==== Front Cell Death Discov Cell Death Discov Cell Death Discovery 2058-7716 Nature Publishing Group UK London 37393356 1532 10.1038/s41420-023-01532-9 Review Article Directional induction of neural stem cells, a new therapy for neurodegenerative diseases and ischemic stroke Nie Luwei 1 Yao Dabao 1 Chen Shiling 1 Wang Jingyi 1 Pan Chao 1 Wu Dongcheng 23 http://orcid.org/0000-0003-4275-6095 Liu Na liuna_2003abc@163.com 1 http://orcid.org/0000-0002-4153-8590 Tang Zhouping ddjtzp@163.com 1 1 grid.33199.31 0000 0004 0368 7223 Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030 Hubei China 2 grid.49470.3e 0000 0001 2331 6153 Department of Biochemistry and Molecular Biology, Wuhan University School of Basic Medical Sciences, Wuhan, 430030 China 3 Wuhan Hamilton Biotechnology Co., Ltd., Wuhan, 430030 China 1 7 2023 1 7 2023 2023 9 21528 4 2023 16 6 2023 22 6 2023 © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Due to the limited capacity of the adult mammalian brain to self-repair and regenerate, neurological diseases, especially neurodegenerative disorders and stroke, characterized by irreversible cellular damage are often considered as refractory diseases. Neural stem cells (NSCs) play a unique role in the treatment of neurological diseases for their abilities to self-renew and form different neural lineage cells, such as neurons and glial cells. With the increasing understanding of neurodevelopment and advances in stem cell technology, NSCs can be obtained from different sources and directed to differentiate into a specific neural lineage cell phenotype purposefully, making it possible to replace specific cells lost in some neurological diseases, which provides new approaches to treat neurodegenerative diseases as well as stroke. In this review, we outline the advances in generating several neuronal lineage subtypes from different sources of NSCs. We further summarize the therapeutic effects and possible therapeutic mechanisms of these fated specific NSCs in neurological disease models, with special emphasis on Parkinson’s disease and ischemic stroke. Finally, from the perspective of clinical translation, we compare the strengths and weaknesses of different sources of NSCs and different methods of directed differentiation, and propose future research directions for directed differentiation of NSCs in regenerative medicine. Subject terms Neuroscience Neural stem cells https://doi.org/10.13039/501100001809 National Natural Science Foundation of China (National Science Foundation of China) 82071330 Tang Zhouping https://doi.org/10.13039/501100003819 Natural Science Foundation of Hubei Province (Hubei Provincial Natural Science Foundation) 2019CFB113 Liu Na issue-copyright-statement© Cell Death Differentiation Association (ADMC) 2023 ==== Body pmcFacts Neural stem cells from different sources can be induced to differentiate into mature and functional neurons or glial cells in vitro. Transplantation of pre-differentiated neural stem cells can differentiate and mature into a specific type of cells, promoting the recovery of neurodegenerative disease or stroke models. Currently, dopaminergic neurons derived from human embryonic stem cells that undergo the neural stem cells stage are being tested in clinical trials in patients with Parkinson’s disease. Open questions For a neurodegenerative disease or stroke, which source of neural stem cells and which directed differentiation method will enable the transplanted cells to meet good manufacturing practices guideline? What is the optimal time window of the differentiation of neural stem cells for transplantation? What is the underlying mechanism of cell replacement in transplanted predifferentiated neural stem cells? Introduction Neurodegenerative diseases (NDs) are a heterogeneous group of disorders that characterized by progressive and selective losses of neurons [1, 2], resulting in loss of sensation, movement and memory impairment, which are represented by Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) [3]. Ischemic stroke, the most common type of stroke, causes neuronal and non-neuronal death in the ischemic core due to decreased blood flow to part of the brain. Given enough time, reversible loss of tissue function in the ischemic penumbra can be permanent [4, 5]. These diseases directly threaten the lives of patients and bring a heavy economic burden to family and society [6]. However, current treatments involved in these diseases are not curative and relatively limited, most of which can relieve symptoms and delay the course of diseases [7, 8]. Nerve repair and regeneration therapy is an ideal way to treat neurological diseases. A great deal of work has been done in this area, mainly from both endogenous and exogenous aspects to promote nerve repair and regeneration [9]. Neural stem cells (NSCs) are a class of multipotent cells defined on the basis of their robust self-renewal capacity and ability to differentiate into various central nervous system (CNS) neuronal and glial cell types [10, 11]. Endogenous neurogenesis mediated by NSCs has been shown in several pathological conditions, such as epilepsy, MS, ischemic stroke, and AD [12], but endogenous repair alone is insufficient. NSCs transplantation strategy, as a type of regenerative medicine, has attracted increasing attention in the treatment of NDs [13]. Moreover, as the field of stem cells advances, the source of NSCs for transplantation has expanded from direct isolation of brain tissue initially to differentiation from pluripotent stem cells (PSCs) and transdifferentiation of somatic cells. So far, NSC-based therapies have been implemented in many rodent models of NDs and ischemic stroke, and several studies proposed potential mechanisms to explain the disease-improving effects of NSCs, including neuroinflammation inhibition, neuronal replacement, immunomodulation and neurotrophic support, which promotes the recovery of ND and stroke models [14–18]. Now clinical trials exploring the feasibility of NSCs treatment for neurological diseases are being conducted. Most studies based on NSCs therapy involve direct transplantation of NSCs from different sources into animal disease models. However, non-negligible challenges of the directly transplanted NSCs are the low survival and irrational differentiation [19–22]. In both NDs and ischemic stroke, chronic or acute activation of innate immune cells in the CNS can be observed [23, 24]. The host micro-environment induced by a neuro-inflammatory response may play a critical role in the survival and differentiation of transplanted NSCs [25–27]. In addition, autophagy, which is involved in inflammatory pathways, has been demonstrated to regulate the differentiation of transplanted NSCs [28]. In animal models of spinal cord injury, transplanted NSCs were influenced by the neurotoxic inflammatory microenvironment and most of them differentiated into astrocytes, resulting in further aggravation [29]. Thus, the inflammatory response may adversely affect the ability of transplanted NSCs to participate in functional recovery. Further, the pathology of NDs is characterized by the selective loss of specific neurons or glial cells in restricted brain regions [30], such as midbrain dopaminergic (DAergic) neuron death in PD, medium spiny γ-aminobutyric acid–mediated (GABAergic) neurons (MSNs) loss in HD, degeneration of cholinergic motor neurons in ALS, and oligodendrocytes loss in MS. Compared with direct transplantation of NSCs, induced differentiation into specific phenotypes may be more amenable to replace lost cells in the CNS. To overcome the limitations of direct transplanted NSCs and given the pathological features of loss of a specific cell type in some NDs, great efforts have been devoted to explore the feasibility of manipulation of NSCs fate prior to transplantation to control the terminal lineage so as to replace lost cells in NDs [31, 32]. Currently, by using chemical-defined systems or ectopic overexpression of critical lineage-specific transcription factors, NSCs from different sources can be directed to differentiate into a specific type of neural lineage cells in vitro, such as DAergic neurons, GABAergic neurons, cholinergic motor neurons, oligodendrocytes, glutamatergic neurons. And subsequent studies have performed in vivo transplantation of predifferentiated cells to investigate their therapeutic role in neurological diseases (Fig. 1).Fig. 1 The directional differentiation of neural stem cells from different sources. Currently, NSCs can be obtained from three ways: isolate from primary CNS tissues, mainly including adult and fetal brain tissue; differentiate from pluripotent stem cells, including iPSCs and ESCs; transdifferentiate from somatic cells, such as blood cells and fibroblasts. NSCs derived from these three sources can be further processed in vitro to control their fate after transplantation in NDs models, thus replacing the lost cells. In NDs, NSCs can be induced to differentiate into DA neurons after transplantation in a PD model, MSNs after transplantation in a HD model, cholinergic motor neurons after transplantation in an ALS model, and oligodendrocytes after transplantation in a MS model. In acute neurodegeneration, NSCs can be induced to differentiate into cortical glutaminergic neurons or oligodendrocytes after transplantation in ischemic stroke models. After transplantation, pretreated NSCs can mature in the host, stably express specific phenotypes, and integrate into neural circuits to improve the symptoms of ND models. ALS Amyotrophic lateral sclerosis, CNS central nervous system, DA dopamine, ESC embryonic stem cell, HD Huntington’s disease, IPSC induced pluripotent stem cell, MS multiple sclerosis, MSN medium spiny γ-aminobutyric acid–mediated neurons, ND neurodegenerative disease, NSC neural stem cell, PD Parkinson’s disease. In this review, we summarized strategies for inducing differentiation of NSCs from different sources in vitro. Then, we outlined the functional improvements and underlying mechanisms of the transplanted preconditioned NSCs in PD and ischemic stroke models. What’s more, we also discussed the limitations of the directional induction of NSCs for clinical translation in NDs and ischemic stroke. The directional differentiation of NSCs from different sources in vitro At present, NSCs can be derived in three different ways: direct extraction from primary CNS tissues, differentiation of PSCs and transdifferentiation from somatic cells [33] (Fig. 2). NSCs are present throughout the developing brain. And in the adult mammalian brain, NSCs can be found in the subgranular zone of hippocampus, the subventricular zone, and even multiple sites along the entire ventricular system [16, 34, 35]. PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can be induced to differentiate into NSCs in vitro via two main methods: embryoid body (EB) formation and adherent monolayer culture [36, 37]. Specifically, the process of neural differentiation of PSCs is multistep, first triggering differentiation toward all the three embryonic germ layers by removing mediators that promote self-renewal, and subsequently inhibiting extraembryonic and meso-endoderm differentiation and favoring neural differentiation by culturing the cells in serum-free medium [38]. In addition, dual SMAD inhibition, which simultaneously inhibits transforming growth factor β and BMP signaling pathways, can reduce cultural variability and improve the efficiency of neural induction [38, 39]. Intriguingly, both of these neural induction methods of human PSCs closely resemble the neural induction processes in vivo, giving rise to NSCs with dorsal forebrain identity [38–40]. In addition, neural induction of PSCs can also be achieved by coculture with stromal cell feeder layers, which can provide clues to restrict the fate of PSCs towards neural lineage [41–43]. Induced NSCs can be directly reprogrammed from somatic cells, such as peripheral blood mononuclear cells (PBMNCs), fibroblasts and other cell types [44, 45]. NSCs from different sources can be induced to differentiate into desired neural lineage cells.Fig. 2 Sources of neural stem cells. Currently, NSCs can be obtained from three ways: 1) isolate from primary CNS tissues, mainly including adult and fetal brain tissue; 2) differentiate from pluripotent stem cells (iPSCs and ESCs) via EB formation or monolayer culture, dual SMAD inhibition can boost the neural induction process; 3) transdifferentiate from somatic cells, such as blood cells and fibroblasts. CNS central nervous system, EB embryoid body, ESC embryonic stem cell, IPSC induced pluripotent stem cell. Understanding the natural development of the nervous system is paramount to manipulating the targeted differentiation of NSCs. The embryonic neural tube undergoes a precise patterning process along the dorso-ventral and antero-posterior axes, resulting in the generation of specific neuronal and glial cell subtypes from NSCs. These intricate developmental events are predominantly orchestrated by organizers, i.e., small groups of cells that release patterning molecules to regulate the fate of NSCs small groups of cells that release patterning molecules to regulate cell fate [38, 46]. Patterning molecules involved in the antero-posterior patterning include fibroblast growth factors (FGF), wingless-type MMTV integration site family (WNT), and retinoic acid (RA), while those affecting dorso-ventral mode include WNTs, bone morphogenic proteins (BMPs), and sonic hedgehog (SHH) [38]. The gradients of morphogens can regulate the intrinsic signaling pathways that define transcription codes [38, 47]. Consequently, it is possible to induce differentiation of NSCs from different sources in vitro by mimicking the regional patterning principles of neural development in vivo, So far, two main approaches of induced differentiation have been developed: chemically defined system and intrinsic transcription factor-mediated method, by which the desired neural lineage cell types can be generated, such as DAergic neurons, MSNs, cholinergic motor neurons, oligodendrocytes, and cortical glutaminergic neurons (Fig. 3). Here, we provide an overview on the progress that has been made in generating several neuronal subtypes as well as oligodendrocytes from different sources of NSCs in vitro (Table 1).Fig. 3 Neurodevelopmental principle for neural lineage subtype specification that guide the directional differentiation of NSCs from different sources in vitro. A Morphogen gradients, including BMP, WNT, FGF, SHH and RA, define transcription codes of various neural lineage subtypes in corresponding brain regions during early neural development both along the rostral-caudal and dorsal-ventral axes. The depicted neural lineage subtypes include the MSN in ventral TEL, the cortical glutaminergic neuron in dorsal TEL, the DAergic in ventral MES, the motor neuron in spinal cord, the OPC in forebrain and spinal cord. B By using the same chemical or TF patterning principles as seen in vivo, NSCs from different sources can be directional differentiated towards neural lineage subtypes in vitro. The methods of inducing differentiation of NSCs in vitro mainly include external chemical defined system and TF-mediated system. Changes in culture environment include two-dimensional and three-dimensional culture. Overexpression of TFs through viral transduction or non-viral mediated transfection, such as electroporation. BMP bone morphogenic protein, DI diencephalon, FGF fibroblast growth factor, MES mesencephalon, MET metencephalon, MSN medium spiny γ-aminobutyric acid–mediated neurons, MYE myelencephalon, NSC neural stem cell, OPC oligodendrocyte precursor cell, RA retinoic acid, SHH sonic hedgehog, TEL telencephalon, TF transcription factor, WNT wingless-type MMTV integration site family, 2D two-dimensional, 3D three-dimensional. Table 1 In vitro differentiation protocols for per neural lineage phenotype and their application in models of neurological diseases. Phenotypes Source of NSCs Differentiation Protocol Differentiation factors Phenotypic markers (% cells) in vitro/vivo Models Functional outcome Reference DAergic neurons Human fetal VM tissue Chemical-defined system BDNF, AA, low oxygen 40–50% MAP2+ 15% TH+/MAP2+ NA NA [55] NA DAergic neurons Human fetal VM tissue (passage 2) Chemical-defined system WNT5 (SHH, FGF8, FGF2 for proliferation) 35%TH+ NA NA [62] NA DAergic neurons Rat embryonic VM tissue Transfected by electroporation Nurr1, Brn4 NA 6-OHDA PD rats Increased DA level; Improved rotational behavior [67] 18%TH+ 14%DAT+ DAergic neurons Rat embryonic VM tissue Transfected by lentivirus TH, Brn4 65.71 ± 5.18%TH+ 32.28 ± 4.39% DAT+ NA NA [66] NA DAergic neurons Rat embryonic VM tissue Chemical-defined system and transfected by lipofectamine SHH, FGF8 and Wnt5a a 20-fold TH+ cells increase 6-OHDA PD mice Increased DA level, improved rotational behavior [60] 9.5% TH+ DAergic neurons Rodents embryonic cortical tissue Transfected by retroviruses Foxa2, Nurr1 37.1% TH+ 55.1% PITX3+/TH+ >78% VMAT2+/TH+ 6-OHDA PD rats Exhibited a mature midbrain DAergic neuronal morphology, improved rotational behavior [73] about 14-fold TH+ cells increase DAergic neurons Rats embryonic cortical tissue Transfected by retroviruses with appropriate vectors and promoters Foxa2, Nurr1, ca-PKA 60% TH+/TUJ1+ 80–90% PITX3+/TH+ VMAT2+/TH+ DAT+/TH+ 6-OHDA PD rats Exhibited an extremely mature midbrain DAergic neuronal morphology, no rotational behavior improvement [74] few TH+ cells <100 cells DAergic neurons Primate ESCs (Co-culture with PA6) Chemical-defined system NA 25 ± 6% TUJ1+ 35 ± 6% TH+/TUJ1+ 6-OHDA PD mice NA [43] 0.7% TH+ DAergic neurons Human ESCs (Co-culture with PA6) Chemical-defined system SHH, FGF8 46 ± 8% MAP+ 80 ± 11% TH+/MAP+ 32% TH+ NA NA [75] DAergic neurons Mouse ESCs (Co-culture with MS5) Chemical-defined system SHH, FGF8 50 ± 10% TH+/TUJ1+ 6-OHDA PD mice improved rotational behavior [42] 10–20% TH+ DAergic neurons Human ESCs (EB) Chemical-defined system SHH, FGF8 50–60% TH+/TUJ1+ 31.8 ± 3.1% TH+ NA NA [77, 79] NA DAergic neurons Human PESCs (EB/Dual SMAD inhibition) Chemical-defined system SHH C25II, FGF8, PUR and CHIR99021 60–80%/70-100% TUJ1+ 20–40%/30-40% TH+ MPTP PD primates Increased DA level, improved rotational behavior [76] 5.2–8.1% TH+ DAergic neurons Human iPSC (EB) Chemical-defined system SHH, FGF8 30 ± 5% TH+ 100% GIRK2+/TH+ 6-OHDA PD rats Improved rotational behavior [78] ~2% TH+ DAergic neurons Human ESCs/iPSCs (Dual SMAD- inhibition) Chemical-defined system CHIR99021, FGF8, PUR and SHH-C25II ±75% TH+ ±50% NURR1+ ±80% FOXA2+ ±60% LMX1A+ 6-OHDA PD mice/rats MPTP PD primates Exhibited excellent DA neuron survival, improved motor deficits. [84] 6% TH+ (rats) DAergic neurons Human ESCs (Dual SMAD inhibition with EB) Chemical-defined system CHIR99021, SHH- C24II NA 6-OHDA PD rats Increased DA level, improved motor deficits, showed similar efficacy and potency to fetal DAergic neurons [82, 178] 54.2 ± 2.5% TH+ 81% LMX1A+/FOXA2+ DAergic neurons Human/primate ESCs/iPSCs (Dual SMAD- inhibition) Chemical-defined system CHIR99021, FGF8b and SHH- C25II 43.6 ± 6.2% TH+ 95.3 ± 2.4% NURR1+/TH+ 96.7 ± 1.8% FOXA2+/TH+ 96.5 ± 2.3% LMX1A+/TH+ 56.3 ± 6.7% GIRK2+/TH+ NA NA [83] DAergic neurons Human iPSCs (Dual SMAD- inhibition) Chemical-defined system CHIR99021, FGF8, and PUR 42 ± 4.4% TH+ 19.9 ± 6.9% NURR1+ 70–75% FOXA2+ 6-OHDA PD rats /MPTP PD primates Improved rotational behavior(rats) increased spontaneous movement, extended dense neurites into the host striatum, increased DA synthesis [81, 85] ±17% TH+ ±28%TH+/NEUN+(rats) 33.3 ± 24.4% TH+(primates) DAergic neurons Human ESCs (Dual SMAD- inhibition) Chemical-defined system CHIR99021, FGF8b, SHH- C25II and SAG 69% TH+ 84% TH+/TUJ1+ >85% GIRK2+/ TH+ 6-OHDA PD mice Displayed A9 characteristics, restored functionality of the reconstructed nigrostriatal circuit, improved motor deficits. [179] 68% TH+/survived DAergic neurons Human iPSC (Dual SMAD- Inhibition with EB) Human iNSC Chemical-defined system CHIR 99021, FGF8, PUR, BMP5 and BMP7 30–50% TH+/TUJ1+ NA NA [86] NA DAergic neurons Human ESCs/iPSC (dual SMAD- Inhibition) Chemical-defined system (3D) CHIR99021, FGF8b and PUR 47% TH+ Fischer rats NA [87] 8.12% TH+/transplanted 46.7% FOXA2/ TH+ DAergic neurons INSCs reprogrammed from PBMNCs Chemical-defined system SAG1, FGF8 57.23% TH+ 62.87% TH+/FOXA2 58.69% TH+/NURR1+ 13.84% TH+ 86.78% FOXA2+/TH+ 91.72% NURR1+/TH+ 98.77% GIRK2+/TH+ 6-OHDA PD mice Improved rotational behavior [88] GABAergic neurons Immortalized striatal human NSC line (STROC05) Chemical-defined system PUR 6.3% DARPP-32+ 46% TUJ+ 27%+ MAP2+ NA NA [99] GABAergic neurons Immortalized striatal human NSC line (ST14A) Chemical-defined system RA, KCl 74% GABA+ QA HD rats maintained neuronal GABAergic phenotype, established pre- and postsynaptic contacts with endogenous striatal cells, improved motor deficits [100] GABAergic neurons Immortalized human NSC line (ReNcell VM) Chemical-defined system VPA 68 ± 4% MAP2+ 90% GABA+/MAP2+ 54% CALB1+/MAP2+ NA NA [101] DKK1, SHH 63 ± 4% MAP2+ 96% GABA+/MAP2+ 84% CALB1+/MAP2+ GABAergic neurons Human ESCs (EB) Chemical-defined system SHH/PUR 90.2 ± 4.2% GABA+/TUJ1+ 89.7 ± 8.3% DARPP32+/TUJ1+ QA HD mice Projected to the anterior substantia nigra and potentially form connections with DAergic neurons, improved motor deficits [102] 62.8 ± 2.6% GABA+ 58.6 ± 3% DARPP-32+/ GABA+ GABAergic neurons Human iPSCs (Co-culture with PA6) Chemical-defined system BDNF 34.1 ± 4.5% DLX2 27.0 ± 1.7%DARPP-32+ 19.1 ± 2.1% CALB1+ QA HD rat Improved motor deficits [41] GABAergic neurons Human ESCs/iPSC (Dual SMAD- Inhibition) Chemical-defined system DKK1, SHH-C25II ±51% MAP2+ ±78% GABA+/MAP2+ ±60.3% CTIP2+/MAP2+ ±86% GABA+/CTIP2+/MAP2+ ±53% CALB1+/MAP2+ ±70.6% CTIP2+/CALB1+/MAP2+ QA HD rat Improved rotational behavior [103] GABAergic neurons Human ESCs (Dual SMAD- Inhibition with EB) Chemical-defined system XAV939, SAG ±87% DARPP32+/MAP2+ ±89.5% GABA+/TUJ1+ 80–100% DARPP-32+/GABA+ 80–100% CALB1+/TUJ1+ QA HD mice Improved motor deficits [104] 48.7 ± 2.8% DARPP32+/hN+ GABAergic neurons Human ESCs/iPSC (Dual SMAD- Inhibition) Chemical-defined system (3D) PUR, DKK1 78%MAP2+ 61% GABA+/MAP2+ 55%DARPP-32+/MAP2+ 70%CTIP2+/MAP2+ 46%CALB1+/MAP2+ 100%CTIP2+/DARPP-32+ R6/2 HD mice Innervated substantia nigra, improved motor deficits. [105] GABAergic neurons Human ESCs/iPSCs (Dual SMAD- Inhibition) Chemical-defined system Activin A 20–50%DARPP-32+ QA HD rats no motor improvement [106] 49 ± 5% DARPP-32+/hN+ 86 ± 4.6%GABA+/hN+ 35 ± 8%CALB1+/hN+ GABAergic neurons Human ESCs/iPSCs (Dual SMAD- Inhibition) Chemical-defined system IWR1 ±6%DARPP-32+/Map2b+ ±6%DARPP-32+/CTIP2+ ±60 %CTIP2+ NA NA [107] NA Cholinergic motor neurons Human fetal cortical NSCs Chemical-defined system FGF2 61% HB9+ 50% H9+/ChAT+ NA NA [114] NA Cholinergic motor neurons HB1.F3 human NSC line Chemical-defined system and transfected by vector Olig2, SHH NA SOD1G93A mutant mice Migrated into ventral horn, and replaced lost host motor neurons, delayed clinical onset and extended life span. [233] Cholinergic motor neurons Mouse ESCs (Co-culture with MS5) Chemical-defined system SHH, RA and FGF2 ±60%HB9+/TUJ1 NA NA [42] NA Cholinergic motor neurons Human ESCs, primate ESCs (Co-culture with MS5) Chemical-defined system SHH, RA 20% HB9+(human) 43% HB9+(primate) NA NA [116] NA Cholinergic motor neurons Human ESCs (EB) Chemical-defined system FGF2, RA and SHH >50% ISL1+/TUJ1+/MAP2+ ±50% HB9+/ISL1/2+ ±21% HB9+ NA NA [120] NA Cholinergic motor neurons Human iPSCs (EB) Chemical-defined system PUR, RA ±60%OLIG2+/SOX3+ ±30%ISL1+/TUJ1+ NA NA [118] NA Cholinergic motor neurons Human iPSCs (EB) Chemical-defined system RA, SHH agonist 20%HB9+ >90%ISL1/2+/HB9+ >50%ChAT+/ISL1/2+/HB9+ NA NA [119] NA Cholinergic motor neurons Human ESCs and iPSCs (EB) Chemical-defined system PUR, RA and SAG, 83 ± 1% TUJ1+ 30 ± 6% ISL1+ 16 ± 5% HB9+ 37 ± 2% ISL1+and HB9+ NA NA [124] NA Cholinergic motor neurons Human ESCs and iPSCs (Dual SMAD Inhibition with EB) Chemical-defined system BIO, PUR and RA 40–50%HB9+ NA NA [122] NA Cholinergic motor neurons Human ESCs and iPSCs Chemical-defined system (Dual SMAD inhibition) SAG, RA and CHIR99021 74% HB9+/ISL1+ NA NA [125] NA Cholinergic motor neurons Human iPSCs (Dual SMAD inhibition) Chemical-defined system CHIR99021, PUR and RA 90 ± 9% MNX1 + 95 ± 3% ISL1+ 91 ± 6%ChAT+/MAP2+ NA NA [126] NA Cholinergic motor neurons Human iPSCs (Dual SMAD inhibition) Transfected by lentivirus NGN2, ISL1, LHX3 88.2 ± 3.5% HB9+ 86.5 ± 4.1%ChAT+ NA NA [121] NA Cholinergic motor neurons Human iNSCs (Reprogrammed from PBMNCs) Chemical-defined system RA, SAG1 14.80 ± 0.90% HB9+ 14.40 ± 1.29% ISL1+ NA NA [130] NA Cholinergic motor neurons Rat iNSCs (Reprogrammed from astrocytes) Chemical-defined system RA, SHH 34.1% ± 2.9% HB9+ NA NA [131] NA oligodendrocytes Human fetal diencephalic/telencephalic tissue Chemical-defined system FGF2, NT3 and PDGF-AA 15–20% O4+ 15–20%GalC+ Lysolecithin MS mice Showed limited myelinating capacity [141] NA oligodendrocytes Human fetal brain tissue Chemical-defined system FGF2, NT3 and PDGF-AA 80.5 ± 2.1%A2B5+ 85.4 ± 3.9%O4+ 90%GalC+ NA NA [140] NA oligodendrocytes Human ESCs (EB) Chemical-defined system RA, SHH, FGF2, NT3, PDGF-AA and IGF1 83.95% PDGFRα+ 91.3%NGN2+ Shiverer MS mice expressed MBP and formed myelin sheaths around nerve fibers [135, 142] NA oligodendrocytes Human ESCs (EB) Chemical-defined system RA, PUR/SAG, FGF2, PDGF-AA, T3, low oxygen Spinal cord 77 ± 13% NGN2+ 38.5 ± 9.0%O4+ 29.9 ± 5.5%MBP+/O4+ Ventral forebrain 91% ± 7% NGN2+ 43% ± 5% O4+ 29.9 ± 5.5%MBP+/O4+ NA NA [143] NA oligodendrocytes Human ESCs and iPSCs (Dual SMAD inhibition) Chemical-defined system RA, SAG, NT3, PDGF-AA and T3 44–70% O4 + Shiverer MS mice Achieved mature oligodendrocyte differentiation and formed dense compact myelin. [145] NA oligodendrocytes Human iPSCs (Dual SMAD inhibition) Transfected by lentivirus SOX10, OLIG2, NKX6.2 62.1 ± 9.5%-79.0 ± 14.8% O4 + 30.37 ± 7.87% MBP+/O4 + Shiverer MS mice myelinated the forebrain, remyelinated the demyelinated spinal cord [146] oligodendrocytes Human iPSCs (Dual SMAD inhibition) Transfected by lentivirus SOX10 50–65% O4 + Shiverer MS mice myelinated neurons [147, 234] 48.13 ± 4.15%MBP+ oligodendrocytes Human ESCs and iPSCs (Dual SMAD inhibition) Chemical-defined system XAV939, PUR, PDGFRα, IGF-1, cAMP and T3 35% O4+ NA NA [149] NA oligodendrocytes Human ESCs Transfected by lentivirus SOX10, OLIG2 19.24 ± 3.18% O4+ 81.58 ± 3.94% FOXG1+/O4+ [148] Cortical glutamatergic neurons Human ESCs and iPSCs (Monolayer) Chemical-defined system Noggin <65% TUJ1+ ±60% VGLUT1+/TUJ1+ <75% TBR1+/TUJ1+ <72% CTIP2+/TUJ1+ <18% CTIP2+/TBR1+/TUJ1+ NA NA [155] NA Cortical glutamatergic neurons Human ESCs and iPSCs (Dual SMAD inhibition with monolayer) Chemical-defined system FGF2, Vitamin A 22–29% TBR1+ 25–30% CTIP2+ 28–36% BRN2+ NA NA [164, 165] NA Cortical glutamatergic neurons Human iPSCs (EB) Chemical-defined system BMP4, WNT3A and cyclopamine 62.2 ± 2.1% TBR1+ ±80% VGLUT1+/TUJ1+ MCAO rats Alleviated sensorimotor deficits, differentiated to glutamatergic neurons and form excitatory, glutamatergic synapses [166, 168, 169] 2.5 ± 0.3% TBR1+ Cortical glutamatergic neurons Human ESCs and iPSCs (EB) Chemical-defined system (3D) None 30-40% TBR1+ ±30% CTIP2+ ±10%SATB2 NA NA [170] NA The phenotypes of neural lineages, sources of neural stem cells, differentiation protocols, drivers of differentiation, representative phenotypic markers (in vitro) for evaluating the differentiation efficiency and culture homogeneity, expression of representative phenotypic markers after transplantation into corresponding neurological disease model, and improvement of functional outcomes after transplantation are broadly reviewed. + represents the percentage of cells stained positive for a specific marker in the differentiation system (in vitro) or in the transplanted population. AA ascorbic acid, BDNF brain derived neurotrophic factor, BIO GSK3β inhibitor 6-bromoindirubin-3′-oxime, BMP5 bone morphogenic protein 5, BMP7 bone morphogenic protein 7, BRN2 brain-specific homeobox/POU domain protein 2 (POU3F2), Brn4 brain-specific homeobox/POU domain protein 4, CALB1 calbindin 1, Ca-PKA constitutively active protein kinase A, CHAT choline acetyltransferase, CHIR99021 GSK3β inhibitor, CTIP2 b-cell CLL/lymphoma 11b(BCL11B)/COUP-TF-interacting protein 2 (COUP-TFII), 3D three-dimensional, DA dopamine, DARPP-32 dopamine and cAMP-regulated neuronal phosphoprotein 32, DAT dopamine transporter, DKK1 dickkopf-1, DLX2 distal-less homeobox 2, ESCs embryonic stem cells, EB embryoid body, EGF epidermal growth factor, FGF2 fibroblast growth factor 2/basic fibroblast growth factor (bFGF), FGF8 fibroblast growth factor 8, FGF8b fibroblast growth factor 8 isoform b, FOXA2 forkhead box protein A2, FOXG1 forkhead box protein G1, GABA γ-aminobutyric acid, GalC Galactocerebrosides, GIRK2 G protein-activated inward rectifier potassium channel 2 (KCNJ6), HB9 homeobox HB9/motor neuron and pancrease homeobox 1 (MNX1), HD Huntington’s disease, hN human nucleus, IGF-1 insulin-like growth factor 1, iNSC induced neural stem cells, iPSCs induced pluripotent stem cells, IWR1 a tankyrase/Wnt inhibitor, ISL1 ISL LIM homeobox 1, ISL1/2 ISL LIM homeobox 1/2, LHX3 LIM homeobox 3, MAP2 microtubule-associated protein 2, MBP myelin basic protein, MPTP 1-methyl-4-phenyl-1236-tetrahydropyridine, MS multiple sclerosis, MS-5 stromal cell line derived from irradiated murine bone marrow cultures, NGN2 neurogenin 2, NKX6-2 NK6 homeobox 2, NSCs neural stem cells, NURR1 nuclear receptor related 1 protein, NT3 neurotrophin-3, 6-OHDA 6-hydroxydopamine, OLIG2 oligodendrocyte transcription factor 2, PA6 stromal cell line derived from newborn calvaria tissue of the C57BL/6 mice, PBMNCs peripheral blood mononuclear cells, PD Parkinson’s disease, PGDF-AA platelet-derived growth factor AA, PGDFα platelet-derived growth factor -alpha receptor, PUR purmorphamine, PITX3 paired-like homeodomain 3, QA quinolinic acid, RA retinoic acid, SAG smoothened agonist, SATB2 special AT-rich sequence-binding protein 2, SHH sonic hedgehog, SHH-C24II recombinant human SHH, SHH-C25II recombinant mouse SHH, SMAD transcription factor and member of the BMP and TGF-β signaling pathways, T3 triiodothyronine, TBR1 T-box brain 1, SOX3 SRY box 3, SOX10 SRY box 10, TH tyrosine hydroxylase, TUJ1 neuron-specific class III beta-tubulin (TUBB3), VGLUT vesicular glutamate transporter, VM ventral midbrain, VPA valproic acid, VMAT2 vesicular monoamine transporter 2, WNT5 wingless-type MMTV integration site family 5, WNT5a wingless-type MMTV integration site family 5a, XAV939 WNT/β-catenin inhibitor. Induction of DAergic neurons from NSCs Differentiation protocols for DAergic neurons, particularly those targeting midbrain DAergic neurons, have garnered considerable interest in the field of regenerative medicine, owing to their potential to treat PD. Midbrain DAergic neurons are thought to originate from mesencephalic floor plate in embryonic development [48, 49]. The correct establishment of midbrain DAergic precursor domains and the subsequent terminal differentiation of ventral midbrain (VM) DAergic neurons are partly attributed to the synergistic action of regulatory networks controlled by SHH, WNT and FGF [50–52]. In more detail, WNT1 represses the transcription factor Nkx2.2 via the upregulation of Otx2 and the WNT1-Lmx1a autoregulatory loop induces the expression of Lmx1a thus repressing Nkx6-1, both of which promotes the establishment of the midbrain DAergic progenitor domain from ventral mesencephalic NSCs. In addition, the two autoregulatory loop (WNT1-Lmx1a and SHH-Foxa2) induce downstream targets, Pitx3 and Nurr1, which are important factors in the terminal differentiation/survival of midbrain DAergic neurons [51, 53]. FGF8 also provides positional information for the development of midbrain DA neurons [51]. Induction of DAergic neurons from NSCs derived from primary CNS tissues Initially, NSCs were extracted from embryonic or adult brain tissue for targeted differentiation of DAergic neurons. NSCs emanating from the mouse or human VM have been shown to naturally develop into DAergic neurons in vitro [54], and the addition of neurotrophins, such as brain derived neurotrophic factor (BDNF) and glial cell-line derived neurotrophic factor (GDNF) [55, 56], cyclic adenosine monophosphate [57], BMP2 [58], or cytokines [56] has been demonstrated to facilitated the yield of DAergic neurons. In addition, mitogenic factors, such as FGF2 or epidermal growth factor can amplify NSCs to increase the initial number of NSCs used for differentiation [54, 59]. Unfortunately, it has long been reported that the number of rodents VM-derived NSCs differentiated into DAergic neurons decreased after subculture [60, 61], but unlike their rodent counterparts, human VM tissue exhibits a greater ability to expand and differentiate into DAergic neurons [62]. To overcome the reduced ability of dopamine differentiation after passages, modifications of culture conditions such as lowering oxygen levels to mimic the hypoxic conditions of brain development, or the addition of ascorbic acid (AA) has been shown to be useful measures to increase the differentiation of human DAergic neurons after passages [63, 64]. A study adjusted the culture conditions of long-term expanded human VM NSCs, and increased the generation of tyrosine hydroxylase (TH)-positive cells by around 40 times (7% of total cell) through the combined application of BDNF, AA, low oxygen, and prolonged differentiation time [55]. Furthermore, another study revealed that by applying midbrain-specific instructive signals, SHH, FGF8, and FGF2 to proliferating human VM NSCs, these cells maintained the ability to generating midbrain DAergic neurons and extended differentiation in the presence of WNT5 [62]. In addition to manipulation in external culture conditions, the expression of internal key transcription factors can also be regulated to promote dopamine neuronal production from VM NSCs [65], for example, NSCs were transfected with Nurr1/TH and Brn4 by electroporation or lentivirus [66, 67]. And one of the first approaches to boost the yield of DAergic neurons from VM NSCs was based on the both external and internal manipulation [60]. Several studies have revealed that DAergic neuron-inducing activity is specific to VM derived NSCs [68]. Compared with VM NSCs, NSCs from other brain regions seem to be hardly to differentiate into functional DA neurons and lack the ability to release DA [69, 70], suggesting that VM NSCs and non-midbrain NSCs differ significantly in their responses to dopamine-induced signals, possibly due to non-midbrain NSCs lacking appropriate “priming” epigenetic states [71]. Lee et al. demonstrated that by co-expression of Nurr1 and Foxa2 via retrovirus transfection, non-midbrain NSCs gave rise to midbrain DA neuron phenotypes at late stages of midbrain development [72, 73]. The Nurr1+Foxa2 project has been modified in cortical-derived NSCs to mimic the physiological expression pattern of developmental factors of VM NSCs via selecting appropriate vectors and promoters, thus inducing the generation of completely mature midbrain DAergic neurons [74]. Despite extensive efforts, the quest for efficient differentiation of DAergic neurons from tissue-derived NSCs remains elusive. Induction of DAergic neurons from NSCs derived from PSCs In contrast to the CNS-derived NSCs, the targeted differentiation of PSCs derived NSCs has progressed rapidly, especially the generation of DAergic neurons. It was initially reported that co-cultured with PA6 or MS-5 feeder cells, mouse or primate ESCs can be induced to differentiate into NSCs and further TH-expressing neurons effectively [42, 43]. Furthermore, SHH and FGF8 can provide lineage-specific instructions to enhance the generation of DAergic neurons [42, 75]. Gradually, studies have shown that based on the EBs formation coupled with the action of SHH and FGF8, TH-positive DAergic neurons can be induced successfully from human PSCs [76–79]. With a better understanding of the pattern molecules and transcriptional networks involved in the generation of DAergic neurons in the midbrain during embryonic development, the differentiation protocol has been modified over time. The activation of WNT signaling involved in early caudalization of the cells in the neural plate, was mimicked by the glycogen synthase kinase 3β inhibitor CHIR99021 in vitro, which resulted in an improved midbrain specification reliably and efficiently [76, 80–85]. Compared with DAergic differentiation via a neural rosette intermediate (i.e., the differentiation protocol using SHH and FGF8 only), the DAergic neurons generated from the floor plate were more efficient, both in number and midbrain markers, that is, the number of cells co-expressing TH and fox2 accounted for about 75% of the culture [84]. It is well known that the BMP/SMAD inhibition is used to promote the neural induction of PSCs, but more recently BMP activation has been found to be helpful in the specification of DAergic neurons [86]. A study has revealed that in vitro application of BMP5/7 during the maturation phase can effectively promote the generation of VM DA neurons [86]. Furthermore, the culture system has expanded from two dimensional (2D) to three dimensional (3D) platforms, where cells can be embedded in biomaterials for 3D culture. Schaffer and colleagues have shown that in a 3D thermoresponsive biomaterial platform, by applying the same small molecules used to induce differentiation as in the 2D system, a higher number of TH-positive neurons (~40%) could be generated rapidly after 25 days of differentiation than in their 2D culture control (20%) [87] or other 2D culture systems (15–30%) [84], and these cells exhibited temporal marker expression profiles that resemble natural VM DAergic development [87]. Induction of DAergic neurons from NSCs derived from somatic transdifferentiation DAergic differentiation of induced NSCs (iNSCs) is infancy and there are not as many DAergic differentiation protocols as the other two types of NSCs s. Induced NSCs from PBMNCs can be induced into mature DAergic neurons through a two-stage method. The first stage mediated the generation of DA progenitors mainly through FGF8 and SAG1, a SHH pathway agonist, and the second stage promoted the maturation of DA neurons through a combination of BDNF, GDNF, TGF-β3, AA and other soluble factors [44, 88, 89] . At the end of differentiation, about 60% of cultured cells co-expressed Foxa2 and TH. Moreover, unlike the characteristics of tissue derived NSCs, induced NSCs retained their dopamine differentiation ability after multiple passages [88]. In addition to NSCs derived from PBMNCs, NSCs transdifferentiated from fibroblasts have also been used for DAergic neuron induction, and the DAergic induction protocols of both were similar. The difference is that the addition of BMP5/7 in the maturity phase further increased the generation of DAergic neurons [86]. Induction of MSNs from NSCs MSNs in the striatum are the most affected cell type in HD, making them the most suitable target cell type for cell replacement therapy [90]. During embryonic development, the lateral ganglionic eminence (LGE) of the ventral telencephalon gives birth to the MSNs [91]. The concentration gradient of SHH, WNT and BMP can affect the pattern of the dorsal ventral axis, that is, SHH promotes ventral localization of the neural tube and WNT and BMP promote dorsal localization [46]. And the LGE specification is patterned under the impact of antagonistic morphogen gradients. In detail, the repression of WNT together with SHH can efficiently induce the ventral fate of the telencephalic precursors. Furthermore, based on the expression of activin receptors and phosphorylated SMAD2 (an activin signaling pathway component) in the developing LGE, it is speculated that the activation of TGF-β pathway is also involved in the generation of striatum MSN [92]. DLX2 and GSX2 are markers of LGE [93], and MSNs are further characterized by expressed dopamine and cAMP-regulated neuronal phosphoprotein 32 (DARPP-32) and a range of other subtype-specific markers [94, 95]. So far, the generation of MSNs has been primary induced from NSCs from the first two sources, and there is no induction protocol for obtaining MSNs from iNSCs. Induction of MSNs from NSCs derived from primary CNS tissues NSCs isolated from fetal ganglion eminences can generate up to 25% of DARPP-32 positive neurons in vitro [96, 97], but similar to midbrain DAergic differentiation, this characteristic declines with culture time [98]. Possibly because of previous exposure to a favorable microenvironment, primary tissue-derived NSCs can be predisposed to adopt a specific phenotype, and these predispositions may be largely lost or offset by in vitro cell expansion. Therefore, it may be necessary to provide external cues to support or even increase the MSN differentiation ability of NSCs after passage, such as morphogens [99] and growth factors [97, 99]. A study explored the MSN differentiation conditions of immortalized striatal human NSC line. They compared the chemical induction systems of SHH, SHH/dickkopf-1 (DKK1)/BDNF, Dibutyryl cAMP/valproic acid (VPA)/BDNF, RA, and Purmorphamine, and found that the hedgehog agonist Purmorphamine most remarkably increased the MSN differentiation of NSCs, doubling the number of MSN in the short-term differentiation and tripling the number of MSN in the long-term differentiation [99]. In addition, sequential RA treatment and KCl depolarization can effectively yield 74% functional GABAergic neurons from the immortalized striatum NSCs [100]. Recently, another study found that striatal GABAergic neurons could be reliably induced from immortalized VM NSCs under hypoxic culturing conditions using two- or three-step differentiation protocol based on VPA or SHH and DKK1, respectively [101]. A majority of cultured cells expressed MSN markers and functional glutamate receptors, in addition to releasing GABA on stimulation [101]. Induction of MSNs from NSCs derived from PSCs It is acknowledged that neural induction via EB formation and subsequent exposure to SHH could drive ventral telencephalic fate in human ESCs. Further exploration found that medium dosage of SHH can pattern NSCs into LGE-like progenitor cells, which generate predominantly DARPP32-expressing GABA neurons, ~75% of the total number of cells in culture [102]. Jeon and colleagues induced the generation of NSCs by co-culturing ESCs and iPSCs derived from a patient with juvenile HD with PA6 stromal cells and subsequently producing 27% of DARPP-32 neurons in the presence of BDNF. Additionally, DARPP-32 can be co-localized with LGE markers DLX2 and GSX2, indicating successful generation of the MSN-like cells [41]. In another protocol, neural induction of human ESC and iPSCs was achieved via dual SMAD inhibition, followed by exposure to SHH and WNT inhibitor DKK1, which patterned NSC towards LGE progenitors [103]. A recent study has optimized this protocol by using small molecules to replace protein components, using a chemical cocktail to quickly and efficiently generate GABAergic MSNs from human ESCs [104]. Similar to the improved differentiation protocol of DAergic neurons, a study used a 3D culture method for neural induction and neural specification of ESCs, then matured on 2D laminin-coated plates. The MSNs generated by this 3D-2D method showed electrophysiological activity compared with those generated by 2D method [105]. Interestingly, the addition of Activin rather than SHH also induced the LGE-like progenitor fate after neural induction via dual SMAD inhibition, and the data showed that activin-mediated LGE fate was independent of SHH signaling [106]. Furthermore, another differentiation protocol did not apply SHH or Activin to induce the LGE-like progenitor fate, but continued to inhibit the BMP and WNT signaling pathways via dual SMAD inhibition and the use of IWR1 to regionalize NSCs after neural induction in ESCs [107]. Induction of cholinergic motor neurons from NSCs Motor neurons can generally be divided into two categories, depending on the location of the cell body: (I) Upper motor neurons that are located in the cerebral cortex, and (II) lower motor neurons that exist in the brainstem and spinal cord [108, 109]. The differences between the two types of motor cells are not limited to their location, but also manifest in neurotransmitters, targeting, and characteristics upon lesion (reviewed in refs. [108, 109]). Spinal MNs are patterned in the highly restricted foci of ventral neural tube in response to morphogens RA, FGFs, and SHH [110]. In more detail, caudalization of the neural tube is primarily facilitated by RA, produced via the activity of retinaldehyde dehydrogenase 2 [111]. And SHH allows specification of ventral part in the neural tube [112]. The temporal and spatial action of these extrinsic morphogens induce the upregulation of the basic helio-loop-helix (bHLH) transcription factor Olig2, which together with another bHLH transcription factor neurogenin 2 directs the expression of MN fate determining genes such as Islet1 and Hb9 [109, 110]. ALS and other motor neuron diseases characterized by motor neuron injury often result in muscle wasting and even paralysis, and the desire to protect and eventually regenerate motor circuits has prompted attempts to generate motor neurons for translational applications [112]. Here we focus our attention exclusively on the induction protocols for NSCs-derived spinal motor neurons, namely cholinergic motor neurons. Induction of cholinergic motor neurons from NSCs derived from primary CNS tissues To date, there has been little exploration of spinal motor neurons differentiation protocols from CNS tissue-derived NSCs. FGF2 is well recognized as a mitogen in the CNS, but it has been shown that FGF2 can direct the differentiation of NSCs into spinal motor neurons [113, 114]. In induction medium supplemented with FGF2, about 60% of human fetal forebrain-derived NSCs differentiated into H9 immunopositive cells on day 10, which supports the dual functions of FGF2, i.e., at high concentrations FGF2 primarily serves as a mitogen for NSCs, while at low concentrations it promotes neurogenesis [114]. Furthermore, immortalized telencephalic NSCs transduced Olig2 via retroviral vector expressed motor neuron-specific phenotypes following treatment with SHH, such as Hb9, Islet1 and choline acetyltransferase [115]. Induction of cholinergic motor neurons from NSCs derived from PSCs Compared with CNS tissue-derived NSCs, the motor neuron differentiation protocol of PSC derived NSCs has been widely investigated. Initial protocols for the differentiation of functional cholinergic motor neurons from ESCs have also heavily relied on the use of stromal feeder cells [42, 116]. Furthermore, studies have differentiated NSCs from PSCs via EB formation, followed by treatment with RA and SHH to induce cholinergic motor neuron generation successfully [117–120]. Gradually, most studies have used dual SMAD inhibition or combined with EB formation to accelerate the neuralization of PSCs [121–124]. In addition, some studies optimized ventral and caudal signaling molecules to promote the induction efficiency of motor neurons, for example, Maury et al. activated WNT signal via exposure to appropriate concentration of CHIR to cooperate with RA in caudal optimization, resulting in 80% of cells expressing the MN progenitor cell marker Olig2 [125]. Similarly, a single concentration of ventral morphogen SHH results in a mix of Olig2-expressing motor neuron progenitors with NKX2.2 -expressing interneuron progenitors residing in the adjacent domains. Du et al. used a combination of SHH (induced the Nkx2.2- and Olig2-expressing progenitors) and CHIR (antagonized the induction of Nkx2.2 expression by SHH) to enrich Olig2+/Nkx2.2−MN progenitors, resulting in a purity of more than 90% of motor neurons [126]. Patani et al. described a retinoid-independent protocol for the cadualization of human ESCs based on activin/nodal signaling inhibition, which resulted in the bias to medial motor columnar pools [117]. In addition to exposure to different combinations of patterning molecules that regulate intrinsic transcription factors to induce the generation of motor neurons, some studies have directly transfected transcription factors Neurog2, Islet1, and Lhx3 into human PSCs-derived NSCs via retroviral vectors to promote motor neuron production, which is simple, reliable and efficient [121, 127–129]. Induction of cholinergic motor neurons from iNSCs Currently, few studies have reported cholinergic motor neurons differentiation protocols starting from iNSCs. INSCs can be reprogrammed from astrocytes or PBMCs, and then patterned by RA and SHH in a chemical defined system to confer caudal and ventral anatomical identities, respectively, finally gave rise to Olig2 expressing progenitors. Finally, these progenitors mature into motor neurons under the action of growth factors [130, 131]. But neither protocol was efficient, producing about 15% and 35% HB9-positive cells, respectively [130, 131]. Induction of oligodendrocytes from NSCs Oligodendrocytes are glial cells that form myelin sheaths around axons in the CNS, supporting rapid nerve conduction and providing trophic and metabolic support to neuronal cells [132]. During neural development, NSCs give rise to oligodendrocyte precursor cells (OPCs), which are patterned in different regions of the neural tube, such as the ventral and dorsal sides of both spinal cord and forebrain [132–134]. Caudalization of the neural tube is modulated by RA, followed by the generation of Olig2-expressing spinal progenitors in response to ventral signal SHH, which are a source of both motoneurons and OPC [132, 135]. After the generation of motor neurons, Olig2-expressing spinal progenitors downregulate neurogenic transcription factors, and give rise to OPCs that express the oligodendroglial transcription factors Nkx2.2 and Sox10 and the surface markers, such as A2B5, platelet-derived growth factor receptor alpha (PDGFRα) and membrane proteoglycan NG2 [132]. These OPCs differentiate into immature oligodendrocytes expressing marker O4 and further become mature oligodendrocytes expressing myelin marker myelin basic protein [132, 134]. Not only the ventral source, a small number of oligodendrocytes also originate from the dorsal neural tube that is independently of SHH, but Olig2 expression is requisite for dorsal OPC specification [136, 137]. Induction of oligodendrocytes from NSCs derived from primary CNS tissues Genetic modification and culture environmental modification of human NSCs has been tested for the sake of obtaining cell populations enriched in oligodendroglia. One of the first approaches to induce oligodendrocytes from human fetal NSCs was based on the overexpression of the bHLH transcription factor Olig2 via lentiviral vectors. This protocol allowed an increased number of A2B5-positive oligodendroglial precursors in vitro, but fully committed O4-positive oligodendrocytes were not detected after 7 days of differentiation [138]. In a chemically defined system, it has been reported that a combination of FGF2, neurotrophin-3 (NT3) and platelet-derived growth factor-AA (PDGF-AA) successfully increased the proportion of oligodendrocytes expressing O4 and GalCer to 15–20% of the total culture cells from embryonic forebrain-derived NSCs [139]. And another protocol used a similar cocktail combination, resulting in highly pure OPCs from human fetal NSCs. In this study, up to 80–90% of culture cells expressed OPC markers O4, Sox10 A2B5, and PDGF-αR, and about 90% of the cells expressed GalCer with further differentiation [140]. The difference in the efficiency of oligodendrocyte production between the two protocols may be due to the origin of NSCs, as well as the difference in the concentration and duration of action of these factors [139, 140]. In addition, a study committed fetal forebrain-derived NSCs to oligodendrocyte phenotypes by adding PDGF-AA, FGF2, SHH, triiodothyronine, and NT-3, followed by the removal of four factors (PDGF-AA, FGF2, SHH, and NT-3) that promoted the expression of final markers of oligodendrocyte differentiation, with more than half of cultured cells expressing myelin basic protein [141]. Induction of oligodendrocytes from NSCs derived from derived from PSCs Thus far, there have been many attempts to generate oligodendrocytes from human PSCs-derived NSCs. One of the first protocols to induce oligodendrocytes from human ESCs was based on the EBs formation in combination with the activation of RA, SHH and FGF2 signaling [133, 142]. Specifically, ESCs-derived NSCs were patterned to progenitor cells expressing Olig2 and Nkx2.2 in the presence of RA and SHH, and subsequent treatment with FGF2 can inhibit motor neuron differentiation to increase pre-OPCs during the neurogenic phase. Finally, the removal of FGF2 and the addition of PDGF-AA, insulin growth factor 1 and NT3 promotes the transition of pre-OPCs to OPCs, and ~80% of cultured cells expressed OPC markers, such as PDGFRα and NG2 [133, 135, 142]. But unfortunately, this induction protocol takes a long time, at least 3 months, to generate OPCs from human PSCs [133, 142]. To address the limitation of long differentiation time, some groups have modified the induction protocol. Franklin et al developed a physiological oxygen tension protocol to generate oligodendrocytes from human ESCs under low oxygen conditions, mimicking the environment of the developing brain [143]. And the results indicated that hypoxic conditions could not only accelerate the overall differentiation process, but also significantly improve oligodendrocyte production [143]. Fossati and colleagues induced the generation of NSCs by using dual SMAD inhibition rapidly and modified the previous oligodendrocyte differentiation conditions slightly, thus speeding up the timetable of glial induction and resulting in most of cultured cells displaying the late OPC marker O4 [144, 145]. However, these optimized protocols shorten the differentiation cycle to 70 days at most, and primary rate-limiting steps are oligodendroglial specification and differentiation, some studies have accelerated the generation of oligodendrocytes by overexpression of transcription factors. More recently, an effective strategy that facilitated the generation of O4- expressing oligodendrocytes to 70% within 28 days of differentiation by using a combination of three transcription factors, Olig2, Sox10, and Nkx6.2 has been reported [146]. Verfaillie and colleagues described that overexpression of a single transcription factor, Sox10, was sufficient to generate similar levels of O4+ cells from human PSCs derived NSCs within 22 days [147]. In addition to the generation of spinal cord OPCs and oligodendrocytes via the use of caudal morphogen RA, several RA-independent approaches that favor telencephalic OPC generation have been reported [143, 148, 149]. A study accelerated the production of RA-independent telencephalic oligodendrocytes by enhancing neural induction using dual SMAD inhibition in conjunction with the tankase inhibitor XAV 939 (antagonizing WNT signaling) [149]. More recently, Xiong and colleagues first promoted the generation of ventral forebrain NSCs of ESCs by dual SMAD inhibition and activation SHH signals, and subsequently demonstrated that overexpression of Sox10 and Olig2 in these cells was sufficient to generate forebrain mature oligodendrocytes at day 40 of differentiation [148]. Induction of cortical glutamatergic neurons from NSCs Glutamatergic pyramidal neurons are the vast majority of excitatory nerve cells in the cerebral cortex that mediate myriad information processing streams and output channels [150]. In brain development, all cortical glutamatergic neurons originate from the embryonic dorsal telencephalon [151], which is patterned by WNTs and BMPs that are derived from the cortical hem [152, 153]. Early dorsal forebrain primordium co-express Pax6 and Otx1/2, and over time, these cells differentiated into cortical glutamatergic neurons, displaying unipolar and pyramidal morphology, and expressing TBR1, CTIP2, and vesicular glutamate transporters [154, 155]. Glutamatergic neurogenesis is also present in adult neurogenic niches, SVZ and SGZ [156, 157]. Interestingly, the pattern of transcription factor expression during adult glutamatergic neurogenesis is akin to the sequential expression of transcription factors in cortical glutamatergic neurons during the embryonic period, indicating that the genetic program specifying the fate of glutamate is spatially and temporally conserved [156]. Induction of cortical glutamatergic neurons from NSCs derived from primary CNS tissues Existing differentiation protocols tend to generate a mixture of cortical neurons from primary CNS tissue derived NSCs, rather than differentiating specifically into cortical glutamate neurons. NSCs isolated from cortex of human fetuses retained their regional identity and differentiated primarily into cortical GABAergic interneurons and glutamatergic neurons after the removal of the mitogen [158]. In addition, immortal fetal cortical NSCs lines also showed similar differentiation characteristics [159, 160], and a cortical human NSCs line CTX0E16 generated about 40% CTIP2-positive cells with typical pyramidal neuron morphology in vitro [159]. More recently, a study indicated that embryonic mouse dorsal cortical derived NSCs developed towards cortical glutaminergic neurons under FGF at below proliferative concentrations, possibly due to the endogenous and transient wave of BMP signals induced by low FGF2 [161]. Induction of cortical glutamatergic neurons from NSCs derived from derived from PSCs As we mentioned earlier, forebrain identity is the default procedure after neural induction of PSCs, and it has been shown that human PSCs predominantly differentiate into dorsal telencephalic NSCs after neural induction without the need for additional patterning morphogens, which is attributed to endogenous WNT signaling [153, 162]. In addition, several differentiation protocols, involving either EB formation or monolayer culture, enhanced neural induction of human PSCs through the use of the BMP inhibitor Noggin, thereby increasing the yield of cortical glutamate-like neuron production [155, 163], and most of these cells generated here exhibited an identity corresponding to deep layers rather than upper layers, which was determined by the expression of layer-specific markers during the process of differentiation [155]. In contrast, another differentiation protocol described that the equivalent proportions of deep and upper layer neurons can be generated from human PSCs when combined with dual inhibition of SMAD signaling and retinoic acid signaling [164, 165]. Nevertheless, recent studies have shown that the use of cyclopamine (an inhibitor of SHH) can promote the population of cortical glutamate neurons by inhibiting ventral differentiation from iPSCs [166–169]. Compared with 2D induction protocols, the generation and maturation of cortical glutamatergic neurons were further promoted by cultivating human PSCs derived NSCs in the PDMS-based 3D culture system [170]. Therapeutic potential of induced directed differentiation of NSCs in neurological disease models As mentioned above, NSCs have been directed to differentiate into specific lineages of cells expressing corresponding transcription factors and markers (Table 1), displaying cellular morphology, as well as manifested by electrophysiological properties in vitro [171, 172]. Furthermore, whether predifferentiated NSCs can survive, stably express the desired cell subtypes, and functionally integrate into the host brain of neurological models are increasingly being investigated (Table 1). The main neurological disease models currently involved include PD, HD, MS, ALS, and ischemic stroke. Here we primarily outlined the therapeutic potential of NSCs-derived specific phenotypes in chronic neurodegenerative disease PD and acute neurodegeneration ischemic stroke. Parkinson’s disease PD is a progressive neurodegenerative disorder characterized pathologically by the degeneration of DAergic neurons in the substantia nigra pars compacta, with a subsequent loss of DAergic axon terminals innervating the striatum, which results in motor disorder [173]. Currently, the main treatment for PD is dopamine replacement therapy. However, it can only relieve the symptoms of PD without delaying the progression of PD [174]. Regarding to the success of targeted differentiation of VM DAergic neurons in vitro, some studies have transplanted these cells into different models of PD to observe their therapeutic effects at the behavioral, cellular, and molecular levels. Most of the studies mentioned above implanted pre-differentiated NSCs into the striatum of 6-OHDA or MPTP-induced PD models, resulting in sensorimotor improvements, such as increased spontaneous activity and reduced circling behavior [60, 67, 73, 76, 78, 81, 82, 84, 85, 88]. Ectopic implantation is considered given that VM tissue grafts placed in the substantia nigra are unable to extend axons long enough to reach their target area, the striatum, to form complex neural circuits [42, 175–177]. After implantation, some of these preconditioned NSCs could survive in the PD animal model and exhibit a phenotype of nigra DAergic neurons, with increased dopamine levels [60, 67, 73, 76, 81, 82, 84, 85, 178]. The development of experimental techniques over the past decade has led to a refined understanding of how transplanted cells integrate with circuitry of the host nervous system [179]. Previous optogenetic and electrophysiological studies have demonstrated that ectopically transplanted NSCs-derived midbrain DAergic neurons are spontaneously active and receive appropriate presynaptic input from the host [180, 181], and the recent availability of the monosynaptic rabies tracing technique allowed us to further investigate the sources and extent of host synaptic inputs comprehensively [182]. For example, it has been shown that despite the ectopic intrastriatal location of NSCs-derived midbrain DAergic neurons, they can receive excitatory and inhibitory inputs from host cortical, striatal and pallidum neuronal subtypes, which are acknowledged to modulate the function of endogenous DAergic neurons in the substantia nigra, and this host afferent pattern helped to explain the proper regulation of DA release in “ectopic” intrastriatal VM-patterned grafts [183]. But the ectopic location of grafted cells may hinder the maximization of their function due to incompatibility with the physiological anatomy. To achieve a more complete circuit repair, homotopic transplantation to the substantia nigra is gradually being performed in rodent PD models [178, 179, 183]. Using VM DAergic cells derived from green fluorescent protein transgenic mouse embryos, earlier studies demonstrated the anatomical and functional reconstruction of nigrostriatal pathway after homotopic transplantation [175, 184]. Subsequently, it was observed that by using species-specific antibodies or genetic labeling, VM DAergic cells derived from NSCs innervated caudate putamen or prefrontal cortex, the main target of endogenous substantia nigra pars compacta (A9) or ventral tegmental area (A10) VM DAergic neurons, indicating that the grafted human VM DAergic neurons are the mixture of A9 and A10 phenotypes [179, 185, 186]. Interestingly, the innervated areas of intrastriatal VM DAergic neuron grafts were almost identical to those of the intranigral grafts, suggesting that the graft path-finding and target projection were largely determined by the cell-intrinsic factors [179, 183]. Therefore, the present studies showed that both ectopic and homotopic transplanted VM DA neurons can perform presynaptic and postsynaptic integration, and it was natural to assume that the reconstructed functional nigra-striatal circuit resulted in motor recovery in PD models, as demonstrated by the use of optogenetic and chemogenetic tools [179, 180, 187]. In addition to the transplant location, the optimal time window of DAergic neuron differentiation for transplantation is also important for the reconstruction of the nigra-striatal circuit [188, 189]. Using VM tissue as the donor, the maturity of the donor cells at transplantation significantly affected the grafts composition and functional outcomes. Specifically, donor tissue isolated before the peak of DA neurogenesis, embryonic day (E) 12, produced more DAergic neurons in the graft, which was attributed to increased DA neuroblasts survival and proliferation at the time of implantation [190–192]. Further studies on embryonic development showed that different DAergic subtypes originated from different progenitor pools and had different birth dates, with A9 DAergic neurons emerging earlier during DA neurogenesis [193–195], thus grafting of younger ventral midbrain donor tissue (E10) enriched A9 population and enhanced motor recovery [189, 193]. This may also be the reason why some studies have selected younger donor tissues for DAergic differentiation of ventral midbrain derived NSCs, and these pretreated VM NSCs grafts showed a higher TH-positive cell survival rate than fetal VM tissue grafts [60]. Currently, despite advanced protocols for the targeted differentiation of NSCs from two other sources into DAergic progenitor cells suitable for transplantation [42, 76, 78, 82, 84, 85, 88, 178, 179] and their rapid transition to clinical trials [196], the optimal stage of differentiation for transplantation has rarely been explored. On the one hand, the more mature the cells are in vitro, the more fragile they are and the more difficult they are to survive after transplantation. On the other hand, a higher degree of stemness is responsible for a greater chance of survival, but may result in insufficient regional regulation to generate mature DAergic neurons in vivo [188]. Therefore, most studies have focused on transplanting their derived VM progenitor cells at intermediate stages of differentiation, aiming to balance the ability to survive and mature [82, 84, 88, 186, 197]. A recent study attempted to transplant progenitor cells at different times of DAergic differentiation to determine the most appropriate transplantation time window, and it was surprising to find that grafts derived from younger progenitor cells consisted of the highest proportion of VM DAergic neurons and the lowest proportion of non-target cell types, showing intensive innervation ability as well as increased DA levels [188]. Although the donor age effect was observed in both human PSC lines, there was some variability across cell lines, highlighting the significance of characterizing cells in vivo on the basis of different cell lines or standardizing differentiation protocols [188]. Furthermore, in this study, some commonly used mesencephalic floor plate markers were used to determine the optimal differentiation time window of transplanted cells, such as FOXA2 and OTX2 [188]. But by using RNA sequencing, another study found that the high DAergic yield and their functional maturation in vivo positively correlated with a specific group of markers associated with the caudal midbrain, rather than the levels of those commonly used markers. According to these markers, a good manufacturing practice differentiation protocol for VM DAergic progenitor cell production was developed through the use timed delivery of FGF8 and a number of other adjustments [197]. In conclusion, the ability of these markers to more precisely predict graft outcome will accelerate the clinical application of stem cells. In the future, a panel of markers can be refined and identified at the progenitor stage in vitro to predict more functional mature A9 DAergic neurons in vivo. Ischemic stroke Ischemic stroke is a cerebrovascular event that, although not classified as a neurodegenerative disease, also presents pathological cell death in the infarct area, including different types of neurons and glial cells [198]. In recent years, the limited treatments for ischemic stroke include endorvascular surgery (called thrombectomy) or intravenous administration of alteplase (called thrombolysis) for the purpose of restoring blood flow [199]. However, due to the narrow therapeutic window, some contraindications, low efficacy to recanalize the large artery via thrombolysis, and even the reperfusion injury after recanalization, only a small percentage of ischemic patients can benefit from these two treatments [200–203]. Possibly inspired by replacement therapy for specific cells lost in NDs, recently there have been studies targeting different lost cell types in ischemic stroke to transplant specific cells for replacement to reconstruct damaged neural circuits [166, 168, 169]. A clinical and imaging study showed that the distribution of damaged cells under the most severe symptoms in stroke patients was often not in the striatum, suggesting that cell replacement strategies should emphasize reconstructing the damaged cortex rather than the striatum [204]. Therefore, some studies have implanted progenitor cells with a cortical glutaminergic phenotype derived from PSCs into the cortex of rat models of ischemic stroke and found that these cells could alleviate sensorimotor impairment at 2 or 6 months after transplantation [166, 169]. Behavioral improvements at the early time point of 2 months post-transplantation were most likely not attributable to neuronal replacement and circuitry integration. The use of human-specific cytoplasmic markers combined with green protein immunostaining revealed that axonal projection of transplanted cells extended to both ipsilateral and contralateral hemispheres [169]. Further by using rabies virus–based transsynaptic tracing and optogenetics, it was found that the contralateral somatosensory cortex received functional monosynaptic input from the transplanted neurons [166]. In addition to functional efferent integration, transplanted cells also received synaptic inputs from the thalamocortex and were able to modulate their own activity in response to physiological sensory stimuli [168]. The functional circuit reconstruction is responsible for the recovery of motor function at late time points after transplantation [166]. Except for cortical glutamatergic excitatory neurons, another study identified the phenotype of transplanted predifferentiated NSCs as GABAergic inhibitory neurons. NSCs isolated from human fetal SVZ were predifferentiated in the presence of BDNF, and the predifferentiated cells were subsequently transplanted into the striatum and cortex of cerebral ischemic rats. Histopathology 28 days-post transplant indicated that these cells stably expressed the GABA phenotype, increased GABA levels, and exerted their trophic effects to promote endogenous neurogenesis, which may lead to faster functional recovery in cerebral ischemic rats than those treated with undifferentiated NSCs [205]. In addition to sensorimotor dysfunction, most stroke survivors suffer from cognitive impairment, which may be related to demyelination of the brain’s white matter, resulting from oligodendrocyte death [206, 207]. Xu et al. proposed a two-step protocol to derive NG2-positive OPCs stably and rapidly from iPSCs, first inhibiting SHH to generate NPC and then overexpressing Olig2 [208]. After transplanting OPCs into the cerebral ventricles of ischemic rats, it was found that these cells could protect host neurons from death under the ischemic environment by suppressing inflammatory and immune responses. Furthermore, these cells rescued learning and memory loss to some extent by facilitating the remyelination process in ischemic stroke rats [208]. Discussion and future directions Based on numerous studies of cell transplantation, it can be inferred that both pre-transplantation history and source of donor cells are critical factors that affect the outcome of transplantation. In terms of pre-transplant history, most of the protocols for targeted differentiation of NSCs from different sources involve external chemical-defined system through patterning cues or intrinsic ectopic overexpression of lineage-specific transcription factors. Currently the external culture system is gradually moving from the initial co-culture or use of poorly defined xenogeneic factors toward a fully chemical-defined, and xeno-free condition, which encourages the establishment of the more robust differentiation protocol [209]. However, most existing targeted differentiation methods tend to generate heterogeneous cultures containing the cell type of interest as well as other undesirable phenotypes. Consequently, further exploration of the use of morphogens, growth factors, and small molecules in concentration, sequence, and duration is warranted. On the other hand, when manipulating gene overexpression, full consideration should be given to their expression patterns during neural development in order to achieve adequate maturation of differentiated cells and long-term phenotypic maintenance [74]. but virus-mediated multigene transduction is somewhat cytotoxic leading to poor implantation of donor cells, and another concern is that viral integration could disrupt normal gene expression, thus may not be suitable for clinical scenarios. In addition to the two approaches to orchestrate the directed induction differentiation of NSCs, increasingly, it has been shown that epigenetic machinery can regulate the interaction between activation and inhibition of various developmental signaling pathways in neural differentiation [210], such as finely tuning genetic programs to coordinate distinct neural lineage differentiation [211]. With the current advances in RNA interference, they could also be used to guide the targeted differentiation of NSCs [212–215]. In addition, Traditional two-dimensional induction methods provide basic soluble regulators to control the fate of NSCs. However, stem cell behaviors are regulated by different physiological, physico-chemical and physico-mechanical cues, so three-dimensional induction involving biological materials is increasingly being emphasized to effectively control the fate of stem cells [216–218]. With the rise of interdisciplinary of medicine and engineering, conducting polymers have been proved to induce the directional differentiation of NSCs through electrical stimulus in vitro [219]. All these efforts are aimed at promoting the targeted differentiation of NSCs and obtaining specific neural lineage cells in vitro. The selection of primary CNS tissues and cell types for the generation of specific neuronal lineage phenotype also requires to be taken into account, as it may affect the efficiency of neuronal lineage differentiation and the effectiveness of transplantation. The potential of primary CNS tissue-derived NSCs varies depending on the developmental stage at which they are obtained and the site from where they were isolated [220]. For instance, under identical culture conditions, NSCs derived from ventral mesencephalic produced more DAergic neurons than those from striatum [68], and A9 neurons are produced earlier than A10 neurons during neural development [189], so NSCs derived from VM at an early stage is more conducive to the generation of A9 DAergic neurons. In contrast, NSCs derived from neural induction of PSCs tend to be at an earlier stage, and they can actively respond to multiple patterning molecules to differentiate into different neural lineages. However, it is gradually recognized that epigenetic differences exist between different PSC strains, leading to deviations in lineage differentiation or different generation efficiencies of the same lineage [163, 188, 221]. These differences highlight the need to further compare the results of directed differentiation of neural lineages across different PSC lines. In contrast to tissue-derived NSCs or ESCs, iPSCs and iNSCs are not subject to ethical concerns. In particular, patient-specific iPSCs or iNSCs are targeted to differentiate into neural lineage cells that will match individual immunity—a goal long pursed in regenerative medicine, but before they can be reasonably used for cell therapy, it is critical to understand and correct any intrinsic defects in these cells [119]. Neural grafts derived from human iNSCs are less likely to produce fast-growing tumors following grafting compared with grafts of PSCs [222, 223]. Currently, several strategies are actively being taken to address the possibility of potentially pathological growth of grafted cells from various sources, such as the use of cell-sorting techniques to remove Off-target contaminating cell types prior to transplantation [81, 224, 225] or the transduction of ligand-activated suicide genes to ablate proliferating cells in vivo [226, 227]. With the rapid development of induced neuron (iN) technology in recent years, it is possible to directly reprogram somatic cells to obtain functional neurons [93]. However, the iN method converts somatic cells directly into non-dividing neurons rather than fate committed neuronal progenitors, and these non-dividing neurons often tend to survive and integrate poorly in the host brain after transplantation [228]. In addition, the specific pathological states presented by different neurological diseases should be fully considered before donor cell transplantation, which may affect the cell transplantation strategy in a degree. In neurodegenerative disorders, the outcome of cell replacement relies on the complexity and precision of the connection patterns that need to be restored [229]. In the case of Parkinson’s disease, only a partial pattern repair can result in significant functional recovery. Ectopic transplantation of DAergic cells, also known as paracrine strategy, can restore the efficient release of dopamine to regulate the substantia nigrostriatal circuitry [60, 67, 73, 76]. Unlike Parkinson’s disease, for local neuronal degeneration due to HD or ALS and global neurodegeneration due to ischemic stroke, it is necessary to re-establish the specific afferent–efferent connections between the graft and host, emphasizing the importance of homotopic transplantation [229]. So far, based on advances in nanotechnology, molecular biology and imaging techniques, transplanted cell tracing technology has shifted from ex vitro detection to in vivo imaging [230], and further combining with viral or genetic labeling strategies can help researchers explore the synaptic connection between transplanted cells and host brain [179]. In addition to transplantation of directionally induced neural stem cells, in vivo cell reprogramming, the in-situ conversion of glial cells to functional new neurons is also a promising neural regeneration strategy [9]. Furthermore, transplanted exogenous stem cells can be genetically engineered to steadily produce growth factors that support the repair of dysfunctional endogenous neurons [231]. For example, human iPSC-derived neural progenitor cells genetically engineered to stably produce GDNF can delivery GDNF after transplantation to protect degenerated neurons in PD models and ALS models [232]. As a result, researchers have poured a great deal of effort in different ways, towards the same goal: to promote nerve repair or regeneration. Recent preclinical studies have demonstrated the safety and efficacy of DAergic neurons derived from PSCs. Based on these promising results, clinical trials are being conducted in different countries [76, 224]. PD clinical trials provide important guidelines for other derived neural lineage cells venturing into these uncharted territories, and we believe that neural lineage cells derived from different sources of NSCs, especially PSCs, will be tested in clinical trials in the near future to develop treatments for related neurological diseases. Author contributions NL and ZT: conceptualization; Luwei Nie: initial draft preparation, wrote the main manuscript text, with the help of DY, SC, JW, CP, and DW; LN prepared table and figures; all authors: literature search, review, commentary, and final approval of the manuscript. Funding This work was funded by National Natural Science Foundation of China (No. 82071330) and the Natural Science Foundation of Hubei Province, China (No. 2019CFB113). Competing interests The authors declare no competing interests. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Kahroba H Ramezani B Maadi H Sadeghi MR Jaberie H Ramezani F The role of Nrf2 in neural stem/progenitors cells: from maintaining stemness and self-renewal to promoting differentiation capability and facilitating therapeutic application in neurodegenerative disease Ageing Res Rev 2021 65 101211 10.1016/j.arr.2020.101211 33186670 2. Zhou J Jangili P Son S Ji MS Won M Kim JS Fluorescent diagnostic probes in neurodegenerative diseases Adv Mater 2020 32 e2001945 10.1002/adma.202001945 32902000 3. Yaribeygi H Panahi Y Javadi B Sahebkar A The underlying role of oxidative stress in neurodegeneration: a mechanistic review CNS Neurol Disord Drug Targets 2018 17 207 15 10.2174/1871527317666180425122557 29692267 4. Tuo QZ Zhang ST Lei P Mechanisms of neuronal cell death in ischemic stroke and their therapeutic implications Med Res Rev 2022 42 259 305 10.1002/med.21817 33957000 5. Back T Pathophysiology of the ischemic penumbra—revision of a concept Cell Mol Neurobiol 1998 18 621 38 10.1023/a:1020629818207 9876870 6. Li X Huang L Lan J Feng X Li P Wu L Molecular mechanisms of mitophagy and its roles in neurodegenerative diseases Pharm Res 2021 163 105240 10.1016/j.phrs.2020.105240 7. Pasteuning-Vuhman S de Jongh R Timmers A Pasterkamp RJ Towards advanced iPSC-based drug development for neurodegenerative disease Trends Mol Med 2021 27 263 79 10.1016/j.molmed.2020.09.013 33121873 8. McCrary MR Jesson K Wei ZZ Logun M Lenear C Tan S Cortical transplantation of brain-mimetic glycosaminoglycan scaffolds and neural progenitor cells promotes vascular regeneration and functional recovery after ischemic stroke in mice Adv Health Mater 2020 9 e1900285 10.1002/adhm.201900285 9. Barker RA Gotz M Parmar M New approaches for brain repair-from rescue to reprogramming Nature 2018 557 329 34 10.1038/s41586-018-0087-1 29769670 10. Andreotti JP Silva WN Costa AC Picoli CC Bitencourt FCO Coimbra-Campos LMC Neural stem cell niche heterogeneity Semin Cell Dev Biol 2019 95 42 53 10.1016/j.semcdb.2019.01.005 30639325 11. Reynolds BA Weiss S Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system Science 1992 255 1707 10 10.1126/science.1553558 1553558 12. Bellenchi GC Volpicelli F Piscopo V Perrone-Capano C di Porzio U Adult neural stem cells: an endogenous tool to repair brain injury? J Neurochem 2013 124 159 67 10.1111/jnc.12084 23134340 13. Zhang B Yan W Zhu Y Yang W Le W Chen B Nanomaterials in neural-stem-cell-mediated regenerative medicine: imaging and treatment of neurological diseases Adv Mater 2018 30 e1705694 10.1002/adma.201705694 29543350 14. Qin C Wang K Zhang L Bai L Stem cell therapy for Alzheimer’s disease: an overview of experimental models and reality Anim Model Exp Med 2022 5 15 26 10.1002/ame2.12207 15. De Gioia R, Biella F, Citterio G, Rizzo F, Abati E, Nizzardo M, et al. Neural stem cell transplantation for neurodegenerative diseases. Int J Mol Sci. 2020;21. 10.3390/ijms21093103. 16. Huang L Zhang L Neural stem cell therapies and hypoxic-ischemic brain injury Prog Neurobiol 2019 173 1 17 10.1016/j.pneurobio.2018.05.004 29758244 17. Corey S Bonsack B Heyck M Shear A Sadanandan N Zhang H Harnessing the anti-inflammatory properties of stem cells for transplant therapy in hemorrhagic stroke Brain Hemorrhages 2020 1 24 33 10.1016/j.hest.2019.12.005 34056567 18. Monsour M Borlongan CV Emerging regenerative medicine for hemorrhagic stroke: an update on stem cell therapies Brain Hemorrhages 2023 4 22 26 10.1016/j.hest.2022.07.001 19. Benner EJ Luciano D Jo R Abdi K Paez-Gonzalez P Sheng H Protective astrogenesis from the SVZ niche after injury is controlled by Notch modulator Thbs4 Nature 2013 497 369 73 10.1038/nature12069 23615612 20. Pous L Deshpande SS Nath S Mezey S Malik SC Schildge S Fibrinogen induces neural stem cell differentiation into astrocytes in the subventricular zone via BMP signaling Nat Commun 2020 11 630 10.1038/s41467-020-14466-y 32005867 21. Tejeda G, Ciciriello AJ & Dumont, CM. Biomaterial strategies to bolster neural stem cell-mediated repair of the central nervous system. Cells Tissues Organs. 2021;1–15. 10.1159/000515351. 22. Bruggeman KF Moriarty N Dowd E Nisbet DR Parish CL Harnessing stem cells and biomaterials to promote neural repair Br J Pharm 2019 176 355 68 10.1111/bph.14545 23. Lambertsen KL Finsen B Clausen BH Post-stroke inflammation-target or tool for therapy? Acta Neuropathol 2019 137 693 714 10.1007/s00401-018-1930-z 30483945 24. Kwon HS Koh SH Neuroinflammation in neurodegenerative disorders: the roles of microglia and astrocytes Transl Neurodegener 2020 9 42 10.1186/s40035-020-00221-2 33239064 25. Mathieu P Battista D Depino A Roca V Graciarena M Pitossi F The more you have, the less you get: the functional role of inflammation on neuronal differentiation of endogenous and transplanted neural stem cells in the adult brain J Neurochem 2010 112 1368 85 10.1111/j.1471-4159.2009.06548.x 20028453 26. Zhang K Lu WC Zhang M Zhang Q Xian PP Liu FF Reducing host aldose reductase activity promotes neuronal differentiation of transplanted neural stem cells at spinal cord injury sites and facilitates locomotion recovery Neural Regen Res 2022 17 1814 20 10.4103/1673-5374.330624 35017443 27. Ideguchi M Shinoyama M Gomi M Hayashi H Hashimoto N Takahashi J Immune or inflammatory response by the host brain suppresses neuronal differentiation of transplanted ES cell-derived neural precursor cells J Neurosci Res 2008 86 1936 43 10.1002/jnr.21652 18335525 28. He Z, Lang L, Hui J, Ma Y, Yang C, Weng W, et al. Brain extract of subacute traumatic brain injury promotes the neuronal differentiation of human neural stem cells via autophagy. J Clin Med. 2022;11. 10.3390/jcm11102709. 29. Yang Y Fan Y Zhang H Zhang Q Zhao Y Xiao Z Small molecules combined with collagen hydrogel direct neurogenesis and migration of neural stem cells after spinal cord injury Biomaterials 2021 269 120479 10.1016/j.biomaterials.2020.120479 33223332 30. Adams KV Morshead CM Neural stem cell heterogeneity in the mammalian forebrain Prog Neurobiol 2018 170 2 36 10.1016/j.pneurobio.2018.06.005 29902499 31. Zhu Y Huang R Wu Z Song S Cheng L Zhu R Deep learning-based predictive identification of neural stem cell differentiation Nat Commun 2021 12 2614 10.1038/s41467-021-22758-0 33972525 32. Lee HR Farhanullah Lee J Jajoo R Kong SY Shin JY Discovery of a small molecule that enhances astrocytogenesis by activation of STAT3, SMAD1/5/8, and ERK1/2 via induction of cytokines in neural stem cells ACS Chem Neurosci 2016 7 90 99 10.1021/acschemneuro.5b00243 26505647 33. Tang Y Yu P Cheng L Current progress in the derivation and therapeutic application of neural stem cells Cell Death Dis 2017 8 e3108 10.1038/cddis.2017.504 29022921 34. Kempermann G Song H Gage FH Neurogenesis in the Adult Hippocampus Cold Spring Harb Perspect Biol 2015 7 a018812 10.1101/cshperspect.a018812 26330519 35. Lin R Iacovitti L Classic and novel stem cell niches in brain homeostasis and repair Brain Res 2015 1628 327 42 10.1016/j.brainres.2015.04.029 25931262 36. Muratore CR Srikanth P Callahan DG Young-Pearse TL Comparison and optimization of hiPSC forebrain cortical differentiation protocols PLoS One 2014 9 e105807 10.1371/journal.pone.0105807 25165848 37. Yan Y Shin S Jha BS Liu Q Sheng J Li F Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells Stem Cells Transl Med 2013 2 862 70 10.5966/sctm.2013-0080 24113065 38. Tao Y Zhang SC Neural subtype specification from human pluripotent stem cells Cell Stem Cell 2016 19 573 86 10.1016/j.stem.2016.10.015 27814479 39. Chambers SM Fasano CA Papapetrou EP Tomishima M Sadelain M Studer L Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling Nat Biotechnol 2009 27 275 80 10.1038/nbt.1529 19252484 40. Zhang SC Wernig M Duncan ID Brustle O Thomson JA In vitro differentiation of transplantable neural precursors from human embryonic stem cells Nat Biotechnol 2001 19 1129 33 10.1038/nbt1201-1129 11731781 41. Jeon I Lee N Li JY Park IH Park KS Moon J Neuronal properties, in vivo effects, and pathology of a Huntington’s disease patient-derived induced pluripotent stem cells Stem Cells 2012 30 2054 62 10.1002/stem.1135 22628015 42. Barberi T Klivenyi P Calingasan NY Lee H Kawamata H Loonam K Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice Nat Biotechnol 2003 21 1200 7 10.1038/nbt870 14502203 43. Kawasaki H Suemori H Mizuseki K Watanabe K Urano F Ichinose H Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity Proc Natl Acad Sci USA 2002 99 1580 5 10.1073/pnas.032662199 11818560 44. Zheng W, Chen Z. Generation of induced neural stem cells from peripheral mononuclear cells and differentiation toward dopaminergic neuron precursors for transplantation studies. J Vis Exp. 2019. 10.3791/59690. 45. Thier M Worsdorfer P Lakes YB Gorris R Herms S Opitz T Direct conversion of fibroblasts into stably expandable neural stem cells Cell Stem Cell 2012 10 473 9 10.1016/j.stem.2012.03.003 22445518 46. Kiecker C Lumsden A The role of organizers in patterning the nervous system Annu Rev Neurosci 2012 35 347 67 10.1146/annurev-neuro-062111-150543 22462542 47. Christie KJ Emery B Denham M Bujalka H Cate HS Turnley AM Transcriptional regulation and specification of neural stem cells Adv Exp Med Biol 2013 786 129 55 10.1007/978-94-007-6621-1_8 23696355 48. Ono Y Nakatani T Sakamoto Y Mizuhara E Minaki Y Kumai M Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells Development 2007 134 3213 25 10.1242/dev.02879 17670789 49. Placzek M Briscoe J The floor plate: multiple cells, multiple signals Nat Rev Neurosci 2005 6 230 40 10.1038/nrn1628 15738958 50. Ye W Shimamura K Rubenstein JL Hynes MA Rosenthal A FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate Cell 1998 93 755 66 10.1016/s0092-8674(00)81437-3 9630220 51. Chung S Leung A Han BS Chang MY Moon JI Kim CH Wnt1-lmx1a forms a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway Cell Stem Cell 2009 5 646 58 10.1016/j.stem.2009.09.015 19951692 52. Mesman S, Smidt MP. Acquisition of the midbrain dopaminergic neuronal identity. Int J Mol Sci. 2020;21. 10.3390/ijms21134638. 53. Prakash N Brodski C Naserke T Puelles E Gogoi R Hall A A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo Development 2006 133 89 98 10.1242/dev.02181 16339193 54. Hovakimyan M Haas SJ Schmitt O Gerber B Wree A Andressen C Mesencephalic human neural progenitor cells transplanted into the neonatal hemiparkinsonian rat striatum differentiate into neurons and improve motor behaviour J Anat 2006 209 721 32 10.1111/j.1469-7580.2006.00654.x 17118060 55. Maciaczyk J Singec I Maciaczyk D Nikkhah G Combined use of BDNF, ascorbic acid, low oxygen, and prolonged differentiation time generates tyrosine hydroxylase-expressing neurons after long-term in vitro expansion of human fetal midbrain precursor cells Exp Neurol 2008 213 354 62 10.1016/j.expneurol.2008.06.014 18652826 56. Jin G Tan X Tian M Qin J Zhu H Huang Z The controlled differentiation of human neural stem cells into TH-immunoreactive (ir) neurons in vitro Neurosci Lett 2005 386 105 10 10.1016/j.neulet.2005.04.065 16046065 57. Sanchez-Pernaute R Studer L Bankiewicz KS Major EO McKay RD In vitro generation and transplantation of precursor-derived human dopamine neurons J Neurosci Res 2001 65 284 8 10.1002/jnr.1152 11494363 58. Yan W Chen ZY Chen JQ Chen HM BMP2 promotes the differentiation of neural stem cells into dopaminergic neurons in vitro via miR-145-mediated upregulation of Nurr1 expression Am J Transl Res 2016 8 3689 99 27725851 59. Studer L Tabar V McKay RD Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats Nat Neurosci 1998 1 290 5 10.1038/1105 10195162 60. Parish CL Castelo-Branco G Rawal N Tonnesen J Sorensen AT Salto C Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice J Clin Investig 2008 118 149 60 10.1172/JCI32273 18060047 61. Chung S Shin BS Hwang M Lardaro T Kang UJ Isacson O Neural precursors derived from embryonic stem cells, but not those from fetal ventral mesencephalon, maintain the potential to differentiate into dopaminergic neurons after expansion in vitro Stem Cells 2006 24 1583 93 10.1634/stemcells.2005-0558 16543488 62. Ribeiro D Laguna Goya R Ravindran G Vuono R Parish CL Foldi C Efficient expansion and dopaminergic differentiation of human fetal ventral midbrain neural stem cells by midbrain morphogens Neurobiol Dis 2013 49 118 27 10.1016/j.nbd.2012.08.006 22940632 63. He XB Kim M Kim SY Yi SH Rhee YH Kim T Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1- and JMJD3-dependent epigenetic control manner Stem Cells 2015 33 1320 32 10.1002/stem.1932 25535150 64. Yan J Studer L McKay RD Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors J Neurochem 2001 76 307 11 10.1046/j.1471-4159.2001.00073.x 11146004 65. Roybon L Hjalt T Christophersen NS Li JY Brundin P Effects on differentiation of embryonic ventral midbrain progenitors by Lmx1a, Msx1, Ngn2, and Pitx3 J Neurosci 2008 28 3644 56 10.1523/JNEUROSCI.0311-08.2008 18385323 66. Tan X Zhang L Zhu H Qin J Tian M Dong C Brn4 and TH synergistically promote the differentiation of neural stem cells into dopaminergic neurons Neurosci Lett 2014 571 23 28 10.1016/j.neulet.2014.04.019 24769320 67. Tan X Zhang L Qin J Tian M Zhu H Dong C Transplantation of neural stem cells co-transfected with Nurr1 and Brn4 for treatment of Parkinsonian rats Int J Dev Neurosci 2013 31 82 87 10.1016/j.ijdevneu.2012.10.007 23085081 68. Okano H Yoshizaki T Shimazaki T Sawamoto K Isolation and transplantation of dopaminergic neurons and neural stem cells Parkinsonism Relat Disord 2002 9 23 28 10.1016/s1353-8020(02)00041-x 12217619 69. Yang H Wang J Wang F Liu X Chen H Duan W Dopaminergic neuronal differentiation from the forebrain-derived human neural stem cells induced in cultures by using a combination of BMP-7 and pramipexole with growth factors Front Neural Circuits 2016 10 29 10.3389/fncir.2016.00029 27147976 70. Jain M Armstrong RJ Tyers P Barker RA Rosser AE GABAergic immunoreactivity is predominant in neurons derived from expanded human neural precursor cells in vitro Exp Neurol 2003 182 113 23 10.1016/s0014-4886(03)00055-4 12821381 71. Rossler R Boddeke E Copray S Differentiation of non-mesencephalic neural stem cells towards dopaminergic neurons Neuroscience 2010 170 417 28 10.1016/j.neuroscience.2010.07.023 20643196 72. Yi SH He XB Rhee YH Park CH Takizawa T Nakashima K Foxa2 acts as a co-activator potentiating expression of the Nurr1-induced DA phenotype via epigenetic regulation Development 2014 141 761 72 10.1242/dev.095802 24496614 73. Lee HS Bae EJ Yi SH Shim JW Jo AY Kang JS Foxa2 and Nurr1 synergistically yield A9 nigral dopamine neurons exhibiting improved differentiation, function, and cell survival Stem Cells 2010 28 501 12 10.1002/stem.294 20049900 74. Kim T Song JJ Puspita L Valiulahi P Shim JW Lee SH In vitro generation of mature midbrain-type dopamine neurons by adjusting exogenous Nurr1 and Foxa2 expressions to their physiologic patterns Exp Mol Med 2017 49 e300 10.1038/emm.2016.163 28280264 75. Vazin T Chen J Lee CT Amable R Freed WJ Assessment of stromal-derived inducing activity in the generation of dopaminergic neurons from human embryonic stem cells Stem Cells 2008 26 1517 25 10.1634/stemcells.2008-0039 18388303 76. Wang YK Zhu WW Wu MH Wu YH Liu ZX Liang LM Human clinical-grade parthenogenetic esc-derived dopaminergic neurons recover locomotive defects of nonhuman primate models of Parkinson’s disease Stem Cell Rep 2018 11 171 82 10.1016/j.stemcr.2018.05.010 77. Ma L Liu Y Zhang SC Directed differentiation of dopamine neurons from human pluripotent stem cells Methods Mol Biol 2011 767 411 8 10.1007/978-1-61779-201-4_30 21822892 78. Swistowski A Peng J Liu Q Mali P Rao MS Cheng L Efficient generation of functional dopaminergic neurons from human induced pluripotent stem cells under defined conditions Stem Cells 2010 28 1893 904 10.1002/stem.499 20715183 79. Yan Y Yang D Zarnowska ED Du Z Werbel B Valliere C Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells Stem Cells 2005 23 781 90 10.1634/stemcells.2004-0365 15917474 80. Gantner CW Cota-Coronado A Thompson LH Parish CL An optimized protocol for the generation of midbrain dopamine neurons under defined conditions STAR Protoc 2020 1 100065 10.1016/j.xpro.2020.100065 33111103 81. Doi D Samata B Katsukawa M Kikuchi T Morizane A Ono Y Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation Stem Cell Rep 2014 2 337 50 10.1016/j.stemcr.2014.01.013 82. Kirkeby A Grealish S Wolf DA Nelander J Wood J Lundblad M Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions Cell Rep 2012 1 703 14 10.1016/j.celrep.2012.04.009 22813745 83. Xi J Liu Y Liu H Chen H Emborg ME Zhang SC Specification of midbrain dopamine neurons from primate pluripotent stem cells Stem Cells 2012 30 1655 63 10.1002/stem.1152 22696177 84. Kriks S Shim JW Piao J Ganat YM Wakeman DR Xie Z Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease Nature 2011 480 547 51 10.1038/nature10648 22056989 85. Kikuchi T Morizane A Doi D Magotani H Onoe H Hayashi T Human iPS cell-derived dopaminergic neurons function in a primate Parkinson’s disease model Nature 2017 548 592 6 10.1038/nature23664 28858313 86. Jovanovic VM Salti A Tilleman H Zega K Jukic MM Zou H BMP/SMAD pathway promotes neurogenesis of midbrain dopaminergic neurons in vivo and in human induced pluripotent and neural stem cells J Neurosci 2018 38 1662 76 10.1523/JNEUROSCI.1540-17.2018 29321139 87. Adil MM Rodrigues GM Kulkarni RU Rao AT Chernavsky NE Miller EW Efficient generation of hPSC-derived midbrain dopaminergic neurons in a fully defined, scalable, 3D biomaterial platform Sci Rep 2017 7 40573 10.1038/srep40573 28091566 88. Yuan Y Tang X Bai YF Wang S An J Wu Y Dopaminergic precursors differentiated from human blood-derived induced neural stem cells improve symptoms of a mouse Parkinson’s disease model Theranostics 2018 8 4679 94 10.7150/thno.26643 30279731 89. Li M Wang Z Zheng T Huang T Liu B Han D Characterization of human-induced neural stem cells and derivatives following transplantation into the central nervous system of a nonhuman primate and rats Stem Cells Int 2022 2022 1396735 10.1155/2022/1396735 36618021 90. Yan S Tu Z Liu Z Fan N Yang H Yang S A Huntingtin Knockin Pig Model recapitulates features of selective neurodegeneration in Huntington’s disease Cell 2018 173 989 1002 e1013 10.1016/j.cell.2018.03.005 29606351 91. Waclaw RR Wang B Pei Z Ehrman LA Campbell K Distinct temporal requirements for the homeobox gene Gsx2 in specifying striatal and olfactory bulb neuronal fates Neuron 2009 63 451 65 10.1016/j.neuron.2009.07.015 19709628 92. Maira M Long JE Lee AY Rubenstein JL Stifani S Role for TGF-beta superfamily signaling in telencephalic GABAergic neuron development J Neurodev Disord 2010 2 48 60 10.1007/s11689-009-9035-6 20339443 93. Fjodorova M Noakes Z Li M How to make striatal projection neurons Neurogenesis 2015 2 e1100227 10.1080/23262133.2015.1100227 27606330 94. Arlotta P Molyneaux BJ Jabaudon D Yoshida Y Macklis JD Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum J Neurosci 2008 28 622 32 10.1523/JNEUROSCI.2986-07.2008 18199763 95. Ouimet CC Langley-Gullion KC Greengard P Quantitative immunocytochemistry of DARPP-32-expressing neurons in the rat caudatoputamen Brain Res 1998 808 8 12 10.1016/s0006-8993(98)00724-0 9795103 96. Ostenfeld T Joly E Tai YT Peters A Caldwell M Jauniaux E Regional specification of rodent and human neurospheres Brain Res Dev Brain Res 2002 134 43 55 10.1016/s0165-3806(01)00291-7 11947936 97. Ivkovic S Ehrlich ME Expression of the striatal DARPP-32/ARPP-21 phenotype in GABAergic neurons requires neurotrophins in vivo and in vitro J Neurosci 1999 19 5409 19 10.1523/JNEUROSCI.19-13-05409.1999 10377350 98. Kelly CM Precious SV Penketh R Amso N Dunnett SB Rosser AE Striatal graft projections are influenced by donor cell type and not the immunogenic background Brain 2007 130 1317 29 10.1093/brain/awm053 17395612 99. El-Akabawy G Medina LM Jeffries A Price J Modo M Purmorphamine increases DARPP-32 differentiation in human striatal neural stem cells through the Hedgehog pathway Stem Cells Dev 2011 20 1873 87 10.1089/scd.2010.0282 21345011 100. Bosch M Pineda JR Sunol C Petriz J Cattaneo E Alberch J Induction of GABAergic phenotype in a neural stem cell line for transplantation in an excitotoxic model of Huntington’s disease Exp Neurol 2004 190 42 58 10.1016/j.expneurol.2004.06.027 15473979 101. Lin L Yuan J Sander B Golas MM In vitro differentiation of human neural progenitor cells into striatal GABAergic neurons Stem Cells Transl Med 2015 4 775 88 10.5966/sctm.2014-0083 25972145 102. Ma L Hu B Liu Y Vermilyea SC Liu H Gao L Human embryonic stem cell-derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice Cell Stem Cell 2012 10 455 64 10.1016/j.stem.2012.01.021 22424902 103. Delli Carri A Onorati M Lelos MJ Castiglioni V Faedo A Menon R Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32+ medium-sized spiny neurons Development 2013 140 301 12 10.1242/dev.084608 23250204 104. Wu M Zhang D Bi C Mi T Zhu W Xia L A chemical recipe for generation of clinical-grade striatal neurons from hESCs Stem Cell Rep 2018 11 635 50 10.1016/j.stemcr.2018.08.005 105. Adil MM Gaj T Rao AT Kulkarni RU Fuentes CM Ramadoss GN hPSC-derived striatal cells generated using a scalable 3D hydrogel promote recovery in a Huntington disease mouse model Stem Cell Rep 2018 10 1481 91 10.1016/j.stemcr.2018.03.007 106. Arber C Precious SV Cambray S Risner-Janiczek JR Kelly C Noakes Z Activin A directs striatal projection neuron differentiation of human pluripotent stem cells Development 2015 142 1375 86 10.1242/dev.117093 25804741 107. Comella-Bolla A Orlandi JG Miguez A Straccia M Garcia-Bravo M Bombau G Human pluripotent stem cell-derived neurons are functionally mature in vitro and integrate into the mouse striatum following transplantation Mol Neurobiol 2020 57 2766 98 10.1007/s12035-020-01907-4 32356172 108. Zayia LC, Tadi P. in StatPearls. 2023. 109. Stifani N Motor neurons and the generation of spinal motor neuron diversity Front Cell Neurosci 2014 8 293 10.3389/fncel.2014.00293 25346659 110. Patani R Generating diverse spinal motor neuron subtypes from human pluripotent stem cells Stem Cells Int 2016 2016 1036974 10.1155/2016/1036974 26823667 111. Sances S Bruijn LI Chandran S Eggan K Ho R Klim JR Modeling ALS with motor neurons derived from human induced pluripotent stem cells Nat Neurosci 2016 19 542 53 10.1038/nn.4273 27021939 112. Davis-Dusenbery BN Williams LA Klim JR Eggan K How to make spinal motor neurons Development 2014 141 491 501 10.1242/dev.097410 24449832 113. Natarajan R Singal V Benes R Gao J Chan H Chen H STAT3 modulation to enhance motor neuron differentiation in human neural stem cells PLoS One 2014 9 e100405 10.1371/journal.pone.0100405 24945434 114. Jordan PM Ojeda LD Thonhoff JR Gao J Boehning D Yu Y Generation of spinal motor neurons from human fetal brain-derived neural stem cells: role of basic fibroblast growth factor J Neurosci Res 2009 87 318 32 10.1002/jnr.21856 18803285 115. Lee HJ Kim KS Ahn J Bae HM Lim I Kim SU Human motor neurons generated from neural stem cells delay clinical onset and prolong life in ALS mouse model PLoS One 2014 9 e97518 10.1371/journal.pone.0097518 24844281 116. Lee H Shamy GA Elkabetz Y Schofield CM Harrsion NL Panagiotakos G Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons Stem Cells 2007 25 1931 9 10.1634/stemcells.2007-0097 17478583 117. Patani R Hollins AJ Wishart TM Puddifoot CA Alvarez S de Lera AR Retinoid-independent motor neurogenesis from human embryonic stem cells reveals a medial columnar ground state Nat Commun 2011 2 214 10.1038/ncomms1216 21364553 118. Karumbayaram S Novitch BG Patterson M Umbach JA Richter L Lindgren A Directed differentiation of human-induced pluripotent stem cells generates active motor neurons Stem Cells 2009 27 806 11 10.1002/stem.31 19350680 119. Dimos JT Rodolfa KT Niakan KK Weisenthal LM Mitsumoto H Chung W Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons Science 2008 321 1218 21 10.1126/science.1158799 18669821 120. Li XJ Du ZW Zarnowska ED Pankratz M Hansen LO Pearce RA Specification of motoneurons from human embryonic stem cells Nat Biotechnol 2005 23 215 21 10.1038/nbt1063 15685164 121. Ren J, Li C, Zhang M, Wang H, Xie Y, Tang Y. A Step-by-step refined strategy for highly efficient generation of neural progenitors and motor neurons from human pluripotent stem cells. Cells. 2021;10. 10.3390/cells10113087. 122. Shimojo D Onodera K Doi-Torii Y Ishihara Y Hattori C Miwa Y Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells Mol Brain 2015 8 79 10.1186/s13041-015-0172-4 26626025 123. Kiskinis E Sandoe J Williams LA Boulting GL Moccia R Wainger BJ Pathways disrupted in human ALS motor neurons identified through genetic correction of mutant SOD1 Cell Stem Cell 2014 14 781 95 10.1016/j.stem.2014.03.004 24704492 124. Amoroso MW Croft GF Williams DJ O’Keeffe S Carrasco MA Davis AR Accelerated high-yield generation of limb-innervating motor neurons from human stem cells J Neurosci 2013 33 574 86 10.1523/JNEUROSCI.0906-12.2013 23303937 125. Maury Y Come J Piskorowski RA Salah-Mohellibi N Chevaleyre V Peschanski M Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes Nat Biotechnol 2015 33 89 96 10.1038/nbt.3049 25383599 126. Du ZW Chen H Liu H Lu J Qian K Huang CL Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells Nat Commun 2015 6 6626 10.1038/ncomms7626 25806427 127. Wu J Tang Y Transcription factor-mediated differentiation of motor neurons from human pluripotent stem cells Methods Mol Biol 2023 2593 245 58 10.1007/978-1-0716-2811-9_16 36513936 128. Akter M Cui H Sepehrimanesh M Hosain MA Ding B Generation of highly pure motor neurons from human induced pluripotent stem cells STAR Protoc 2022 3 101223 10.1016/j.xpro.2022.101223 35300000 129. Sepehrimanesh M Ding B Generation and optimization of highly pure motor neurons from human induced pluripotent stem cells via lentiviral delivery of transcription factors Am J Physiol Cell Physiol 2020 319 C771 C780 10.1152/ajpcell.00279.2020 32783653 130. Tang X Yu M Liao C Lan S Fan Y Induced neural stem cells-derived spinal cord progenitor cells as stable stage for rapid and efficient generation of human oligodendrocytes and motor neurons Connect Tissue Res 2021 62 206 14 10.1080/03008207.2019.1670651 32380866 131. Shao Z Luo Q Liu D Mi Y Zhang P Ju G Induced differentiation of neural stem cells of astrocytic origin to motor neurons in the rat Stem Cells Dev 2011 20 1163 70 10.1089/scd.2010.0262 21087155 132. Cristobal CD Lee HK Development of myelinating glia: an overview Glia 2022 70 2237 59 10.1002/glia.24238 35785432 133. Goldman SA Kuypers NJ How to make an oligodendrocyte Development 2015 142 3983 95 10.1242/dev.126409 26628089 134. Bergles DE Richardson WD Oligodendrocyte development and plasticity Cold Spring Harb Perspect Biol 2015 8 a020453 10.1101/cshperspect.a020453 26492571 135. Hu BY Du ZW Li XJ Ayala M Zhang SC Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects Development 2009 136 1443 52 10.1242/dev.029447 19363151 136. Fogarty M Richardson WD Kessaris N A subset of oligodendrocytes generated from radial glia in the dorsal spinal cord Development 2005 132 1951 9 10.1242/dev.01777 15790969 137. Cai J Qi Y Hu X Tan M Liu Z Zhang J Generation of oligodendrocyte precursor cells from mouse dorsal spinal cord independent of Nkx6 regulation and Shh signaling Neuron 2005 45 41 53 10.1016/j.neuron.2004.12.028 15629701 138. Maire CL Buchet D Kerninon C Deboux C Baron-Van Evercooren A Nait-Oumesmar B Directing human neural stem/precursor cells into oligodendrocytes by overexpression of Olig2 transcription factor J Neurosci Res 2009 87 3438 46 10.1002/jnr.22194 19739249 139. Neri M Maderna C Ferrari D Cavazzin C Vescovi AL Gritti A Robust generation of oligodendrocyte progenitors from human neural stem cells and engraftment in experimental demyelination models in mice PLoS One 2010 5 e10145 10.1371/journal.pone.0010145 20405042 140. Wang C Luan Z Yang Y Wang Z Wang Q Lu Y High purity of human oligodendrocyte progenitor cells obtained from neural stem cells: suitable for clinical application J Neurosci Methods 2015 240 61 66 10.1016/j.jneumeth.2014.10.017 25445251 141. Monaco MC, Maric D, Bandeian A, Leibovitch E, Yang W & Major EO Progenitor-derived oligodendrocyte culture system from human fetal brain. J Vis Exp. 2012. 10.3791/4274. 142. Hu BY Du ZW Zhang SC Differentiation of human oligodendrocytes from pluripotent stem cells Nat Protoc 2009 4 1614 22 10.1038/nprot.2009.186 19834476 143. Stacpoole SR Spitzer S Bilican B Compston A Karadottir R Chandran S High yields of oligodendrocyte lineage cells from human embryonic stem cells at physiological oxygen tensions for evaluation of translational biology Stem Cell Rep 2013 1 437 50 10.1016/j.stemcr.2013.09.006 144. Douvaras P Fossati V Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells Nat Protoc 2015 10 1143 54 10.1038/nprot.2015.075 26134954 145. Douvaras P Wang J Zimmer M Hanchuk S O’Bara MA Sadiq S Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells Stem Cell Rep 2014 3 250 9 10.1016/j.stemcr.2014.06.012 146. Ehrlich M Mozafari S Glatza M Starost L Velychko S Hallmann AL Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors Proc Natl Acad Sci USA 2017 114 E2243 E2252 10.1073/pnas.1614412114 28246330 147. Garcia-Leon JA Garcia-Diaz B Eggermont K Caceres-Palomo L Neyrinck K Madeiro da Costa R Generation of oligodendrocytes and establishment of an all-human myelinating platform from human pluripotent stem cells Nat Protoc 2020 15 3716 44 10.1038/s41596-020-0395-4 33097924 148. Ma L Mei Y Xu P Cheng Y You Z Ji X Fast generation of forebrain oligodendrocyte spheroids from human embryonic stem cells by transcription factors iScience 2022 25 105172 10.1016/j.isci.2022.105172 36217550 149. Piao J Major T Auyeung G Policarpio E Menon J Droms L Human embryonic stem cell-derived oligodendrocyte progenitors remyelinate the brain and rescue behavioral deficits following radiation Cell Stem Cell 2015 16 198 210 10.1016/j.stem.2015.01.004 25658373 150. Matho KS Huilgol D Galbavy W He M Kim G An X Genetic dissection of the glutamatergic neuron system in cerebral cortex Nature 2021 598 182 7 10.1038/s41586-021-03955-9 34616069 151. Rakic P The radial edifice of cortical architecture: from neuronal silhouettes to genetic engineering Brain Res Rev 2007 55 204 19 10.1016/j.brainresrev.2007.02.010 17467805 152. MuhChyi C Juliandi B Matsuda T Nakashima K Epigenetic regulation of neural stem cell fate during corticogenesis Int J Dev Neurosci 2013 31 424 33 10.1016/j.ijdevneu.2013.02.006 23466416 153. Li XJ Zhang X Johnson MA Wang ZB Lavaute T Zhang SC Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells Development 2009 136 4055 63 10.1242/dev.036624 19906872 154. Sperandeo A Tamburini C Noakes Z de la Fuente DC Keefe F Petter O Cortical neuronal hyperexcitability and synaptic changes in SGCE mutation-positive myoclonus dystonia Brain 2022 10.1093/brain/awac365 155. Espuny-Camacho I Michelsen KA Gall D Linaro D Hasche A Bonnefont J Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo Neuron 2013 77 440 56 10.1016/j.neuron.2012.12.011 23395372 156. Hodge RD Kahoud RJ Hevner RF Transcriptional control of glutamatergic differentiation during adult neurogenesis Cell Mol Life Sci 2012 69 2125 34 10.1007/s00018-011-0916-y 22249196 157. Sequerra EB Miyakoshi LM Froes MM Menezes JR Hedin-Pereira C Generation of glutamatergic neurons from postnatal and adult subventricular zone with pyramidal-like morphology Cereb Cortex 2010 20 2583 91 10.1093/cercor/bhq006 20154014 158. Kallur T Darsalia V Lindvall O Kokaia Z Human fetal cortical and striatal neural stem cells generate region-specific neurons in vitro and differentiate extensively to neurons after intrastriatal transplantation in neonatal rats J Neurosci Res 2006 84 1630 44 10.1002/jnr.21066 17044030 159. Anderson GW Deans PJ Taylor RD Raval P Chen D Lowder H Characterisation of neurons derived from a cortical human neural stem cell line CTX0E16 Stem Cell Res Ther 2015 6 149 10.1186/s13287-015-0136-8 26296747 160. Cacci E Villa A Parmar M Cavallaro M Mandahl N Lindvall O Generation of human cortical neurons from a new immortal fetal neural stem cell line Exp Cell Res 2007 313 588 601 10.1016/j.yexcr.2006.11.001 17156776 161. Micali N Kim SK Diaz-Bustamante M Stein-O’Brien G Seo S Shin JH Variation of human neural stem cells generating organizer states in vitro before committing to cortical excitatory or inhibitory neuronal fates Cell Rep 2020 31 107599 10.1016/j.celrep.2020.107599 32375049 162. Gaspard N Bouschet T Hourez R Dimidschstein J Naeije G van den Ameele J An intrinsic mechanism of corticogenesis from embryonic stem cells Nature 2008 455 351 7 10.1038/nature07287 18716623 163. Kim JE O’Sullivan ML Sanchez CA Hwang M Israel MA Brennand K Investigating synapse formation and function using human pluripotent stem cell-derived neurons Proc Natl Acad Sci USA 2011 108 3005 10 10.1073/pnas.1007753108 21278334 164. Shi Y Kirwan P Livesey FJ Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks Nat Protoc 2012 7 1836 46 10.1038/nprot.2012.116 22976355 165. Shi Y Kirwan P Smith J Robinson HP Livesey FJ Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses Nat Neurosci 2012 15 477 86 10.1038/nn.3041 22306606 166. Palma-Tortosa S Tornero D Hansen G Monni E Hajy M Kartsivadze S Activity in grafted human iPS cell-derived cortical neurons integrated in stroke-injured rat brain regulates motor behavior Proc Natl Acad Sci USA 2020 117 9094 100 10.1073/pnas.2000690117 32253308 167. Cao SY Hu Y Chen C Yuan F Xu M Li Q Enhanced derivation of human pluripotent stem cell-derived cortical glutamatergic neurons by a small molecule Sci Rep 2017 7 3282 10.1038/s41598-017-03519-w 28607372 168. Tornero D Tsupykov O Granmo M Rodriguez C Gronning-Hansen M Thelin J Synaptic inputs from stroke-injured brain to grafted human stem cell-derived neurons activated by sensory stimuli Brain 2017 140 692 706 10.1093/brain/aww347 28115364 169. Tornero D Wattananit S Gronning Madsen M Koch P Wood J Tatarishvili J Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery Brain 2013 136 3561 77 10.1093/brain/awt278 24148272 170. Chen C Dong X Fang KH Yuan F Hu Y Xu M Develop a 3D neurological disease model of human cortical glutamatergic neurons using micropillar-based scaffolds Acta Pharm Sin B 2019 9 557 64 10.1016/j.apsb.2019.03.004 31193866 171. Vergano-Vera E Diaz-Guerra E Rodriguez-Traver E Mendez-Gomez HR Solis O Pignatelli J Nurr1 blocks the mitogenic effect of FGF-2 and EGF, inducing olfactory bulb neural stem cells to adopt dopaminergic and dopaminergic-GABAergic neuronal phenotypes Dev Neurobiol 2015 75 823 41 10.1002/dneu.22251 25447275 172. Ghareghani M Sadeghi H Zibara K Danaei N Azari H Ghanbari A Melatonin increases oligodendrocyte differentiation in cultured neural stem cells Cell Mol Neurobiol 2017 37 1319 24 10.1007/s10571-016-0450-4 27987059 173. Bloem BR Okun MS Klein C Parkinson’s disease Lancet 2021 397 2284 303 10.1016/s0140-6736(21)00218-x 33848468 174. Han F Hu B Stem cell therapy for Parkinson’s disease Adv Exp Med Biol 2020 1266 21 38 10.1007/978-981-15-4370-8_3 33105493 175. Gaillard A Decressac M Frappe I Fernagut PO Prestoz L Besnard S Anatomical and functional reconstruction of the nigrostriatal pathway by intranigral transplants Neurobiol Dis 2009 35 477 88 10.1016/j.nbd.2009.07.003 19616502 176. Mendez I Sanchez-Pernaute R Cooper O Vinuela A Ferrari D Bjorklund L Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson’s disease Brain 2005 128 1498 510 10.1093/brain/awh510 15872020 177. Mukhida K Baker KA Sadi D Mendez I Enhancement of sensorimotor behavioral recovery in hemiparkinsonian rats with intrastriatal, intranigral, and intrasubthalamic nucleus dopaminergic transplants J Neurosci 2001 21 3521 30 10.1523/JNEUROSCI.21-10-03521.2001 11331381 178. Grealish S Diguet E Kirkeby A Mattsson B Heuer A Bramoulle Y Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson’s disease Cell Stem Cell 2014 15 653 65 10.1016/j.stem.2014.09.017 25517469 179. Xiong M Tao Y Gao Q Feng B Yan W Zhou Y Human stem cell-derived neurons repair circuits and restore neural function Cell Stem Cell 2021 28 112 126.e116 10.1016/j.stem.2020.08.014 32966778 180. Steinbeck JA Choi SJ Mrejeru A Ganat Y Deisseroth K Sulzer D Optogenetics enables functional analysis of human embryonic stem cell-derived grafts in a Parkinson’s disease model Nat Biotechnol 2015 33 204 9 10.1038/nbt.3124 25580598 181. Tonnesen J Parish CL Sorensen AT Andersson A Lundberg C Deisseroth K Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model PLoS One 2011 6 e17560 10.1371/journal.pone.0017560 21394212 182. Bjorklund A Parmar M Dopamine cell therapy: from cell replacement to circuitry repair J Parkinsons Dis 2021 11 S159 S165 10.3233/JPD-212609 33814467 183. Adler AF Cardoso T Nolbrant S Mattsson B Hoban DB Jarl U hESC-Derived dopaminergic transplants integrate into Basal Ganglia circuitry in a preclinical model of Parkinson’s disease Cell Rep 2019 28 3462 3473.e3465 10.1016/j.celrep.2019.08.058 31553914 184. Thompson LH Grealish S Kirik D Bjorklund A Reconstruction of the nigrostriatal dopamine pathway in the adult mouse brain Eur J Neurosci 2009 30 625 38 10.1111/j.1460-9568.2009.06878.x 19674082 185. Aldrin-Kirk P Akerblom M Cardoso T Nolbrant S Adler AF Liu X A novel two-factor monosynaptic TRIO tracing method for assessment of circuit integration of hESC-derived dopamine transplants Stem Cell Rep 2022 17 159 72 10.1016/j.stemcr.2021.11.014 186. Niclis JC Gantner CW Hunt CPJ Kauhausen JA Durnall JC Haynes JM A PITX3-EGFP reporter line reveals connectivity of dopamine and non-dopamine neuronal subtypes in grafts generated from human embryonic stem cells Stem Cell Rep 2017 9 868 82 10.1016/j.stemcr.2017.08.002 187. Chen Y Xiong M Dong Y Haberman A Cao J Liu H Chemical control of grafted human PSC-derived neurons in a mouse model of Parkinson’s disease Cell Stem Cell 2016 18 817 26 10.1016/j.stem.2016.03.014 27133795 188. de Luzy IR Pavan C Moriarty N Hunt CPJ Vandenhoven Z Khanna A Identifying the optimal developmental age of human pluripotent stem cell-derived midbrain dopaminergic progenitors for transplantation in a rodent model of Parkinson’s disease Exp Neurol 2022 358 114219 10.1016/j.expneurol.2022.114219 36055392 189. Kauhausen J Thompson LH Parish CL Cell intrinsic and extrinsic factors contribute to enhance neural circuit reconstruction following transplantation in Parkinsonian mice J Physiol 2013 591 77 91 10.1113/jphysiol.2012.243063 23045338 190. Torres EM Monville C Gates MA Bagga V Dunnett SB Improved survival of young donor age dopamine grafts in a rat model of Parkinson’s disease Neuroscience 2007 146 1606 17 10.1016/j.neuroscience.2007.03.037 17478050 191. Gates MA Torres EM White A Fricker-Gates RA Dunnett SB Re-examining the ontogeny of substantia nigra dopamine neurons Eur J Neurosci 2006 23 1384 90 10.1111/j.1460-9568.2006.04637.x 16553799 192. Freeman TB Sanberg PR Nauert GM Boss BD Spector D Olanow CW The influence of donor age on the survival of solid and suspension intraparenchymal human embryonic nigral grafts Cell Transpl 1995 4 141 54 10.1177/096368979500400118 193. Bye CR Thompson LH Parish CL Birth dating of midbrain dopamine neurons identifies A9 enriched tissue for transplantation into parkinsonian mice Exp Neurol 2012 236 58 68 10.1016/j.expneurol.2012.04.002 22524988 194. Blaess S Bodea GO Kabanova A Chanet S Mugniery E Derouiche A Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei Neural Dev 2011 6 29 10.1186/1749-8104-6-29 21689430 195. Joksimovic M Anderegg A Roy A Campochiaro L Yun B Kittappa R Spatiotemporally separable Shh domains in the midbrain define distinct dopaminergic progenitor pools Proc Natl Acad Sci USA 2009 106 19185 90 10.1073/pnas.0904285106 19850875 196. Schweitzer JS Song B Herrington TM Park TY Lee N Ko S Personalized iPSC-derived dopamine progenitor cells for Parkinson’s disease N. Engl J Med 2020 382 1926 32 10.1056/NEJMoa1915872 32402162 197. Kirkeby A Nolbrant S Tiklova K Heuer A Kee N Cardoso T Predictive markers guide differentiation to improve graft outcome in clinical translation of hESC-based therapy for Parkinson’s disease Cell Stem Cell 2017 20 135 48 10.1016/j.stem.2016.09.004 28094017 198. Datta A Sarmah D Mounica L Kaur H Kesharwani R Verma G Cell death pathways in ischemic stroke and targeted pharmacotherapy Transl Stroke Res 2020 11 1185 202 10.1007/s12975-020-00806-z 32219729 199. Campbell BCV Khatri P Stroke Lancet 2020 396 129 42 10.1016/s0140-6736(20)31179-x 32653056 200. Patel P Yavagal D Khandelwal P Hyperacute management of ischemic strokes: JACC focus seminar J Am Coll Cardiol 2020 75 1844 56 10.1016/j.jacc.2020.03.006 32299596 201. Stoll G Pham M Beyond recanalization—a call for action in acute stroke Nat Rev Neurol 2020 16 591 2 10.1038/s41582-020-00417-0 32968233 202. Chen J Duris K Yang X Effect of cerebral microbleeds on hemorrhagic transformation and functional prognosis after intravenous thrombolysis of cerebral infarction Brain Hemorrhages 2022 3 117 9 10.1016/j.hest.2021.05.004 203. Jiang Y Han J Spencer P Li Y Vodovoz SJ Ning M-M Diabetes mellitus: a common comorbidity increasing hemorrhagic transformation after tPA thrombolytic therapy for ischemic stroke Brain Hemorrhages 2021 2 116 23 10.1016/j.hest.2020.11.004 204. Delavaran H Sjunnesson H Arvidsson A Lindvall O Norrving B van Westen D Proximity of brain infarcts to regions of endogenous neurogenesis and involvement of striatum in ischaemic stroke Eur J Neurol 2013 20 473 9 10.1111/j.1468-1331.2012.03877.x 23057628 205. Abeysinghe HC Bokhari L Quigley A Choolani M Chan J Dusting GJ Pre-differentiation of human neural stem cells into GABAergic neurons prior to transplant results in greater repopulation of the damaged brain and accelerates functional recovery after transient ischemic stroke Stem Cell Res Ther 2015 6 186 10.1186/s13287-015-0175-1 26420220 206. Wang Y Liu G Hong D Chen F Ji X Cao G White matter injury in ischemic stroke Prog Neurobiol 2016 141 45 60 10.1016/j.pneurobio.2016.04.005 27090751 207. Shi L Sun Z Su W Xu F Xie D Zhang Q Treg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke Immunity 2021 54 1527 1542 e1528 10.1016/j.immuni.2021.04.022 34015256 208. Xu J Zhao J Wang R Zhang Y Shen L Xiao Q Shh and Olig2 sequentially regulate oligodendrocyte differentiation from hiPSCs for the treatment of ischemic stroke Theranostics 2022 12 3131 49 10.7150/thno.69217 35547747 209. Niclis JC Gantner CW Alsanie WF McDougall SJ Bye CR Elefanty AG Efficiently specified ventral midbrain dopamine neurons from human pluripotent stem cells under xeno-free conditions restore motor deficits in parkinsonian rodents Stem Cells Transl Med 2017 6 937 48 10.5966/sctm.2016-0073 28297587 210. Vieira MS Santos AK Vasconcellos R Goulart VAM Parreira RC Kihara AH Neural stem cell differentiation into mature neurons: Mechanisms of regulation and biotechnological applications Biotechnol Adv 2018 36 1946 70 10.1016/j.biotechadv.2018.08.002 30077716 211. Cacci E Negri R Biagioni S Lupo G Histone methylation and microRNA-dependent regulation of epigenetic activities in neural progenitor self-renewal and differentiation Curr Top Med Chem 2017 17 794 807 10.2174/1568026616666160414124456 27086782 212. Shi Z Zhou H Lu L Pan B Wei Z Liu J MicroRNA-29a regulates neural stem cell neuronal differentiation by targeting PTEN J Cell Biochem 2018 119 5813 20 10.1002/jcb.26768 29637609 213. Roese-Koerner B Stappert L Koch P Brustle O Borghese L Pluripotent stem cell-derived somatic stem cells as tool to study the role of microRNAs in early human neural development Curr Mol Med 2013 13 707 22 10.2174/1566524011313050003 23642053 214. Stappert L Borghese L Roese-Koerner B Weinhold S Koch P Terstegge S MicroRNA-based promotion of human neuronal differentiation and subtype specification PLoS One 2013 8 e59011 10.1371/journal.pone.0059011 23527072 215. Low WC Yau WW Stanton LW Marcy G Goh E Chew SY Directing neuronal differentiation of primary neural progenitor cells by gene knockdown approach DNA Cell Biol 2012 31 1148 60 10.1089/dna.2011.1557 22339269 216. Karimi M Bahrami S Mirshekari H Basri SM Nik AB Aref AR Microfluidic systems for stem cell-based neural tissue engineering Lab Chip 2016 16 2551 71 10.1039/c6lc00489j 27296463 217. Darvishi M Ghasemi Hamidabadi H Sahab Negah S Moayeri A Tiraihi T Mirnajafi-Zadeh J PuraMatrix hydrogel enhances the expression of motor neuron progenitor marker and improves adhesion and proliferation of motor neuron-like cells Iran J Basic Med Sci 2020 23 431 8 10.22038/ijbms.2020.39797.9434 32489557 218. Zimmermann JA Schaffer DV Engineering biomaterials to control the neural differentiation of stem cells Brain Res Bull 2019 150 50 60 10.1016/j.brainresbull.2019.05.007 31103526 219. Chao KY Huang WY Ho CY Wan D Wang HC Yang CY Biodegradable aniline-derived electroconductive film for the regulation of neural stem cell fate J Mater Chem B 2021 9 1325 35 10.1039/d0tb02171g 33443514 220. Temple S The development of neural stem cells Nature 2001 414 112 7 10.1038/35102174 11689956 221. Nishizawa M Chonabayashi K Nomura M Tanaka A Nakamura M Inagaki A Epigenetic variation between human induced pluripotent stem cell lines is an indicator of differentiation capacity Cell Stem Cell 2016 19 341 54 10.1016/j.stem.2016.06.019 27476965 222. Gao M Yao H Dong Q Zhang H Yang Z Yang Y Tumourigenicity and immunogenicity of induced neural stem cell grafts versus induced pluripotent stem cell grafts in syngeneic mouse brain Sci Rep 2016 6 29955 10.1038/srep29955 27417157 223. Chen Z Cell therapy for Parkinson’s disease: new hope from reprogramming technologies Aging Dis 2015 6 499 503 10.14336/AD.2014.1201 26618051 224. Doi D Magotani H Kikuchi T Ikeda M Hiramatsu S Yoshida K Pre-clinical study of induced pluripotent stem cell-derived dopaminergic progenitor cells for Parkinson’s disease Nat Commun 2020 11 3369 10.1038/s41467-020-17165-w 32632153 225. Samata B Doi D Nishimura K Kikuchi T Watanabe A Sakamoto Y Purification of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1 Nat Commun 2016 7 13097 10.1038/ncomms13097 27739432 226. Itakura G Kawabata S Ando M Nishiyama Y Sugai K Ozaki M Fail-safe system against potential tumorigenicity after transplantation of iPSC derivatives Stem Cell Rep 2017 8 673 84 10.1016/j.stemcr.2017.02.003 227. Tieng V Cherpin O Gutzwiller E Zambon AC Delgado C Salmon P Elimination of proliferating cells from CNS grafts using a Ki67 promoter-driven thymidine kinase Mol Ther Methods Clin Dev 2016 6 16069 10.1038/mtm.2016.69 27990449 228. Thompson LH Andersson E Jensen JB Barraud P Guillemot F Parmar M Neurogenin2 identifies a transplantable dopamine neuron precursor in the developing ventral mesencephalon Exp Neurol 2006 198 183 98 10.1016/j.expneurol.2005.11.025 16438966 229. Rossi F Cattaneo E Opinion: neural stem cell therapy for neurological diseases: dreams and reality Nat Rev Neurosci 2002 3 401 9 10.1038/nrn809 11988779 230. Xue CR Wang K Zhang MZ Wang Z Song YY Yu HJ Tracking neural stem cells in vivo: achievements and limitations Stem Cell Rev Rep 2022 18 1774 88 10.1007/s12015-022-10333-z 35122628 231. Gowing G Svendsen S Svendsen CN Ex vivo gene therapy for the treatment of neurological disorders Prog Brain Res 2017 230 99 132 10.1016/bs.pbr.2016.11.003 28552237 232. Akhtar AA Gowing G Kobritz N Savinoff SE Garcia L Saxon D Inducible expression of GDNF in transplanted iPSC-derived neural progenitor cells Stem Cell Rep 2018 10 1696 704 10.1016/j.stemcr.2018.03.024 233. Hwang DH Kim BG Kim EJ Lee SI Joo IS Suh-Kim H Transplantation of human neural stem cells transduced with Olig2 transcription factor improves locomotor recovery and enhances myelination in the white matter of rat spinal cord following contusive injury BMC Neurosci 2009 10 117 10.1186/1471-2202-10-117 19772605 234. Garcia-Leon JA Kumar M Boon R Chau D One J Wolfs E SOX10 single transcription factor-based fast and efficient generation of oligodendrocytes from human pluripotent stem cells Stem Cell Rep 2018 10 655 72 10.1016/j.stemcr.2017.12.014