kobe Japan

See more in PubMed

Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–72. PubMed

Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, et al.. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318(5858):1917–20. PubMed

Doss MX, Sachinidis A. Current challenges of iPSC-based disease modeling and therapeutic implications. Cells. 2019;8(5):403. PubMed PMC

Rowe RG, Daley GQ. Induced pluripotent stem cells in disease modelling and drug discovery. Nat Rev Genet. 2019;20(7):377–88. PubMed PMC

Elitt MS, Barbar L, Tesar PJ. Drug screening for human genetic diseases using iPSC models. Hum Mol Genet. 2018;27(R2):R89–98. PubMed PMC

Israel MA, Yuan SH, Bardy C, Reyna SM, Mu Y, Herrera C, Hefferan MP, Van Gorp S, Nazor KL, Boscolo FS, Carson CT, et al.. Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells. Nature. 2012;482(7384):216–20. PubMed PMC

Okubo T, Nagoshi N, Kohyama J, Tsuji O, Shinozaki M, Shibata S, Kase Y, Matsumoto M, Nakamura M, Okano H. Treatment with a gamma-secretase inhibitor promotes functional recovery in human iPSC-derived transplants for chronic spinal cord injury. Stem Cell Reports. 2018;11(6):1416–32. PubMed PMC

Lin GQ, He XF, Liang FY, Guo Y, Sunnassee G, Chen J, Cao XM, Chen YY, Pan GJ, Pei Z, Tan S. Transplanted human neural precursor cells integrate into the host neural circuit and ameliorate neurological deficits in a mouse model of traumatic brain injury. Neurosci Lett. 2018;674:11–17. PubMed

Hicks AU, Lappalainen RS, Narkilahti S, Suuronen R, Corbett D, Sivenius J, Hovatta O, Jolkkonen J. Transplantation of human embryonic stem cell-derived neural precursor cells and enriched environment after cortical stroke in rats: cell survival and functional recovery. Eur J Neurosci. 2009;29(3):562–74. PubMed

Bühnemann C, Scholz A, Bernreuther C, Malik CY, Braun H, Schachner M, Reymann KG, Dihné M. Neuronal differentiation of transplanted embryonic stem cell-derived precursors in stroke lesions of adult rats. Brain. 2006;129(Pt 12):3238–48. PubMed

Daadi MM, Lee SH, Arac A, Grueter BA, Bhatnagar R, Maag AL, Schaar B, Malenka RC, Palmer TD, Steinberg GK. Functional engraftment of the medial ganglionic eminence cells in experimental stroke model. Cell Transplant. 2009;18(7):815–26. PubMed

Ben-Menachem-Zidon O, Ben-Menahem Y, Ben-Hur T, Yirmiya R. Intra-hippocampal transplantation of neural precursor cells with transgenic over-expression of IL-1 receptor antagonist rescues memory and neurogenesis impairments in an Alzheimer’s disease model. Neuropsychopharmacology. 2014;39(2):401–14. PubMed PMC

Blurton-Jones M, Kitazawa M, Martinez-Coria H, Castello NA, Muller FJ, Loring JF, Yamasaki TR, Poon WW, Green KN, LaFerla FM. Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease. Proc Natl Acad Sci U S A. 2009;106(32):13594–99. PubMed PMC

Li X, Zhu H, Sun X, Zuo F, Lei J, Wang Z, Bao X, Wang R. Human neural stem cell transplantation rescues cognitive defects in APP/PS1 model of Alzheimer’s disease by enhancing neuronal connectivity and metabolic activity. Front Aging Neurosci. 2016;8:282. PubMed PMC

Hefferan MP, Galik J, Kakinohana O, Sekerkova G, Santucci C, Marsala S, Navarro R, Hruska-Plochan M, Johe K, Feldman E, Cleveland DW, et al.. Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation. PLoS One. 2012;7(8):e42614. PubMed PMC

De Feo D, Merlini A, Laterza C, Martino G. Neural stem cell transplantation in central nervous system disorders: from cell replacement to neuroprotection. Curr Opin Neurol. 2012;25(3):322–33. PubMed

Åkesson E, Sundström E. Human neural progenitor cells in central nervous system lesions. Best Pract Res Clin Obstet Gynaecol. 2016;31:69–81. PubMed

Cossetti C, Alfaro-Cervello C, Donegà M, Tyzack G, Pluchino S. New perspectives of tissue remodelling with neural stem and progenitor cell-based therapies. Cell Tissue Res. 2012;349(1):321–29. PubMed PMC

Trueman RC, Klein A, Lindgren HS, Lelos MJ, Dunnett SB. Repair of the CNS using endogenous and transplanted neural stem cells. Curr Top Behav Neurosci. 2013;15:357–98. PubMed

Chohan MO, Moore H. Interneuron progenitor transplantation to treat CNS dysfunction. Front Neural Circuits. 2016;10:64. PubMed PMC

Karumbayaram S, Lee P, Azghadi SF, Cooper AR, Patterson M, Kohn DB, Pyle A, Clark A, Byrne J, Zack JA, Plath K, et al.. From skin biopsy to neurons through a pluripotent intermediate under good manufacturing practice protocols. Stem Cells Transl Med. 2012;1(1):36–43. PubMed PMC

Li Y, Nguyen HV, Tsang SH. Skin biopsy and patient-specific stem cell lines. Methods Mol Biol. 2016;1353:77–88. PubMed PMC

Agu CA, Soares FA, Alderton A, Patel M, Ansari R, Patel S, Forrest S, Yang F, Lineham J, Vallier L, Kirton CM. Successful generation of human induced pluripotent stem cell lines from blood samples held at room temperature for up to 48 hr. Stem Cell Reports. 2015;5(4):660–71. PubMed PMC

Ohmine S, Dietz AB, Deeds MC, Hartjes KA, Miller DR, Thatava T, Sakuma T, Kudva YC, Ikeda Y. Induced pluripotent stem cells from GMP-grade hematopoietic progenitor cells and mononuclear myeloid cells. Stem Cell Res Ther. 2011;2(6):46. PubMed PMC

Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76. PubMed

Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 2007;448(7151):313–17. PubMed

Giorgetti A, Montserrat N, Aasen T, Gonzalez F, Rodriguez-Piza I, Vassena R, Raya A, Boue S, Barrero MJ, Corbella BA, Torrabadella M, et al.. Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell. 2009;5(4):353–57. PubMed PMC

Ye Z, Zhan H, Mali P, Dowey S, Williams DM, Jang YY, Dang CV, Spivak JL, Moliterno AR, Cheng L. Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders. Blood. 2009;114(27):5473–80. PubMed PMC

Haridhasapavalan KK, Borgohain MP, Dey C, Saha B, Narayan G, Kumar S, Thummer RP. An insight into non-integrative gene delivery approaches to generate transgene-free induced pluripotent stem cells. Gene. 2019;686:146–59. PubMed

Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(8):348–62. PubMed PMC

Churko JM, Burridge PW, Wu JC. Generation of human iPSCs from human peripheral blood mononuclear cells using non-integrative Sendai virus in chemically defined conditions. Methods Mol Biol. 2013;1036:81–88. PubMed PMC

Soares FA, Pedersen RA, Vallier L. Generation of human induced pluripotent stem cells from peripheral blood mononuclear cells using sendai virus. Methods Mol Biol. 2016;1357:23–31. PubMed

Komuta Y, Ishii T, Kaneda M, Ueda Y, Miyamoto K, Toyoda M, Umezawa A, Seko Y. In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells. Biol Open. 2016;5(6):709–19. PubMed PMC

Riedel M, Jou CJ, Lai S, Lux RL, Moreno AP, Spitzer KW, Christians E, Tristani-Firouzi M, Benjamin IJ. Functional and pharmacological analysis of cardiomyocytes differentiated from human peripheral blood mononuclear-derived pluripotent stem cells. Stem Cell Reports. 2014;3(1):131–41. PubMed PMC

Nakagawa M, Taniguchi Y, Senda S, Takizawa N, Ichisaka T, Asano K, Morizane A, Doi D, Takahashi J, Nishizawa M, Yoshida Y, et al.. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep. 2014;4:3594. PubMed PMC

Strnadel J, Carromeu C, Bardy C, Navarro M, Platoshyn O, Glud AN, Marsala S, Kafka J, Miyanohara A, Kato T, Jr, Tadokoro T, et al.. Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs. Sci Transl Med. 2018;10(440):eaam6651. PubMed

Bohaciakova D, Hruska-Plochan M, Tsunemoto R, Gifford WD, Driscoll SP, Glenn TD, Wu S, Marsala S, Navarro M, Tadokoro T, Juhas S, et al.. A scalable solution for isolating human multipotent clinical-grade neural stem cells from ES precursors. Stem Cell Res Ther. 2019;10(1):83. PubMed PMC

Pruszak J, Sonntag KC, Aung MH, Sanchez-Pernaute R, Isacson O. Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cells. 2007;25(9):2257–68. PubMed PMC

Pruszak J, Ludwig W, Blak A, Alavian K, Isacson O. CD15, CD24, and CD29 define a surface biomarker code for neural lineage differentiation of stem cells. Stem Cells. 2009;27(12):2928–40. PubMed PMC

Sundberg M, Jansson L, Ketolainen J, Pihlajamäki H, Suuronen R, Skottman H, Inzunza J, Hovatta O, Narkilahti S. CD marker expression profiles of human embryonic stem cells and their neural derivatives, determined using flow-cytometric analysis, reveal a novel CD marker for exclusion of pluripotent stem cells. Stem Cell Res. 2009;2(2):113–24. PubMed

Peh GS, Lang RJ, Pera MF, Hawes SM. CD133 expression by neural progenitors derived from human embryonic stem cells and its use for their prospective isolation. Stem Cells Dev. 2009;18(2):269–82. PubMed

Golebiewska A, Atkinson SP, Lako M, Armstrong L. Epigenetic landscaping during hESC differentiation to neural cells. Stem Cells. 2009;27(6):1298–308. PubMed

Yuan SH, Martin J, Elia J, Flippin J, Paramban RI, Hefferan MP, Vidal JG, Mu Y, Killian RL, Israel MA, Emre N, et al.. Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One. 2011;6(3):e17540. PubMed PMC

Trujillo CA, Gao R, Negraes PD, Gu J, Buchanan J, Preissl S, Wang A, Wu W, Haddad GG, Chaim IA, et al.. Complex oscillatory waves emerging from cortical organoids model early human brain network development. Cell Stem Cell. 2019;25(4):558–569.e7. PubMed PMC

Eura N, Matsui TK, Luginbühl J, Matsubayashi M, Nanaura H, Shiota T, Kinugawa K, Iguchi N, Kiriyama T, Zheng C, Kouno T, et al.. Brainstem organoids from human pluripotent stem cells. Front Neurosci. 2020;14:538. PubMed PMC

Svendsen CN, ter Borg MG, Armstrong RJ, Rosser AE, Chandran S, Ostenfeld T, Caldwell MA. A new method for the rapid and long term growth of human neural precursor cells. J Neurosci Methods. 1998;85(2):141–52. PubMed

Mazzini L, Gelati M, Profico DC, Sgaravizzi G, Projetti Pensi M, Muzi G, Ricciolini C, Rota Nodari L, Carletti S, Giorgi C, Spera C, et al.. Human neural stem cell transplantation in ALS: initial results from a phase I trial. J Transl Med. 2015;13:17. PubMed PMC

Riley J, Federici T, Polak M, Kelly C, Glass J, Raore B, Taub J, Kesner V, Feldman EL, Boulis NM. Intraspinal stem cell transplantation in amyotrophic lateral sclerosis: a phase I safety trial, technical note, and lumbar safety outcomes. Neurosurgery. 2012;71(2):405–416; discussion 416. PubMed

Curtis E, Martin JR, Gabel B, Sidhu N, Rzesiewicz TK, Mandeville R, Van Gorp S, Leerink M, Tadokoro T, Marsala S, Jamieson C, et al.. A first-in-human, phase I study of neural stem cell transplantation for chronic spinal cord injury. Cell Stem Cell. 2018;22(6):941–950.e6. PubMed

Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, McKay RD. Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev. 1996;10(24):3129–40. PubMed

Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K, Steward O. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci. 2005;25(19):4694–4705. PubMed PMC

Kumamaru H, Lu P, Rosenzweig ES, Kadoya K, Tuszynski MH. Regenerating corticospinal axons innervate phenotypically appropriate neurons within neural stem cell grafts. Cell Rep. 2019;26(9):2329–2339.e4. PubMed PMC

Usvald D, Vodicka P, Hlucilova J, Prochazka R, Motlik J, Kuchorova K, Johe K, Marsala S, Scadeng M, Kakinohana O, Navarro R, et al.. Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs. Cell Transplant. 2010;19(9):1103–22. PubMed

Kakinohana O, Juhasova J, Juhas S, Motlik J, Platoshyn O, Galik J, Hefferan M, Yuan SH, Vidal JG, Carson CT, van Gorp S, et al.. Survival and differentiation of human embryonic stem cell-derived neural precursors grafted spinally in spinal ischemia-injured rats or in naive immunosuppressed minipigs: a qualitative and quantitative study. Cell Transplant. 2012;21(12):2603–19. PubMed

van Gorp S, Leerink M, Kakinohana O, Platoshyn O, Santucci C, Galik J, Joosten EA, Hruska-Plochan M, Goldberg D, Marsala S, Johe K, et al.. Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation. Stem Cell Res Ther. 2013;4(3):57. PubMed PMC

Sevc J, Goldberg D, van Gorp S, Leerink M, Juhas S, Juhasova J, Marsala S, Hruska-Plochan M, Hefferan MP, Motlik J, Rypacek F, et al.. Effective long-term immunosuppression in rats by subcutaneously implanted sustained-release tacrolimus pellet: effect on spinally grafted human neural precursor survival. Exp Neurol. 2013;248:85–99. PubMed

Kobayashi Y, Okada Y, Itakura G, Iwai H, Nishimura S, Yasuda A, Nori S, Hikishima K, Konomi T, Fujiyoshi K, Tsuji O, et al.. Pre-evaluated safe human iPSC-derived neural stem cells promote functional recovery after spinal cord injury in common marmoset without tumorigenicity. PLoS One. 2012;7(12):e52787. PubMed PMC

Nori S, Okada Y, Yasuda A, Tsuji O, Takahashi Y, Kobayashi Y, Fujiyoshi K, Koike M, Uchiyama Y, Ikeda E, Toyama Y, et al.. Grafted human-induced pluripotent stem-cell-derived neurospheres promote motor functional recovery after spinal cord injury in mice. Proc Natl Acad Sci U S A. 2011;108(40):16825–30. PubMed PMC

Marsala M, Kakinohana O, Yaksh TL, Tomori Z, Marsala S, Cizkova D. Spinal implantation of hNT neurons and neuronal precursors: graft survival and functional effects in rats with ischemic spastic paraplegia. Eur J Neurosci. 2004;20(9):2401–14. PubMed

Cizkova D, Kakinohana O, Kucharova K, Marsala S, Johe K, Hazel T, Hefferan MP, Marsala M. Functional recovery in rats with ischemic paraplegia after spinal grafting of human spinal stem cells. Neuroscience. 2007;147(2):546–60. PubMed PMC