Direct reprogramming of terminally differentiated cells into neurons: A novel and promising strategy for Alzheimer's disease treatment

Progress in Neuropsychopharmacology & Biological Psychiatry 98 (2020) 109820

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Direct reprogramming of terminally differentiated cells into neurons: A novel and promising strategy for Alzheimer's disease treatment

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Hanie Yavarpour-Balia, Maryam Ghasemi-Kasmanb,c, , Amir Shojaeid a

Student Research Committee, Babol University of Medical Sciences, Babol, Iran Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran c Neuroscience Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran d Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Alzheimer's disease Glial cells Neural repair Reprogramming Neuron

Glial activation is a common pathological process of the central nervous system (CNS) in disorders such as Alzheimer's disease (AD). Several approaches have been used to reduce the number of activated astrocytes and microglia in damaged areas. In recent years, various kinds of fully differentiated cells have been successfully reprogrammed to a desired type of cell in lesion areas. Interestingly, internal glial cells, including astrocytes and NG2 positive cells, were efficiently converted to neuroblasts and neurons by overexpression of some transcription factors (TFs) or microRNAs (miRNAs). Notably, some specific subtypes of neurons have been achieved by in vivo reprogramming and the resulting neurons were successfully integrated into local neuronal circuits. Furthermore, somatic cells from AD patients have been converted to functional neurons. Although direct reprogramming of a patient's own internal cells has revolutionized regenerative medicine, but there are some major obstacles that should be examined before using these induced cells in clinical therapies. In the present review article, we aim to discuss the current studies on in vitro and in vivo reprogramming of somatic cells to neurons using TFs, miRNAs or small molecules in healthy and AD patients.

1. Introduction Alzheimer's disease (AD) is a neurodegenerative multifactorial syndrome, associated with clinically progressive loss of cognitive function and memory, as well as pathologic accumulation of amyloid plaques and neurofibrially tangles (NFTs) (Selkoe, 1999). AD is the sixth leading cause of death in the United States (US) and it has been shown that its incidence in the US will increase to 7.7 million cases in 2030 (Citron, 2004). The high global prevalence and the socioeconomic burden associated with the disease present major challenges for public health in the 21st century (Marchesi, 2005). Moreover, the multifactorial and complex nature of AD make it difficult for scientists to find an appropriate treatment that suppresses all or most of the major aspects of the disease. Genetics, age, environmental factors, metabolic dysfunction, and inflammation play important roles in the pathogenesis of AD (Ashford and Mortimer, 2002; Mortimer et al., 1998). However, the amyloid cascade, production and deposition of amyloid beta (Aβ) and phosphorylation of tau protein are the most well-documented hypothesis (Drachman, 2014; Marchesi, 2005). In this regard, researchers are trying to find substances that target amyloid cascades such as β-

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secretase inhibitor (BACE1), γ-secretase inhibitor or modulators and other factors that contribute to decreasing amyloid production and enhance its clearance (Huang and Mucke, 2012). The positive effects of these strategies have only been demonstrated in experimental studies, and in clinical trials are yet to be proceeded. Drugs that are available and approved for AD are mainly categorized into two groups; N-methylD-aspartate receptor (NMDAR) and acetylcholinesterase inhibitors (AchEI), which are used to reduce excitotoxity of glutamate and inhibit AchE activity in the synaptic cleft, respectively (Francis et al., 2005; Gómez-Isla et al., 1997). However, these pharmacological approaches could not be curative and their effects lessen over time. Therefore, discovering a novel approach with better efficacy has attracted considerable attention among researchers. Several lines of evidence have documented that considerable neuronal death occurs in AD, especially in the amygdala, basal forebrain, cortical area, and hippocampus (Bartus et al., 1982; Schliebs and Arendt, 2006; West et al., 1994; Whitehouse et al., 1982). Stem cell therapy is regarded as an ideal candidate for treating neurodegenerative disorders such as AD due to their ability in differentiating to different neuronal subtypes, such as cholinergic and GABAergic neurons, and their capacity to migrate to

Corresponding author at: Health Reserach Institute, Babol University of Medical Sciences, P.O.Box 4136747176, Babol, Iran. E-mail address: [email protected] (M. Ghasemi-Kasman).

https://doi.org/10.1016/j.pnpbp.2019.109820 Received 16 July 2019; Received in revised form 11 November 2019; Accepted 12 November 2019 Available online 16 November 2019 0278-5846/ © 2019 Elsevier Inc. All rights reserved.

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the different strategies for generating neurons both in vitro and in vivo. Terminally differentiated cells are converted to induced pluripotent stem cells (iPSCs), and after which they can be differentiated into neurons in vitro. In addition, somatic cells can be directly reprogrammed towards neurons using overexpression of TFs, miRNAs, or small molecules. Newly generated neuronal cells can also be achieved by differentiation of neural stem cells (NSCs) or neural progenitors (NPs) in vitro. In vivo approaches include enhancement of adult neurogenesis or stem cells transplantation. Moreover, glial cells, including astrocytes, microglia, and NG2 expressing cells, can be directly converted to neurons by overexpression specific TFs or miRNAs in vivo. In this review article, we highlight current reports on in vitro and in vivo direct reprogramming of normal and AD somatic cells towards neuronal cells using TFs, miRNAs, and small molecules.

the lesion site (Reubinoff et al., 2001; Zhang et al., 2001). Several studies have reported the enhancement of cognitive performance after neural stem cells (NSCs) transplantation. Moreover, these transplanted cells have been shown to reduce amyloid plaques, enhance their clearance, and attenuate other neuropathological aspects of AD. Interestingly, the ability of stem cells to be genetically modified, nominates them as a vehicle for delivering NGF. In this regard, autologous modified fibroblasts carrying NGF were implanted into the forebrain of an AD patient in a clinical trial study. Surprisingly, the results demonstrated attenuating AD aspects both in neuropathology and symptoms (Tuszynski, 2002; Tuszynski et al., 1990). Based on this ability of stem cells, their role as a vehicle for several therapeutic agents, such as cathepsin B (Sakamoto et al., 2008) and nepriliysin has also been evaluated (Blurton-Jones et al., 2014). Despite several advantages of cellbased therapy, some major concerns remain that limit the application of stem cells in clinical therapies. One of major concern is related to the source of stem cells. Stem cells can be obtained from the embryo, but there are some ethical concerns for using embryonic stem cells (ESCs) in clinical therapies. Additionally, heterologous stem cell is often associated with graft rejection (Nauta et al., 2006). Among the various strategies that have been experimented, the generation of induced pluripotent stem cells (iPSCs) from fully differentiated cells has been introduced as a promising strategy in regenerative medicine. For the first time, fibroblasts were reprogrammed to iPSCs by ectopic expression of four embryonic transcription factors (TFs), including Sox2, Oct4, Klf4, and c-Myc (Takahashi and Yamanaka, 2006). Subsequently, numerous studies used different sets of TFs to produce iPSCs. Despite the various advantages of iPSCs, transplanting these cells led to teratoma formation. Moreover, c-Myc, Oct4, and Klf4 are known as oncogenes and can integrate in the genome of host cells (Laurent et al., 2011; Lengner, 2010; Marchetto et al., 2009). To overcome these limitations, several research groups focused on transdifferentiating somatic cells to a desired type of cell, without passing through the pluripotent stage (Abeliovich and Doege, 2009; Zhou et al., 2008). Numerous reports showed that different types of somatic cells can be effectively reprogrammed to neuronal cells (Fig. 1). It has been demonstrated that by overexpressing some specific TFs and microRNAs (miRNAs) or applying of small molecules, fully differentiated cells were successfully converted to neurons. Fig. 2 illustrates

2. Direct conversion of terminally differentiated cells to neurons using TFs Previous reports showed that TFs are major regulators of both early and late aspects of neuronal identity (Lodato and Arlotta, 2015; Lodato et al., 2014). Currently, several efforts have been made to reprogram somatic cells to neurons using TFs. Vierbuchen et al., employed a set of three important TFs, including Ascl1 (Mash1), Brn2 (Pou3f2), and Mytl1, that successfully produced induced-neural (iN) cells from mouse embryonic and postnatal fibroblasts. Interestingly, Ascl1 alone was sufficient to convert fibroblasts into iN cells. The iN cells expressed various neuronal markers and had the ability for firing action potentials and forming functional synapses with neighboring neurons (Vierbuchen et al., 2010). Moreover, functional neurons were generated from human fibroblasts using a basic helix-loop-helix TFs, including NeuroD1, Ascl1, Brn2, and Mytl1. Although a high similarity between induced and endogenous neurons was observed, but iN cells could not generate functional synapses. Interestingly, by introducing NeuroD1 to these three TFs, both fetal and postnatal human fibroblasts were successfully converted into functional iN cells. The iN cells displayed typical neuronal morphologies and expressed some specific neuronal markers. Additionally, the induced cells formed functional synapses and were integrated into pre-existing neuronal networks (Pang et al., 2011). Xu et al. used a combination of four TFs to convert primary fibroblasts to induced serotonergic (i5HT) neurons. After screening a variety of TFs, that were involved in the generation of 5HT positive neurons or direct conversion of fibroblasts to iN cells, they identified a combination of four TFs, including Ascl1, Lmx1a, FoxA2, and FEV, that could directly convert human fibroblasts to i5HT expressing cells. The induced serotonergic neurons exhibited spontaneous electrophysiological activity and had active serotonergic synaptic transmission. Furthermore, the reprogrammed cells displayed Ca2+-dependent release and selective uptake of serotonin. Interestingly, the process of reprogramming had significantly increased through p53 knockdown and hypoxia (Xu et al., 2016). A previous study indicated that overexpression of Brn4/Pou3f4, c-Myc, Klf4, Sox2, and E47/Tcf3 led to generation of iNSCs. The iNSCs were identical to the brain-tissue derived NSCs in several aspects such as morphology, gene expression profile, epigenetic status, self-renewing, and differentiation capacity. Furthermore, up-regulation of posterior genes and down-regulation of anterior genes markers showed a posterior regionalization of the iNSCs. Interestingly, the iNSCs differentiated to neurons, astrocytes, and oligodendrocytes when transplanted into the mouse subventricular zone (Han et al., 2012). In addition, adult dermal fibroblasts directly converted into NPC-like cells (iNPCs) under the ectopic expression of five TFs, which were Pou3f2, Bmi1, Sox2, Nr2e1, and c-Myc. The iNPs possess similar properties to primary NPCs, such as proliferation, self-renewal, and differentiation. Moreover, among these TFs, Bmi1 was essential for the direct conversion of dermal fibroblasts into iNPCs (Tian et al., 2012). Neuronal restricted progenitors (NRPs), also known as neuroblasts, are considered as another source of cells suitable for reprogramming (Mayer-Proschel

Fig. 1. Direct reprogramming of different types of somatic cells to neuron. 2

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Fig. 2. Different approaches for generating a neuron.

following CNS injuries (Göritz et al., 2011). In this regard, Karow et al. reported a direct conversion of adult human pericytes to human pericyte-derived induced neuronal cells (hPdiNs) using retrovirus-mediated co-expression of Mash1 and Sox2. Surprisingly, the iN cells had the ability to fire action potentials and were integrated into neural networks. Interestingly, most fibroblasts had ability to generate iN cells with glutamatergic, dopaminergic, or cholinergic identity, whereas hPdiNs mostly exhibited GABAergic phenotypes. This data suggested that the nature of the initial cell plays a crucial role in the specific neuronal subtype that is generated (Karow et al., 2012). iNPCs were also successfully generated from neonatal and adult peripheral blood using Oct4. The blood-derived (BD)-NPCs had the ability for survival, as well as tripotent neural differentiation in vitro, and efficiently differentiated into neurons. Additionally, iNPCs produced glial lineage cells and other neuronal subtypes (Lee et al., 2015). Sheng et al. reported that sertoli cells could be directly reprogrammed into multipotent neural stem/progenitor cells by overexpressing nine TFs, specifically cMyc, Brn2, Hes1, Pax6, Ascl1, Id1, Ngn2, Sox2, and Klf4. The iNSCs showed various similarities with normal neural stem cells (NSCs) including gene expression profile, self-renewing and their potency for differentiating into glial lineage cells and neurons. Furthermore, these iN cells could survive and formed synapses after being transplanted into the dentate gyrus region of the hippocampus. Interestingly, growing lines of evidence suggest that non-neuronal cells can also be converted into distinct neuronal subtypes in vivo (Buffo et al., 2005; Grande et al., 2013; Niu et al., 2013; Ohori et al., 2006; Sheng et al., 2012). Torper et al. showed that using viral delivery of Ascl1, Brn2a, and Myt1l, fully differentiated mouse and human cells were converted to neurons in vivo. Furthermore, iNs successfully achieved from endogenous mouse astrocytes using viral delivery (Torper et al., 2013). It has been demonstrated that astrocytes can be converted to functional neurons by expressing a single TF named Ascl1, both in vitro and in vivo. Liu et al. showed that postnatal dorsal midbrain

et al., 1997; Yang et al., 2000). The positive aspect of NRPs is their ability for converting to neurons, but not glial lineage cells (Mo et al., 2007). To generate human-induced neuronal restricted progenitors (hiNRPs) from human fetal fibroblasts, Zou et al. used c-Myc, Sox2, and either Brn2 or Brn4. The hiNRPs displayed considerable neuronal characteristics such as morphology, expression of neuronal specific markers, self-renewal ability, and a genome-wide transcriptional outline. The results of this study showed that during reprogramming, Sox2 and c-Myc convert fibroblasts to progenitor cells with proliferation capacity, while Brn2 or Brn4 has a role in differentiating induced cells to neuronal cells. The hiNRP cells had the capability to differentiate into various functional neuronal subtypes, including dopaminergic (DA), cholinergic, GABAergic, and serotonergic neurons (Zou et al., 2014). Caiazzo et al. demonstrated a direct conversion of fibroblasts to DA neurons using forced expression of three TFs, namely Mash1, Lmx1a, and Nurr1 (Nr4a2). Reprogrammed cells were identical to brain-derived DA neurons in gene expression and electrophysiological activity. The induced neurons released dopamine and could be modulated via D2 receptors (Caiazzo et al., 2011). Similarly, a set of five TFs, that is, Sox2, Mash1, Pitx3, Ngn2, and Nurr1, were employed to reprogram human fibroblasts into DA neuron-like cells. The induced cells expressed various DA neuron markers and showed DA neuron-specific electrophysiological properties (Liu et al., 2012). In addition to fibroblasts, other cell sources have also been used as initial cells for reprogramming. In another report, human fetal neural stem cells were directly converted to iPSCs using Oct4 overexpression. The induced cells resembled human embryonic stem cells in regard to their epigenetic status, pluripotency, and global gene expression profiles in vitro and in vivo. In comparison with iN cells, which are directly generated from fibroblasts, induced NPCs were expanded in vitro and had maintained their ability to differentiate into different neuronal subtypes and glial lineage cells (Kim et al., 2009). Recently, local pericytes have been introduced as a major proliferating cell source in glial scar formation 3

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renewal of NSCs (Rybak et al., 2008; Zhao et al., 2010). MiRNAs target high-mobility group A2 (HMGA2) protein promote rapid and efficient formation of human induced neural stem cells (hiNSCs), and facilitate direct conversion of human senescent cells and blood cells to other desired cells (Nishino et al., 2008; Yu et al., 2015). It has been reported that inhibiting let-7 or expression of its target as HMGA2 enhances Sox2-mediated direct reprogramming of old differentiated cells, human adult fibroblasts, and blood cells to iNSCs (Victor et al., 2014). It has been shown that decreased expression of a single RNA binding polypyrimidine-tract-binding (PTB) remarkably enhances differentiation or direct conversion of different cell types to neuronal cells. In a previous study, Xue et al. demonstrated a reprogramming of different cell types to neuronal-like cells in PTB-depleted cells (Xue et al., 2009). In another study, Xue et al. illustrated that massive reprogramming towards generation of neuronal cells occurs by regulating PTB expression (Xue et al., 2013). Direct reprogramming of fibroblasts to striatal neurons was achieved using four TFs, CTIP2, MYT1L, DLX1, and DLX2. Interestingly, these TFs synergize with miR-9/9-124 to form striatal medium spiny neurons (MSNs). The MSNs efficiently survived and could be integrated into the local neural circuit after being transplanted into murine striatum (Victor et al., 2014). It has been well documented that miR-124 plays a crucial role in the differentiation of adult neural progenitor cells and is regarded as one of the most abundant miRNAs in the brain (Lagos-Quintana et al., 2002). Liu et al. showed that miR-124a overexpression significantly decreased the proliferation of neural progenitor cells and increased the neuronal differentiation in vitro. It has been demonstrated that Notch signaling pathway plays an important role in neurogenesis in both physiological and pathophysiological conditions. Surprisingly, miR-124a repressed the expression of Notch singling's ligand as Jagged-1 (JAG1) in neural progenitor cells derived from SVZ of adult rats. In addition, overexpression of miR-124a decreased the levels of JAG1 and led to the inactivation of Notch signals in neural progenitor cells. Furthermore, the level of p27Kip1, a downstream target gene of the Notch signaling pathway, was markedly upregulated in miR-124a transfected cells (Liu et al., 2011; Papagiannakopoulos and Kosik, 2009). In another study, it was reported that transfection of non-neuronal HeLa cells by miR-124 and miR-1 shifted the miRNA profile towards neuronal pattern and suppressed the expression of non-neuronal genes, such as small C-terminal domain phosphate 1 (SCP1) in induced cells (Lim et al., 2005). According to previous in vitro studies, miR-124 mediates neuronal differentiation (Krichevsky et al., 2006; Makeyev et al., 2007). It has been shown that during CNS development, timely down-regulation of SCP1 is essential for neurogenesis and miR-124 increases the neurogenesis process at least in part through down-regulating SCP1 (Visvanathan et al., 2008). SRY-box TF (Spox9) is another target of miR-124. It has been demonstrated that overexpression of Sox9 in the SVZ of adult mice abolishes neuronal differentiation, while neuronal formation increases following knock down of Sox9. Furthermore, it has been shown that miR-124 is regarded as an important moderator of neurogenesis in adult mice. Overexpression of miR-124 maintained the proliferation capacity of SVZ cells and promoted the neuronal cell differentiation. Conversely, neuronal regeneration was delayed following knocking down of miR-124 (Cheng et al., 2009). Overexpression of miR-124 and miR-137 induced neuronal differentiation in mouse oligodendroglioma tumor stem cells (mOSCs) and GBM stem cells. Interestingly, an upregulation in miR-137 and miR-124 has been observed during neuronal differentiation of adult mNSCs. It has been demonstrated that miR-137 and miR-124 inhibit the proliferation capability of GBM cell lines by promoting G0/G1 cell cycle arrest and inhibiting growth-factor-derived hGSCs. Additionally, miR-124 and miR-137 suppressed levels of cyclindependent kinase 6 (CDK6) and CDK6 protein, and phosphorylated retinoblastoma in GBM cells (Silber et al., 2008). It has been well documented that miR-302 family possesses a crucial role in regulating pluripotency and differentiation in hESCs. Furthermore, at the transcription level, Oct4 and NR2F2 (COUP-TFII) are pivotal for

astrocytes could be successfully converted to functional glutamatergic or GABAergic neurons. The induced neurons were mostly capable of generating action potentials. Furthermore, when GFAP-adeno associated virus (AAV) Ascl1 expressing vector was used to infect astrocytes in somatosensory cortex, striatum, and dorsal midbrain of postnatal and adult mice, iN cells scored positive for expression of neuronal markers and their action potential firing pattern was identical to endogenous midbrain neurons. The iN cells also efficiently formed synapses with neighboring neuronal cells (Liu et al., 2015). Niu et al. have achieved a novel strategy to produce induced adult neuroblasts (iANBs) from astrocytes. They suggested that endogenous Sox2 is not sufficient for fate change and self-regeneration of astrocytes; however, astrocytes could be directly converted into DCX positive cells using ectopic expression of Sox2. Nevertheless, induced cells rarely become mature in physiological conditions. Interestingly, exogenous expression of brain-derived neurotrophic factor (BDNF) and noggin (NOG) remarkably improved neuronal survival and differentiation potency (Niu et al., 2013). In addition, independent evidence from another study showed the capacity of Sox2 for inducting glia-to-neuron fate switch. In this study, NG2 expressing cells were selected for reprogramming to neural cells. It has been shown that retroviral expression of Sox2 or in combination with Ascl1 induces neurogenesis in the cerebral cortex following traumatic brain injury in adult mice. The induced cells were morphologically mature and became negative for DCX. Despite the ability of Sox2 to generate neurons in the lesion area, overexpression of Sox2 failed to convert oligodendrocytes and astrocytes to DCX positive cells in the unlesioned cortex (Heinrich et al., 2014). Glioblastoma multiform (GBM) originates from glial lineage cells in the brain (Ostrom et al., 2016). In order to prevent the invasive proliferation of GBM-forming cells, reprogramming glial lineage cells into neuronal cells has emerged as an effective therapeutic strategy in GBM (Carén et al., 2015; Guichet et al., 2013). In this regard, Cheng et al. used a single TF, Ascl1, to convert glioma cells into terminally neural cells. Forced expression of Ascl1 resulted in generation of tubulin beta-3 (TUBB3) positive neural cells and a heterogeneous population of excitatory and inhibitory neurons. In order to achieve similar results in vivo, the Ascl1-infected glioma cells were transplanted into mouse striatum. The results suggested that terminally differentiated neurons could be generated in vivo, and that induced cells inhibited tumor growth (Cheng et al., 2019). Table 2 illustrates some current literatures on in vivo reprogramming of terminally differentiated cells to neuronal cells. 3. Direct reprogramming of terminally differentiated cells to neurons using microRNAs MicroRNAs (miRNAs) play an important role in the process of gene regulation when they are endogenous and short non-coding RNAs. MiRNAs modulate various cellular processes using RNA interferencebased mechanisms (Silber et al., 2008; Yang et al., 2017). The application of viral particles for overexpression of reprogramming factors leads to some integrations in the host genome that limits the usage of viral particles in clinical therapies (Ghasemi-Kasman et al., 2015). Recently, microRNAs have appeared as an alternative strategy in direct reprogramming. Since a single miRNA can simultaneously target multiple pathways, their repressive action on gene expression process is powerful. Furthermore, cultured cells are easily incubated with chemically synthesized miRNAs (miRNA mimics) through lipid-based transfection and display low toxicity in vivo. Additionally, due to the small size of a single miRNA, packing multiple transcripts in the same delivery vector enhances the efficiency of reprogramming as well as the functional homogeneity of the converted cells (Jayawardena et al., 2012). Overall, the use of miRNAs seems to be a safer method for the goal of using reprogrammed cells in clinical therapy. Here, we present studies on microRNAs-mediated direct conversion of somatic cells to neurons or neuroblasts in vitro and in vivo. It has been reported that let-7 has a role in specification and self4

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in the process of neural conversion. NG2N also acted as a pioneer factor and FD altered the epigenetic state of the cells (Smith et al., 2016). A chemical cocktail containing SP600125, CHIR99021, rolipram, NaB, LPA, rolipram, and A83–01 in combination with Oct4 could reprogram adult dermal fibroblasts (AHDF) to hiNSCs colonies expressing PAX6, PLZF, and OTX2. Moreover, transduced cells expressed Ki67, a proliferation marker, and growth rate was comparable to control hNSCs. Furthermore, results demonstrated the close similarity between the global gene expression profile of hiNSCs and control group. Interestingly, induced cells could be differentiated to mature neurons and spontaneous excitatory post-synaptic current was observed. In vivo study showed that the transplanted cells could express neuronal lineage markers and a subset of cell clusters were positive for an astrocyte marker (Zhu et al., 2014). A combination of four small molecules as SB431542, FSK, CHIR99021, and ISX9 converted mouse fibroblasts to neuroblasts and neural cells. It has been demonstrated that these cocktails of small molecules performed direct conversion by activating two TFs, NeuroG2 and NeuroD1. Chemically induced neurons (CiNs) exhibited gene expression pattern consistent with functional neurons. Interestingly, CiNs had electrophysiological properties and functional synapses. Moreover, ISX9 was necessary for activating neuronal-specific genes (Li et al., 2015). VPA, CHIR99021, and Repsox (VCR) are other components of chemical cocktail that reprogrammed astrocytes directly into neurons in the culture. It has been shown that chemical conversion is mediated by activating NeuroG2 and NeuroD1 TFs in astrocytes. Surprisingly, these chemical cocktails could convert adult mice astrocytes to neurons in vitro (Cheng et al., 2015). However, these components of small molecules could not induce obvious changes on cultured human astrocytes. In this regard, Gao et al. treated cultured astrocytes with three small molecules, ISX-9, FSK, and iBet151, all of which are frequently used in neuronal differentiation and reprogramming. These different sets of small molecules generated induced cells that were similar to typical neurons and expressed neuronal markers. Importantly, the induced cells displayed electrophysiological activities and formed functional synaptic connections. In addition, genome-wide transcriptome profiling of iN cells after transplantation into mouse brains was consistent with human embryonic stem cell-derived neurons. Furthermore, the engrafted cells survived and fired action potentials (Gao et al., 2017). It has been reported that a defined set of nine chemical compounds, including Puro, CHIR99021, SAG, DAPT, Tzav, VPA, TTNPB, SB431542, and LDN193189, directly reprogrammed human astrocytes into neurons after sequential administration of the compounds. Interestingly, small molecules-reprogrammed-human neurons were survived for more than 5 months in culture condition and displayed functional synaptic activities. Furthermore, iN cells were integrated into brain circuits and survived for more than 1 month (Zhang et al., 2015). Han et al. used another combination of small molecules, which included A83-01, PD0325901, CHIR99021, vitamin C, RG108, BIX-01294, and VPA, to convert mouse embryonic fibroblasts to NSCs. The morphology, self-renewal, gene expression pattern, and excitability of small molecule-induced neural stem (SMINS) cells were similar to endogenous NSCs. The induced cells could also be differentiated to astrocytes, neurons, and oligodendrocytes in vitro and in vivo. It has been shown that PD0325901, RG108, and BIX-01294 were necessary for efficient reprogramming of mouse embryonic fibroblasts (Han et al., 2016). Pfisterer et al. used a combination of six small molecules containing PP2, BML210, FSK, amino resveratrol sulfate, PGE2 and kenpaullone to promote the efficiency of reprogramming. These small molecules trigger multiple signaling pathways, which had previously been reported to enhance of the efficiency of reprogramming in iPSCs and hiNs (Pfisterer et al., 2016). Lee et al. used a chemical cocktail containing DAPT, I-BET 151, ISX9, CHIR99021, and FSK, to directly convert U87MG human glioblastoma cells into terminally differentiated neurons. Small molecules-treated U87MG glioblastoma cells could be successfully reprogrammed into early neurons. The morphology and immunofluorescent characteristics of reprogrammed cells were similar

maintaining an undifferentiated state. A study of interactions between miR-302 and these two TFs in pluripotent hESCs revealed that Oct4 transcriptionally represses NR2F2 gene, but this gene can be activated when Oct4 is unbounded from the promoter. In addition, Oct4 activated miR-302 post-transcriptionally represses the NR2F2 gene. Functional analysis has demonstrated that NR2F2 has an important role in the proper induction of different neural genes, such as Pax6, during initial specification of neural ectoderm. In order to shift cells towards a specific cell fate, NR2F2 decreases the level of Oct4 during differentiation (Rosa and Brivanlou, 2011). Transfection of adult SVZ-derived NSCs with miR-137 enhanced neuronal differentiation and no effect on astrocyte differentiation was observed (Silber et al., 2008). MiR-137 also promotes proliferation and inhibits differentiation of adult hippocampal NSCs. The possible function of miR-137 was also evaluated in embryonic brains in an in vivo report (Szulwach et al., 2010). Results indicated that miR-137 has a significant role in controlling the fate of determination and accelerating neural differentiation of embryonic NSCs. Interestingly, a premature differentiation and outward migration was observed in transduced NSCs in the ventricular zone of embryonic mouse brain following in utero electroporation of miR-137. It has been shown that TLX, an upstream regulator of miR-137, recruits LSD1 to the genomic regions of miR-137 and represses the expression level of miR137 (Shi et al., 2004; Sun et al., 2010). It has been postulated that regulatory role of miR-137 in neural differentiation is mediated by repressing LSD1 expression (Sun et al., 2011). MiR-302/367 cluster has been introduced as one of the most highly expressed miRNAs in pluripotent stem cells (Abad et al., 2013; Anokye-Danso et al., 2011; Lu et al., 2014). Ghasemi-Kasman et al. reported a direct conversion of astrocytes to neuroblasts using miR-302/367 both in vitro and in vivo (Ghasemi-Kasman et al., 2017; Ghasemi-Kasman et al., 2015). The results of this study indicated that miR-302/367, in conjugation with valproic acid (VPA), converted human astrocytes to neuroblasts, which could potentially produce neurons. Moreover, a remarkable population of neuroblasts and neurons were observed around the injection site. Additionally, no expression levels of pluripotency markers were detected, which suggest that direct reprogramming occurred without passing through pluripotency stage (Ghasemi-Kasman et al., 2015). 4. Direct reprogramming of terminally differentiated cells to neuron-like cells using small molecules In recent years, small molecules have emerged as a novel strategy in somatic cell reprogramming (Ichida et al., 2009; Li et al., 2009; Shi et al., 2008; Staerk et al., 2011; Zhu et al., 2010). In this approach, small molecules activate signaling pathways that are associated with neural TFs. It has been shown that small molecules can directly convert fully differentiated cells to neurons (Hou et al., 2013; Li et al., 2015). In this section, we have reviewed some current literatures on direct conversion of terminally differentiated cells to neurons using small molecules. A previous report indicated that the combination of neurogenin 2 (NG2N), a key TF in neurogenesis, and two chemical compounds, that is, dorsomorphin (DM) and forskolin (FSK), efficiently reprogrammed human fetal lung fibroblasts to cholinergic neurons. FSK could generate neuronal-like cells in a dose-dependent manner and DM could synergize with FSK to increase the survival and maturation of neurons. Importantly, DM independently had no significant effect on the conversion of somatic cells to neuronal-like cells. Interestingly, the results indicated that while FSK and DM induce rapid morphological changes, the morphology or proliferation of cultured fetal fibroblasts did not remarkably change in NGN2 incubated cells. Furthermore, adult human skin fibroblasts were also converted to cholinergic neurons by introducing SOX11 and FGF2 growth factors. Similarly, Smith et al. performed a systematic analysis of the individual and synergistic actions of NEUROG2 and small molecules including FSK and DM during reprogramming. It was demonstrated that FD (forskolin and dorsomorphin) could improve chromatin occupancy and H3K27 acetylation 5

GABAergic GABAergic and glutamatergic

Fibroblasts

6

Induced neural progenitor cells (iNPCs) iNPCs➔ differentiated in vivo to glial cells (astrocytes and oligodendrocytes) and multiple neuronal subtypes including dopaminergic and nociceptive neurons multipotent neural stem/progenitor cells (iNSCs) iNSC➔ differentiated into astrocytes, oligodendrocytes, and different subtypes of neurons: dopaminergic, GABAergic, cholinergic) ➔ most of induced neurons were GABAergic Engrafting cells into mouse dentate gyrus ➔ cells survived, migrated, and differentiated into neurons

Peripheral blood cells

Sertoli cells

GABAergic

Pericytes

Dopaminergic (DA)

In vivo➔neurons Dopaminergic (DA)

hiNRPs ➔ neuronal cells (cholinergic, serotonergic, dopaminergic, and GABAergic)

Human induced neuronal restricted progenitors (hiNRPs)

Induced neural progenitor cells (iNPCs)

iNSC➔ astrocytes, oligodendrocytes, and Neurons (GABAergic, glutamatergic, cholinergic, and dopaminergic)

Induced neural stem cells (iNSCs)

Induced serotonergic (i5HT)

GABAergic, glutamatergic, and Catecholaminergic neurons

Induced neuron

Initial cells

Transcription factor-based reprogramming

Table 1 In vitro reprogramming of somatic cells to neuronal cells.

NeuN, Synapsin

Nestin, Pax6, Olig2, Sox2, DCX NeuN, MAP2, GABA, Synapsin

PAX6, Nestin, Sox2, CD133 Neuronal markers (Nurr1, Tuj1, MAP2, and NeuN) Astrocytes marker (GFAP)

Tuj1, MAP2

Tuj1, MAP2, Syn1, Neurofilament light subunit, Neuron-specific enolase NeuN Tuj1, Mash1, Sox2, Ngn2, Nurr1, Pitx3 Various DA neuron-specific markers (TH, DDC, DAT) DA markers (VMAT2, ALDH1A1, calbindin and DAT), Tuj1

Nestin, DCX

Tuj1, MAP2 Synapsin, vGLUT1, GABA, Tbr1, MAP2, NeuN Tbr1, Peripherin, vGLUT1, vGLUT2, β-IIItubulin, DCX, MAP2, NCAM, Synapsin Neuronal markers Tuj1, Sertonergic markers (AADC, ALDH1A1, SERT, TPH2, VMAT2) NSCs markers (Sox2, Mash1/ Ascl1) Some of which expressed fibroblasts markers (Ctgf, Acta2) Neuronal markers (Tuj1, Dcx) Astrocytes markers (GFAP, S100β), oligodendrocytes marker (NG2 CXCR4 (chemotactic protein) Nestin (NPC marker)

Neural markers

Ascl1, Ngn2, Hes1, Id1, Pax6, Brn2, Sox2, c-Myc, Klf4

Oct4

Mahs1, Sox2

Mash1, Nurr1, lmx1a

Mash1, Ngn2, Sox2, Nurr1, and Pitx3

Sox2, c-Myc, Brn2 or Brn4

Pou3f2, Nr2e1, Sox2, cMyc, Bmi1

Obtained from 5-day-old pups

Neonatal and adult peripheral blood cells

Prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients, Mouse embryonic fibroblasts Pericytes of adult human brain

Lateral ventricle Human fibroblasts

Human fetal fibroblasts

Human fetal fibroblasts

Adult dermal fibroblasts

Sheng et al. (2012)

Karow et al. (2012) Lee et al. (2015)

Caiazzo et al. (2011)

Liu et al. (2012)

Zou et al. (2014)

Tian et al. (2012)

Han et al. (2012)

Xu et al. (2016)

Pang et al. (2011)

Vierbuchen et al. (2010)

Ref.

(continued on next page)

Expressed multiple NSCs-specific markers, selfrenewal, and differentiation into glial cells, electrophysiological activity, iNSCs could survived and generated synapses following transplantation into the dentate gyrus

Fired repetitive action potential, exhibited neuronal morphology Produced iNPCs and differentiated into neurons exhibiting the functional membrane properties and activities of mature neurons.

Induced DAs exhibited gene expression similar to endogenous DA, releasing dopamine, and had pacemaker activity modulated via D1 receptors

Exhibited DA neuron-specific electrophysiological profiles, showed DA uptake

Exhibited similar proliferation, self-renewal and differentiation, and chemotactic properties similar to primary NPCs. Exhibited distinct neuronal characteristics, including cell morphology, multiple neuronal marker expression Self-renewal capacity, and a genome-wide transcriptional profile and capability for differentiation into various functional neurons.

Human primary fibroblasts

Ascl1, Foxa2, Lmx1b, FEV

Mouse fibroblasts

Displayed typical neuronal morphologies, expressed multiple neuronal markers, generated functional synapses. Expressed markers for mature serotonergic neurons, had Ca2+-dependent 5HT release and selective 5HT uptake, exhibited spontaneous action potentials and spontaneous excitatory postsynaptic currents. Exhibited cell morphology, gene expression, epigenetic features, differentiation potential, and self-renewing capacity, as well as in vitro and in vivo NSCs.

Primary human fetal/postnatal fibroblasts

Brn4/Pou3f4, Sox2, Klf4, c-Myc, E47/Tcf3

Express multiple neuron-specific proteins, generated action potentials, formed functional synapses

Major outcomes

Mouse embryonic and postnatal fibroblasts

Environment

Ascl1, Brn2, Myt1l Brn2, Myt1l, Zic1, Olig2, Ascl1 Ascl1, Brn2, Mytl1, NeuroD1

Reprogramming factors

H. Yavarpour-Bali, et al.

Progress in Neuropsychopharmacology & Biological Psychiatry 98 (2020) 109820

GABAergic and glutamatergic neurons

Glioma cells

DCX, Tuj1, NeuN

Neural cells

Neural cells

Neuroblasts

Human embryonic stem cells

Mouse embryonic neural stem cells Adult human astrocytes

7

Mouse embryonic fibroblasts

Human astrocytes

Sox2 Astrocytes marker (GFAP), neural markers (MAP2, NeuN)

VPA, BIX-01294, RG108, PD0325901, CHIR99021, vitamin C, A83–01

LDN193189, SB431542, TTNPB, Tzv, CHIR99021, VPA, DAPT, SAG, Purmo

DCX, MAP2, NeuN, Tuj1, SYN1, Tau

Neurons (mainly glutamatergic neurons and 70% positive for VGULT1), some of them were cholinergic neurons Transplanted into the lateral ventricle of postnatal mice ➔ survived and were functional Functional neurons (mainly glutamatergic neurons) and some fraction of which was GABAergic (GAD67) Injecting these iN into mouse brain➔ were functional and survived Neural stem cells (NSCs) DCX, Tuj1, MAP2, NeuN

VCR cocktail (Valproic acid, Chir99021, and Repsox) and Forskolin, i-Bet151, and ISX-9

DCX, Tuj1, NeuN

Mouse fibroblasts

A83–01, CHIR99021, NaB, LPA, rolipram, SP600125 in combination with ectopic expression of Oct4

Forskolin (FSK) and dorsomorphin (DM) in combination with SOX11

Forskolin and dorsomorphin

Neurons and neuroblasts

hiNSCs ➔ transplanted into mouse brain ➔ survived, migrated, developed to GABAergic and glutamatergic neurons Glutamatergic (45.8%), GABAergic (20.8%)

Astrocytes from 1-day postnatal mouse brain Human astrocytes

Human induced neural stem cells (hiNSCs)

Postnatal and adult skin fibroblasts from healthy and diseased human patients Adult dermal fibroblasts

Motor neuron markers (HB9, ISL1/2) ChAT, VAChT Pan neuronal marker (MAP2), presynaptic marker (SYT1), early motor neuron marker (HB9), Tuj1, ChAT PAX6, PLZF, OTX2, PAX6, Nestin, Sox1, Proliferation marker: Ki67 Tuj1, DCX, NeuN

Reprogramming factors

miR-302/367 cluster in conjugation with VPA

miR-302 linked with two TFs (Oct4 and NR2F2) miR-137

Han et al. (2016)

Zhang et al. (2015)

Li et al. (2015) Cheng et al. (2015) Gao et al. (2017)

Zhu et al. (2014)

(continued on next page)

Up regulated ➔ Dll1, Notch2, Hey1, and Pou3f3 (involved in the Notch signaling pathway), Shh (in the

Inhibited Notch signaling, GSK3β, and BMP/TGFβ signaling pathways, Down-regulated GFAP transcriptional level, increased NGN2 transcriptional level

↑ Expression of NGN2, ASCL1, MAP2, SYN1 ↓ Expression of astrocytes genes S100β, ALDH1L1, and GFAP

↑ Expression of NeuroG2 and NeuroD1

↑ Expression of NeuroD1 and Ngn2

↑PAX6 expression

Liu et al. (2013)

Ref.

Ghasemi-Kasman et al. (2015)

Sun et al. (2011)

Rosa and Brivanlou, 2011

Xue et al. (2013)

Increased NEUROG2 chromatin binding intensity

Major mechanisms

PTB-regulated expression of REST cofactor SCP1 (activation of miR-124/REST) MiR-302 fine-tuned the balance between Oct4 and NR2F2 Formed a feedback regulatory loop with TLX and LSD1 Expressed neuron-specific proteins, generated action potentials

Victor et al. (2014)

↑ The expression of CTIP2, DLX1, DLX2, MYT1L (CDM)

Cheng et al. (2019)

Ref.

Ref.

Terminally differentiated into neurons

Major outcomes

Major mechanisms

Forskolin, ISX9, CHIR99021, SB431542 VPA, CHIR99021, Repsox

Induced cholinergic neurons (hiCN)

Human fetal lung fibroblasts

Neural marker

Synapsin1, vGLUT1, NeuN, Tuj1 Pax6

MiR-9/9, miR-124 Transcription factors (CTIP2, DLX1, DLX2, and MYT1L) miR-124

Reprogramming factors

Mouse striatum

Environment

MAP2, NF-H, GAD67, VGLUT1

Induced neurons

Initial cells

Small molecules-based reprogramming

Tuj1

Neurons analogous to striatal medium spiny neurons (MSNs) In vivo differentiation ➔ GABAergic GABAergic

Fibroblasts

Ascl1

Reprogramming factors

EGFP, MAP2, DARPP-32, FOXP1, CTIP2

Induced neurons

Neural marker

Tuj1, MAP2, DCX

Neural markers

Initial cells

miRNAs-based reprogramming

Induced neuron

Initial cells

Transcription factor-based reprogramming

Table 1 (continued)

H. Yavarpour-Bali, et al.

Progress in Neuropsychopharmacology & Biological Psychiatry 98 (2020) 109820

Progress in Neuropsychopharmacology & Biological Psychiatry 98 (2020) 109820

H. Yavarpour-Bali, et al.

Triggered CamK activity, derepressed myocyte enhancer factor-2 (MEF2) Shh signaling pathway), Bmp2 and Bmp15 (in the BMP signaling pathway) Down regulating of ➔ Hey2 and Heyl (involved in the Notch signaling pathway), Nog (in the BMP signaling)

Ref.

5. Direct conversion of fully differentiated cells to neuron-like cells in Alzheimer's disease

Forskolin, ISX9, CHIR99021 I-BET 151, DAPT

Direct reprogramming of terminally differentiated cells of patient's to neurons has been introduced as a promising therapeutic strategy in AD (Choi and Tanzi, 2012; Qiang et al., 2011; Sproul et al., 2012). Guo et al. showed that overexpression of NeuroD1 in cortical reactive glial cells of stab-injured or AD animal model generated neurons. Surprisingly, astrocytes were converted into glutamatergic neurons while transfected NG2 positive cells were reprogrammed to both excitatory and inhibitory neurons. Further analysis showed that induced cells could be successfully integrated into local neural circuits. Furthermore, it has been shown that overexpression of NeuroD1 converted cultured human cortical astrocytes into neuron-like cells (Guo et al., 2014). In another study, Hu et al. used seven small molecules including Y-27632, VPA, GO6983, SP600125, Repsox, FSK, and CHIR99021 to reprogram fibroblasts to human chemical-induced neurons (hciNs). Microarray analysis revealed the similarity of gene expression patterns in hciNs and control neurons. Furthermore, fibroblasts from four familial AD patients (FAD) could be successfully converted into hciNs using the small molecules mentioned above (Hu et al., 2015). To decipher the possibility of miR302/367-based conversion in AD, miR-302/367 cluster was injected into the hippocampus. Behavioral analysis showed the enhancement of working and spatial memory in miR-302/367 + VPA treated mice in experimental model of AD. Furthermore, immunostaining results indicated that the majority of transfected astrocytes expressed NeuN, a mature neuron marker. Interestingly, the membrane of iN cells had electrophysiological properties similar to endogenous neurons (Ghasemi-Kasman et al., 2018). Table 3 depicts the current reports on direct conversion of differentiated cells to neuron-like cells in AD.

6. Conclusions Notably, a wide range of somatic cells have been successfully converted to neurons by overexpression of specific TFs or miRNAs, as well as exposure of cultured cells to appropriate small molecules. Recently, direct conversion of resident glial cells to neuron-like cells has emerged as a promising strategy in regenerative medicine. Besides normal cells, fully differentiated cells from AD patients have also been reprogrammed to functional neurons. Despite the remarkable advantages of direct reprogramming, there are still several major issues that should be addressed before using reprogrammed neurons in clinical therapies. Reprogramming factors are mostly introduced to the starting cells using viral particles that significantly increase the risk of mutagenesis in the host genome. Additionally, the efficiency of reprogramming and the number of reprogrammed neurons is frequently low. It has been postulated that by reducing the number of TFs and applying some small molecules, the safety and efficiency of reprogramming will likely increase. Furthermore, other major concerns are whether newly converted cells can survive for a long time and efficiently integrate into local host circuits. The ability of iN cells in firing repetitive action potentials and their potency in functional recovery following brain injuries are also considered as main obstacles that should be solved by further studies. Nevertheless, generation of patient-subtype-specific neurons by in situ conversion of internal reactive glial cells may provide a promising cell source for improving neural repair in the CNS.

Lee et al. (2018)

Human glioblastoma cells

Oligodendrocyte marker (Olig2)

NSCs transplanted into lateral ventricle of nude pups➔ oligodendrocytes, neurons, and astrocytes Terminally differentiated neurons

DCX, Tuj1

Major mechanisms Reprogramming factors Neural marker Induced neurons Initial cells

Small molecules-based reprogramming

Table 1 (continued)

to neuronal cells. The expression of MAP2, Brn2, Ascl1, and Ngn2 genes also increased following small molecules treatment. Interestingly, the ability of iN cells to form of tumor-like spheroids highly decreased following reprogramming (Lee et al., 2018). Tables 1 and 2 summarize recent evidences on conversion of somatic cells to neuron-like cells.

8

9

DCX, NeuN

DCX, NeuN

Neuroblasts, Neuron

Neuroblasts, Neuron

Adult mouse astrocytes

Neural marker

Induced cells

Initial cells

MicroRNA-based reprogramming

MiR-302/367 in conjugation with (VPA) MiR-302/367 in conjugation with (VPA)

Reprogramming factors

Ascl1

Lentivirus

Lentivirus

Vector

Lentivirus

Brn2, Myt1l, Ascl1

NeuN, MAP2, SYN, vGlut, GAD65/67 NeuN, Gad1, VGLUT2

Both GABAergic and glutamatergic

Lentivirus

Sox2 + BDNF/Noggin or VPA

DCX

Induced adult neuroblasts (iANBs) that were differentiated to GABAergic and glutamatergic neurons Induced neurons

Hippocampus

Intact striatum

Environment

Dorsal midbrain, striatum, and somatosensory cortex of postnatal and adult mice

Intact striatum

Intact striatum

Mouse brain

Cortex, stab wound

Mouse striatum

Environment

Torper et al. (2013) Torper et al. (2013)

Action potential firing, synaptic input, integrated into local circuitry Exhibited neuronal morphology and markers, action potential firing, synaptic inputs

Ghasemi-Kasman et al. (2015) Ghasemi-Kasman et al. (2017)

Ref.

Cheng et al. (2019) Heinrich et al. (2014) Karow et al. (2012) Niu et al. (2013)

Ref.

Terminally differentiated neurons, inhibited tumor growth Electrophysiological properties, low frequency synaptic input Immature, express functional transmitter receptors Action potential firing, synaptic inputs

Major outcomes

Enhanced neurogenesis in kainic acid (KA)- induced hippocampal neurodegeneration

Expressed neuronal markers

Major outcomes

Adeno-associated virus (AAV) vector

Retrovirus

Sox2, Mahs1

βIII-tubulin

Adult human brain pericytes Astrocytes

Retrovirus

Lentivirus

GABAergic neurons

Induced neurons

NG2 cells

Ascl1

βIII-tubulin

Vector

Sox2

Neural cells

Glioma cells

Reprogramming factors

Neural marker

DCX, NeuN

Induced cells

Initial cells

Transcription factors-based reprogramming

Table 2 In vivo reprogramming of somatic cells to neuronal cells.

H. Yavarpour-Bali, et al.

Progress in Neuropsychopharmacology & Biological Psychiatry 98 (2020) 109820

Progress in Neuropsychopharmacology & Biological Psychiatry 98 (2020) 109820

Ghasemi-Kasman et al. (2018)

Guo et al. (2014)

Hu et al. (2015)

Declaration of competing interest The authors declare no potential conflicts of interest. Acknowledgements The authors would like to apologize to the all authors whose outstanding study has not been discussed here due to space limitations. There are no funders to report for the present study.

Decreased fibroblast-specific genes and increased the expression of neuronal markers Exhibited neuronal morphology, action potential firing, and integrated into neural circuits Induced neurons improved spontaneous alternation and spatial memory in animal model of AD

Ethical statement All authors have seen and approved the final version of the manuscript being submitted. They warrant that the article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere. This manuscript is also in accordance with the Authorship statement of ethical standards for manuscripts submitted to Progress in Neuropsychopharmacology & Biological Psychiatry Journal.

Cortex of stab-injured or Alzheimer's disease (AD) model mice Dentate gyrus of hippocampus

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MiR-302/367 in conjugation with VPA

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DCX, NeuN

Retrovirus NeuroD1

Adult human astrocytes

Glutamatergic neurons Both GABAergic and Glutamatergic neurons Neuroblasts, Neuron Postnatal cortical astrocytes Postnatal NG2 cells

DCX, NeuN

In vitro N/A VPA, CHIR99021, Repsox, Forskolin, SP600125, GO6983, Y-27632 Human chemical induced neurons (hciNs), mostly glutamatergic Normal and familial AD patients fibroblasts

DCX, Tuj1, MAP2, vGLUT1

Induced cells Initial cells

Table 3 Direct reprogramming of somatic cells to neurons in AD.

Neural marker

Reprogramming factor

Vector

Environment

Major outcomes

Ref.

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