Acquisition of functional neurons by direct conversion: Switching the developmental clock directly

Journal Pre-proof Acquisition of functional neurons by direct conversion: Switching the developmental clock directly Shuangquan Chen, Juan Zhang, Dongming Zhang, Jianwei Jiao PII:

S1673-8527(19)30168-7

DOI:

https://doi.org/10.1016/j.jgg.2019.10.003

Reference:

JGG 731

To appear in:

Journal of Genetics and Genomics

Received Date: 16 August 2019 Revised Date:

20 September 2019

Accepted Date: 8 October 2019

Please cite this article as: Chen, S., Zhang, J., Zhang, D., Jiao, J., Acquisition of functional neurons by direct conversion: Switching the developmental clock directly, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2019.10.003. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Copyright © 2019, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.

Acquisition of functional neurons by direct conversion: Switching the developmental clock directly

Shuangquan Chen b,1, Juan Zhang a,1, Dongming Zhang a,1, Jianwei Jiao a,c,d,e*

a

Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

b

School of Pharmaceutical Sciences, Tsinghua University, Beijing100084, China

c

University of Chinese Academy of Sciences, Beijing 100049, China

d

Co-Innovation Center of Neuroregeneration, Nantong University, Nantong 226001, China;

e

Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China.

1

These authors contributed equally to this work.

*Corresponding author. Email address: [email protected] (J. Jiao)

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Abstract

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Identifying approaches for treating neurodegeneration is a thorny task but is important for a

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growing number of patients. Researchers have focused on discovering the underlying molecular

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mechanisms of reprogramming and optimizing the technologies for acquiring neurons. Direct

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conversion is one of the most important processes for treating neurological disorders. Induced

6

neurons (iNs) derived from direct conversion, which bypass the pluripotency stage, are more

7

effective, more quickly obtained and safer than those produced via induced pluripotent stem cells

8

(iPSCs). Based on iPSC strategies, scientists have derived methods to obtain functional neurons by

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direct conversion, such as neuron-related transcriptional factors (TFs), small molecules,

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microRNAs, and epigenetic modifiers. In this review, we discuss the present strategies for the direct

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conversion of somatic cells into functional neurons and the potentials of direct conversion for

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producing functional neurons and treating neurodegeneration.

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Key words: direct conversion; fibroblasts; induced neurons; neurodegeneration

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1. Introduction

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Reprogramming is the process of turning back a cell’s developmental clock, and it has been studied

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for 60 years (Gurdon JB, 1958; Haag et al., 2018). Takahashi and Yamanaka (2006) first used four

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transcriptional factors (TFs), Oct4, Sox2, Klf4 and c-Myc, to successfully reprogram fibroblasts

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into induced pluripotent stem cells (iPSCs). Although induced pluripotent stem cells (iPSCs)

22

provide a solution for ethical concerns and immunological rejection, there still present many

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problems, such as tumorigenesis, inefficiency and instability (Jaenisch and Young, 2008; Polo et al.,

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2010).

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Direct conversion, a process directly converting terminal, fully differentiated cells to another

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type of terminal special cells that not only bypasses the pluripotency stage but also simplifies the

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transdifferentiation procedure, is an attractive approach for generating specific cell types

28

(Vierbuchen et al., 2010; Velasco et al., 2014). This process allows for conversion between two

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totally unrelated cell types, providing an interesting approach for repairing or replacing injured

30

organs and curing neurodegenerative diseases (Ebrahimi, 2016).

31

Various neurological diseases are caused by injury to specific subtypes of neurons; thus, there is

32

a demand for various subtypes of neurons. In fact, the earliest transdifferentiation originated in 1987.

33

Using the TF MyoD, researchers were able to derive myoblasts from mouse embryonic fibroblasts

34

(MEFs) (Davis et al., 1987). Nevertheless, this technology did not advance rapidly until iPSCs

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emerged. In 2010, induced neurons (iNs) were first derived from mouse fibroblasts by Vierbuchen

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and colleagues through the use of three neuron-related TFs (Brn2, Ascl1, and Myt1L; BAM)

37

(Vierbuchen et al., 2010). When NeuroD1 was added to the three TFs, human fibroblasts were able

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to be converted into neurons (Pang et al., 2011). Subsequently, the field of direct conversion has

39

grown. Many strategies for achieving specific cell types, such as neurons, neural progenitors, neural

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crests, pancreatic β cells, myocardial cells, chondrocytes, hepatocytes, endotheliocytes, and

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epithelial cells, have been introduced (Ieda et al., 2010; Kim et al., 2011a; Jayawardena et al., 2012;

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Kim et al., 2014a; Kim et al., 2014b; Kulangara et al., 2014; Lee et al., 2015; Kaminski et al., 2016;

43

Lee et al., 2017; Van Pham et al., 2017).

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Theoretically, direct conversion can provide a safer and quicker method for addressing

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neurological disorders. This method has great implications for the clinical treatment of

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neurodegeneration. In this review, we focus on the recent advancements in the acquisition of 1

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functional neurons through direct conversion.

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2. Strategies for the direct conversion of fibroblasts into functional neurons

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2.1 Transcription factors

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In different neurological diseases, different subtypes of neurons are impaired and need to be

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targeted for repair. Originally, studies on direct conversion activated “master control genes (MCGs)”

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to control cell fate (Lewis, 1992). The earliest evidence of direct conversion was in 1987, when

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Davis and colleagues converted fibroblasts into myoblasts using the MCG MyoD (Davis et al.,

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1987). However, in 2010, Vierbuchen and colleagues reported that the transdifferentiation of

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fibroblasts into functional neurons can be achieved using three neuron-related TFs (Brn2, Ascl1,

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and Myt1L) (Vierbuchen et al., 2010). They demonstrated that these three neuron-related TFs can

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directly convert mouse fibroblasts into functional neurons. The iNs exhibit features of functional

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membranes, namely, action potentials (APs) and spontaneous action potentials (Vierbuchen et al.,

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2010). One or a few TFs whose encoding genes act as MCGs are sufficient to trigger the activation

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of many other genes, leading to cell fate changes (Nizzardo et al., 2013).

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In fact, subsequent research has shown that the overexpression of the three TFs Ascl1, Brn2 and

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Ngn2 can also induce the transdifferentiation of fibroblasts into functional neurons (Meng et al.,

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2012). Furthermore, researchers have demonstrated that the single reprogramming factor Ascl1 can

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generate iNs (Chanda et al., 2014). Additionally, human fibroblasts can also be converted into

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neurons upon the combination of the neural differential-related TF NeuroD1 with the three TFs

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Brn2, Ascl1 and Myt1L (Pang et al., 2011). Moreover, patient-specific iNs can be generated by a

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direct conversion strategy (Wang et al., 2014), and Ngn2 can enhance the generation of

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patient-specific iNs (Zhao et al., 2015), providing more approaches for treating degenerative

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diseases.

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Another way to activate neuron-related TFs is by inhibiting the RNA binding protein PTB,

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which results in the expression of neuron-related TFs and microRNAs (Xue et al., 2013). In

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addition, the non-neural progenitor TF Ptf1a is sufficient to convert mouse and human fibroblasts

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into induced neural stem cells (iNSCs) (Xiao et al., 2018). Thus, focusing on non-neural TFs is also

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a new idea for transdifferentiation.

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Based on the preceding studies, many researchers have successfully derived various types of

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specific functional neurons, such as motor neurons, medium spiny neurons, peripheral sensory 2

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neurons, and dopaminergic neurons, via transdifferentiation (Table 1) (Caiazzo et al., 2011; Kim et

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al., 2011b; Blanchard et al., 2015; Hu et al., 2015; Tian et al., 2015).

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Compared to the use of only TFs, the combination of TFs and small molecules is more

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convenient and provides more advantages (Ladewig et al., 2012; Pfisterer et al., 2016; Smith et al.,

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2016). For example, Ladewig et al. (2012) showed that three small molecules, CHIR99021 (an

82

inhibitor of GSK3), SB431542 (an inhibitor of the TGF-β receptor) and LDN193189 (an inhibitor

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of the BMP receptor), increase the efficiency of generating Ascl1/Ngn2-iNs from human fibroblasts.

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Smith et al. (2016) reported that small molecules facilitate Ngn2-mediated transdifferentiation via

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modulating chromatin accessibility. The combination of TFs and small molecules also facilitates the

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generation of target cells, such as striatal neurons, serotonergic neurons, cholinergic neurons,

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cortical pyramidal neurons, from human fibroblasts (Berry et al., 2011; Pfisterer et al., 2011;

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Ladewig et al., 2012; Liu et al., 2013; Victor et al., 2014; Dai et al., 2015; Pfisterer et al., 2016; Xu

89

et al., 2016; Miskinyte et al., 2017).

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2.2 Small-molecule compounds

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Compared to other strategies, small molecules hold many unique advantages. As drugs, they are

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acceptable for clinical use. They can modulate specific targets, including transcription, metabolism,

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signaling and epigenetic modification, which represent valuable approaches for probing cell fate

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determinants and generating specific functional neurons (Schugar et al., 2008).

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As compounds, small molecules exhibit distinct advantages in the process of direct

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reprogramming (Table1). First, they are convenient to use for direct conversion. They provide rapid

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and reversible effects. Meanwhile, their effects are confined to target cells or tissues and do not act

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on other cells or tissues. Second, they are cost effective. The preparation of TFs requires more time

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and relatively complex procedures. Small molecules are easier to manufacture, quantify and

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produce. Third, they allow a high degree of temporal and spatial control. Additionally, the

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compounds are highly permeating, which allows their effects to be reversible. The concentration

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and combination can be fine-tuned (Yu et al., 2014; Xie et al., 2017). Different concentrations and

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combinations can be used for various approaches for specific demands (Li et al., 2013).

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Small molecules normally promote the expression or activation of master neuronal TFs, such as

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ISX9 (an isoxazole), which is necessary to induce the activation of neuronal genes (Ngn2, NeuroD1,

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NF-H, Tau, and Syn2) in fibroblasts by regulating signaling pathways that facilitate neural 3

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differentiation via neurotransmitter-evoked Ca2+ signaling pathways (Schneider et al., 2008; Li et al.,

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2015). Dai et al. (2015) reported a highly efficient approach based on inhibition of the SMAD

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signaling and the MEK-ERK pathway to directly convert human fibroblasts into functional neurons

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using a chemical cocktail of six types of small molecules, namely, CHIR99021 (an inhibitor of

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GSK3β), SB431542 (a TGF-β receptor inhibitor), LDN193189 (a BMP receptor inhibitor),

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PD0325901 (a MEK-ERK inhibitor), pifithrin-α (a p53 inhibitor) and forskolin. Another study

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revealed that forskolin and dorsomorphin can increase Ngn2-based direct reprogramming by

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increasing H3K27 acetylation and chromatin accessibility (Smith et al., 2016). Stimulating Wnt

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signaling with CHIR99021 and inhibiting TGF-β signaling with SB431542 significantly enhance

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the conversion of fibroblasts into neurons after the transduction of Ngn2 and Ascl1 (Ladewig et al.,

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2012). CHIR99021 has been previously shown to be critical for the phosphorylation of Ngn2 and

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motoneuron specification (Ma et al., 2008). When CHIR99021 is combined with Ngn2 for

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transdifferentiation, no HB9-positive cells are detected (Liu et al., 2013). Therefore, small-molecule

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compounds may contribute to the acquisition of specific neurons.

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Using a cocktail of four chemicals (forskolin, CHIR99021, ISX9, and I-BET151), Li et al.

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(2015) successfully generated functional neurons (also called chemically induced neurons (CiNs))

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from mouse fibroblasts. Hu et al. (2015) reported that direct neuronal conversion from human

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fibroblasts can be achieved with a chemical cocktail (named VCRFSGY) including seven types of

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small-molecule compounds (V, VPA; C, CHIR99021; R, RepSox; F, forskolin; S, SP600625; G,

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GO6983; Y, Y-27632) and iNs need to be cultured in CFD (C, CHIR99021; F, forskolin; D,

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dorsomorphin) for maturation. Furthermore, a strategy was derived to generate human functional

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neurons from patients with familial Alzheimer’s disease, which provides a promising clinical

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approach for modeling neurological disorders and for regenerative medicine (Hu et al., 2015).

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As a nascent approach for direct conversion, small molecules present significant challenges, but

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they are valuable for facilitating the application of molecule-based neuronal differentiation for the

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treatment of degenerative diseases.

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2.3 Epigenetic modifiers

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There have been extensive studies on DNA methylation by DNA methyltransferases (Smith and

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Meissner, 2013), which exerts a profound effect on the stability of genomes, transcriptional

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processes, development and reprogramming (Ko et al., 2010). A previous report has shown that 4

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primary neurons exhibit extensive DNA demethylation (Yu et al., 2015). Although there are some

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reports focusing on the relationship between epigenetic modifications and reprogramming, fewer

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focus on the relationship between epigenetic modifications and direct conversion (Table1) (Gu et al.,

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2011; Gao et al., 2013; Yu et al., 2015).

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Zhang and colleagues (2016) presented a new strategy for direct conversion. Their research

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suggests that DNA demethylation contributes to direct conversion of fibroblasts into functional

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neurons. 5hmC is correlated with neuron-specific gene expression in the central nervous system

144

(Kriaucionis and Heintz, 2009; Mellen et al., 2012). Epigenetic modification mediated by 5hmC is

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critical in neurodevelopment (Szulwach et al., 2011). Thus, the generation of 5hmC may be pivotal

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for the modification of neuronal differentiation by epigenetics (Zhu et al., 2016). Tet3, which is well

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known for its functional role in DNA demethylation, has been shown to guide the direct conversion

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of mouse embryonic fibroblasts to functional neurons (Zhang et al., 2016).

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In addition to DNA demethylation, DNA methylation is essential for iN generation. De novo

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methylation by DNMT3A suppresses the donor fibroblast and the competing myogenic programs,

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which is necessary for TF-induced direct reprogramming (Luo et al., 2019).

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2.4 MicroRNAs

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Since neuron-related TFs can convert fibroblasts into functional neurons, scientists have developed

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neuron-related microRNA approaches. The neuronal-specific microRNAs miR-9/9* and miR-124

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can promote the transdifferentiation of human fibroblasts into neurons. Although the induced cells

156

only express the neuronal marker MAP2, this approach widens the field of direct conversion (Yoo

157

et al., 2011). Combined with TFs, microRNAs can facilitate efficiency and maturation (Table1)

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(Ambasudhan et al., 2011; Pang et al., 2011; Yoo et al., 2011). Reports have also shown that the

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repression of the release of a single RNA binding protein, PTB, silences neuronal lineage-specific

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genes, including microRNAs (Xue et al., 2013). Inhibiting PTB-mediated effects of microRNAs on

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multiple components of the REST complex, is a key event in neuronal cell generation (Xue et al.,

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2013).

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2.5 Others

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In addition to the major factors above, other indirect paths can be used to achieve direct conversion.

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For example, Li et al. (2017) reported that chemically induced cells in an extraembryonic endoderm

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(XEN)-like state derived from fibroblasts can be directly converted into functional neurons without 5

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entering the pluripotent stage. They improved the previous protocol for inducing chemically

168

induced pluripotent stem cells (CiPSCs) from fibroblasts; VC6TFAE, which contains seven types of

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small-molecule compounds (VPA, TD114-2, 616452, tranylcypromine, forskolin, AM580, and

170

EPZ004777), was used to generate the desired cell type (Li et al., 2017). Junsang Yoo and

171

colleagues (2017) found that, in addition to chemical and biological methods, electromagnetic fields

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(EMFs) can be used for direct reprogramming. Gold nanoparticles (AuNPs) can facilitate the

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conversion of fibroblasts and astrocytes into iNs under specific EMF frequencies (Yoo et al., 2017).

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The EMF system is a non-invasive method and thus may be more suitable for application. EMFs

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combined with a small-molecule strategy may be an ideal approach for reprogramming somatic

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cells into neurons.

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In addition to fibroblasts, astrocytes and microglia are good donor cells for generating iNs. In 2007,

178

Berninger and colleagues (2007) determined that astrocytes can be converted into functional

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neurons. Generation of subtype-specific neurons, such as glutamatergic and GABAergic neurons,

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from astrocytes can also be achieved through the overexpression of the neurogenic TFs Neurog2

181

and Dlx2 (Heinrich et al., 2010). Gong Chen’s group demonstrated that after brain injury, reactive

182

astrocytes can be reprogrammed into functional neurons in vivo through overexpressing a single TF,

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NeuroD1 (Guo et al., 2014). In order to improve the in vivo glia-to-neuron conversion for

184

therapeutic applications, they used an adeno-associated virus (AAV) Cre-FLEX system to express

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NeuroD1 and regenerated 30%‒40% of lost neurons in an ischemic injury mouse model (Chen et al.,

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2019). Recently, Matsuda et al. (2019) demonstrated that mouse microglia can be converted into

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neurons both in vitro and in vivo through the expression of NeuroD1 (Matsuda et al., 2019). Glia

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cells, the major cells in the brain, accumulate at injured sites in the central nervous system (CNS).

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They may be suitable for restoring neurons through direct conversion in vivo without exhausting

190

resources (Zhang et al., 2018). Further work is needed to address the functionality of these

191

converted neuronal cells and to improve the conversion efficiency in mouse models of

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neurodegeneration.

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The model of direct conversion strategies shown in Fig. 1 illustrates approaches for directly

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converting mouse and human fibroblasts into functional neurons. Various combinations of TFs,

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small molecules, epigenetic modifiers, and microRNAs may produce a number of new cell types.

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On the other hand, iNSCs or astrocytes can also differentiate into target neurons. These strategies 6

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are derived from practices and new discoveries. As these strategies are developed, the field of direct

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conversion may be used for clinical applications.

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3. Discussion

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Achieving functional target cell types and promising safe and effective clinical treatments are

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fundamental to regenerative medicine. Cell lineage reprogramming brings promise to the

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neurodegenerative diseases, especially those that require neuronal-specific cell types.

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Researchers have described methods that drive the transdifferentiation of fibroblasts into

204

neurons (Yang et al,. 2011). They have further explored the hierarchical mechanisms of direct

205

conversion from fibroblasts into neurons through the use of Ascl1, Brn2 and Myt1L: Ascl1first

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occupies the genomic sites of fibroblasts and then recruits Brn2 to these sites; Zfp238 is the target

207

gene of Ascl1, which induces transdifferentiation (Wapinski et al., 2013). In recent years, Ascl1

208

have been studied very frequently in the field of neuronal induction. During neurogenesis, Ascl1

209

can bind to closed chromatin to promote chromatin accessibility and then activate gene expression

210

(Raposo et al., 2015). For transdifferentiation, neuronal and cell cycle genes are also activated by

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Ascl1 overexpression in fibroblasts. The initial transcriptional response of all cells to Ascl1 is

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nearly homogenous; however, the later events play a main role in reprogramming efficiency

213

(Treutlein et al., 2016). The study of single cell RNA sequencing at multiple time points during the

214

conversion of fibroblasts into neurons has suggested that the molecular reprogramming path is

215

remarkably continuous. Cells go through a unique intermediate transcriptional stage that is

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unrelated to donor and target cell programs rather than a canonical neural progenitor cell stage

217

(Treutlein et al., 2016).

218

It seems difficult to achieve highly efficient conversion with the use of only TFs compared with

219

other methods for reprogramming fibroblasts into neurons (Table 1). The main reason may be that

220

most research uses viruses to ectopically express TFs. Thus, it is impossible to control the

221

expression levels and temporal effects of TFs in donor cells. In addition, signaling pathways,

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metabolome and proteome are dramatically changed during transdifferentiation. TFs cannot

223

reprogram cells in multiple “dimensions” at once. Moreover, using viruses also limits the

224

application of iNs in translational medicine for safety reasons. The combination of TFs with

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microRNAs or small molecules can significantly increase the conversion efficiency of iNs and even

226

some specific neuronal subtypes (Ambasudhan et al., 2011; Yoo et al., 2011; Ladewig et al., 2012; 7

227

Liu et al., 2013; Victor et al., 2014). Thus, regulating donor cells in multiple dimensions is more

228

advantageous for the acquisition of iNs. Recent studies have demonstrated the mechanism of

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transdifferentiation by small molecules that target metabolic processes, epigenetic modifications,

230

and signaling pathways. However, the precise targets of most small-molecule compounds during

231

transdifferentiation remain unclear. In addition, one small molecule may have multiple targets in

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different donor cells. For example, forskolin is a very common small molecule used for

233

reprogramming. Smith and colleagues (2016) revealed that forskolin can promote chromatin

234

remodeling and enhance H3K27 acetylation. However, another group found that forskolin improves

235

the neural transdifferentiation of mesenchymal stem cells (MSCs) through downregulation of the

236

neuron restrictive silencer factor (NRSF) (Thompson et al., 2019). Using pure chemical compound

237

cocktails is an ideal way to safely obtain neuronal cells, which can generate reprogrammed

238

functional neurons from normal fibroblasts as well as from fibroblasts of Alzheimer’s patients (Hu

239

et al., 2015; Li et al., 2015). Ma et al. (2019) employed RNA-sequencing to explore the

240

transcriptome dynamics of chemical reprogramming of human astrocytes into neurons. They

241

identified several signaling pathways that might be critical during conversion process, and also

242

identified several new genes, such as neuronatin (NNAT), jagged canonical notch ligand 1 (JAG1)

243

and repulsive guidance molecule A (RGMA), which are critical to coordinate the chemical

244

reprogramming process.

245

The generation of some neuronal subtypes via these similar approaches is possible. miR-9/9*

246

and miR-124 (miR-9/9*-124) promotes the conversion of human fibroblasts into glutamatergic

247

neurons (Yoo et al., 2011); when miR-124 is combined with Myt1L and Brn2, the induction of

248

glutamatergic neurons from human fibroblasts can also be achieved (Ambasudhan et al., 2011). The

249

combination of Bcl11b (also called Ctip2), Myt1L, Dlx1 and Dlx2 can guide the transdifferentiation

250

of human postnatal and adult fibroblasts into striatal neurons (Victor et al., 2014). Dopaminergic

251

neurons derived from mouse and human fibroblasts can be obtained upon the overexpression of

252

three TFs, Ascl1, Nurr1 (also called Nr4a2) and Lmx1a (Caiazzo et al., 2011). Seven TFs (Ascl1,

253

Brn2, Myt1L, Hb9, Isl1, Lhx3, and Ngn2) have been identified to facilitate the production of spinal

254

motor neurons from both mouse and human fibroblasts (Son et al., 2011).

255

Great achievements in neuronal direct conversion have been made since 2010. Different

256

approaches, including TFs, microRNAs and small-molecule cocktails, have been used to change the 8

257

fate of cells from fibroblasts to neurons (Vierbuchen et al., 2010; Pang et al., 2011; Yoo et al., 2011;

258

Hu et al., 2015; Li et al., 2015). However, some questions still exist. Although he hierarchical

259

mechanism of direct conversion has been clarified (Wapinski et al., 2013), the underlying

260

mechanism of direct reprogramming still needs to be better understood (Xu et al., 2015). It is more

261

difficult to transdifferentiate human cells into neurons than to transdifferentiate mouse cells into

262

neurons. In addition, the conversion efficiency decreases as the age of the donor increases (Tanabe

263

et al., 2015). Furthermore, for clinical application, high-quality and high-pure human-derived

264

induced neurons must be acquired. The safety of the cell source used for transplantation for the

265

treatment of neurodegenerative diseases should be considered a main concern (Xu et al., 2015).

266

It is difficult to ensure that all transplanted induced cells are integrated into local neural circuits.

267

According to previous studies, grafted iNs can reverse symptoms in mouse model of some diseases,

268

such as Parkinson's disease. These iNs are able to integrate into and survive in brain networks and

269

have also been characterized by electrophysiological examination, immunostaining, etc. However,

270

few studies have determined the efficiency of iN survival in iN-grafted brains, and the interaction

271

between the microenvironment and iNs has not been fully elucidated (Caiazzo et al., 2011; Kim et

272

al., 2011b; Torper et al., 2013; Cassady et al., 2014; Tian et al., 2015). In addition, different

273

molecules and factors that affect grafted cell fate are largely unidentified. The direct conversion

274

from fibroblasts into functional neurons provides us with a good model for drug screening and for

275

the study of neurodegenerative diseases (Graf, 2011). However, potential risks should be avoided.

276

There is still a long journey to clinical application in this field.

277 278

Acknowledgments

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This work was supported by grants from the CAS Strategic Priority Research Program

280

(XDA16020602), the National Key R&D Program of China (2019YFA0110300), the National

281

Science Foundation of China (81825006, 31730033 and 31621004), and the K.C.Wong Education

282

Foundation.

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References

285

Ambasudhan, R., Talantova, M., Coleman, R., Yuan, X., Zhu, S., Lipton, S.A., Ding, S., 2011.

286

Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. 9

287

Cell Stem Cell 9, 113-118.

288

Berninger, B., Costa, M.R., Koch, U., Schroeder, T., Sutor, B., Grothe, B., Gotz, M., 2007.

289

Functional properties of neurons derived from in vitro reprogrammed postnatal astroglia. J.

290

Neurosci. 27, 8654-8664.

291

Berry, N., Gursel, D.B., Boockvar, J.A., 2011. Direct conversion of human fibroblasts to functional

292

neurons in one step. Neurosurgery 69, N18.

293

Blanchard, J.W., Eade, K.T., Szucs, A., Lo Sardo, V., Tsunemoto, R.K., Williams, D., Sanna, P.P.,

294

Baldwin, K.K., 2015. Selective conversion of fibroblasts into peripheral sensory neurons. Nat.

295

Neurosci. 18, 25-35.

296

Caiazzo, M., Dell'Anno, M.T., Dvoretskova, E., Lazarevic, D., Taverna, S., Leo, D., Sotnikova, T.D.,

297

Menegon, A., Roncaglia, P., Colciago, G., Russo, G., Carninci, P., Pezzoli, G., Gainetdinov, R.R.,

298

Gustincich, S., Dityatev, A., Broccoli, V., 2011. Direct generation of functional dopaminergic

299

neurons from mouse and human fibroblasts. Nature 476, 224-227.

300

Cassady, J.P., D'Alessio, A.C., Sarkar, S., Dani, V.S., Fan, Z.P., Ganz, K., Roessler, R., Sur, M.,

301

Young, R.A., Jaenisch, R., 2014. Direct lineage conversion of adult mouse liver cells and B

302

lymphocytes to neural stem cells. Stem Cell Rep. 3, 948-956.

303

Chanda, S., Ang, C.E., Davila, J., Pak, C., Mall, M., Lee, Q.Y., Ahlenius, H., Jung, S.W., Sudhof,

304

T.C., Wernig, M., 2014. Generation of induced neuronal cells by the single reprogramming factor

305

ASCL1. Stem Cell Rep. 3, 282-296.

306

Chen, Y.-C., Ma, N.-X., Pei, Z.-F., Wu, Z., Do-Monte, F.H., Keefe, S., Yellin, E., Chen, M.S., Yin,

307

J.-C., Lee, G., Toribio, A.M., Hu, Y., Bai, Y.-T., Lee, K., Quirk, G.J., Chen, G., 2019. A NeuroD1

308

AAV-based gene therapy for functional brain repair after ischemic injury through in vivo

309

astrocyte-to-neuron conversion. Mol. Ther. doi.org/10.1016/j.ymthe.2019.09.003.

310

Dai, P., Harada, Y., Takamatsu, T., 2015. Highly efficient direct conversion of human fibroblasts to

311

neuronal cells by chemical compounds. J. Clin. Biochem. Nutr. 56, 166-170.

312

Davis, R.L., Weintraub, H., Lassar, A.B., 1987. Expression of a single transfected cDNA converts

313

fibroblasts to myoblasts. Cell 51, 987-1000.

314

Ebrahimi, B., 2016. Engineering cell fate: Spotlight on cell-activation and signaling-directed

315

lineage conversion. Tissue Cell 48, 475-487.

316

Gao, Y., Chen, J., Li, K., Wu, T., Huang, B., Liu, W., Kou, X., Zhang, Y., Huang, H., Jiang, Y., Yao, 10

317

C., Liu, X., Lu, Z., Xu, Z., Kang, L., Chen, J., Wang, H., Cai, T., Gao, S., 2013. Replacement of

318

Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and

319

hydroxymethylation in reprogramming. Cell Stem Cell 12, 453-469.

320

Graf, T., 2011. Historical origins of transdifferentiation and reprogramming. Cell Stem Cell 9,

321

504-516.

322

Gu, T.P., Guo, F., Yang, H., Wu, H.P., Xu, G.F., Liu, W., Xie, Z.G., Shi, L., He, X., Jin, S.G., Iqbal,

323

K., Shi, Y.G., Deng, Z., Szabo, P.E., Pfeifer, G.P., Li, J., Xu, G.L., 2011. The role of Tet3 DNA

324

dioxygenase in epigenetic reprogramming by oocytes. Nature 477, 606-610.

325

Guo, Z., Zhang, L., Wu, Z., Chen, Y., Wang, F., Chen, G., 2014. In vivo direct reprogramming of

326

reactive glial cells into functional neurons after brain injury and in an Alzheimer's disease model.

327

Cell Stem Cell 14, 188-202.

328

Gurdon JB, E.T., Fischberg M., 1958. Sexually mature individuals of Xenopus laevis from the

329

transplantation of single somatic nuclei. Natue 182, 2.

330

Haag, S.M., Gulen, M.F., Reymond, L., Gibelin, A., Abrami, L., Decout, A., Heymann, M., van der

331

Goot, F.G., Turcatti, G., Behrendt, R., Ablasser, A., 2018. Targeting STING with covalent

332

small-molecule inhibitors. Nature 559, 269-273.

333

Heinrich, C., Blum, R., Gascon, S., Masserdotti, G., Tripathi, P., Sanchez, R., Tiedt, S., Schroeder,

334

T., Gotz, M., Berninger, B., 2010. Directing astroglia from the cerebral cortex into subtype specific

335

functional neurons. PLoS Biol. 8, e1000373.

336

Herdy, J., Schafer, S., Kim, Y., Ansari, Z., Zangwill, D., Ku, M., Paquola, A., Lee, H., Mertens, J.,

337

Gage, F.H., 2019. Chemical modulation of transcriptionally enriched signaling pathways to

338

optimize the conversion of fibroblasts into neurons. eLife 8, e41356.

339

Hu, W., Qiu, B., Guan, W., Wang, Q., Wang, M., Li, W., Gao, L., Shen, L., Huang, Y., Xie, G., Zhao,

340

H., Jin, Y., Tang, B., Yu, Y., Zhao, J., Pei, G., 2015. Direct conversion of normal and Alzheimer's

341

disease human fibroblasts into neuronal cells by small molecules. Cell Stem Cell 17, 204-212.

342

Ieda, M., Fu, J.D., Delgado-Olguin, P., Vedantham, V., Hayashi, Y., Bruneau, B.G., Srivastava, D.,

343

2010. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell

344

142, 375-386.

345

Jaenisch, R., Young, R., 2008. Stem cells, the molecular circuitry of pluripotency and nuclear

346

reprogramming. Cell 132, 567-582. 11

347

Jayawardena, T.M., Egemnazarov, B., Finch, E.A., Zhang, L., Payne, J.A., Pandya, K., Zhang, Z.,

348

Rosenberg, P., Mirotsou, M., Dzau, V.J., 2012. MicroRNA-mediated in vitro and in vivo direct

349

reprogramming of cardiac fibroblasts to cardiomyocytes. Circ. Res. 110, 1465-1473.

350

Kaminski, M.M., Tosic, J., Kresbach, C., Engel, H., Klockenbusch, J., Muller, A.L., Pichler, R.,

351

Grahammer, F., Kretz, O., Huber, T.B., Walz, G., Arnold, S.J., Lienkamp, S.S., 2016. Direct

352

reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors. Nat.

353

Cell Biol. 18, 1269-1280.

354

Kim, H.S., Kim, J., Jo, Y., Jeon, D., Cho, Y.S., 2014a. Direct lineage reprogramming of mouse

355

fibroblasts to functional midbrain dopaminergic neuronal progenitors. Stem Cell Res. 12, 60-68.

356

Kim, J., Efe, J.A., Zhu, S., Talantova, M., Yuan, X., Wang, S., Lipton, S.A., Zhang, K., Ding, S.,

357

2011a. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc. Natl. Acad. Sci. U. S.

358

A. 108, 7838-7843.

359

Kim, J., Su, S.C., Wang, H., Cheng, A.W., Cassady, J.P., Lodato, M.A., Lengner, C.J., Chung, C.Y.,

360

Dawlaty, M.M., Tsai, L.H., Jaenisch, R., 2011b. Functional integration of dopaminergic neurons

361

directly converted from mouse fibroblasts. Cell Stem Cell 9, 413-419.

362

Kim, Y.J., Lim, H., Li, Z., Oh, Y., Kovlyagina, I., Choi, I.Y., Dong, X., Lee, G., 2014b. Generation

363

of multipotent induced neural crest by direct reprogramming of human postnatal fibroblasts with a

364

single transcription factor. Cell Stem Cell 15, 497-506.

365

Ko, M., Huang, Y., Jankowska, A.M., Pape, U.J., Tahiliani, M., Bandukwala, H.S., An, J., Lamperti,

366

E.D., Koh, K.P., Ganetzky, R., Liu, X.S., Aravind, L., Agarwal, S., Maciejewski, J.P., Rao, A., 2010.

367

Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature 468,

368

839-843.

369

Kriaucionis, S., Heintz, N., 2009. The nuclear DNA base 5-hydroxymethylcytosine is present in

370

Purkinje neurons and the brain. Science 324, 929-930.

371

Kulangara, K., Adler, A.F., Wang, H., Chellappan, M., Hammett, E., Yasuda, R., Leong, K.W., 2014.

372

The effect of substrate topography on direct reprogramming of fibroblasts to induced neurons.

373

Biomaterials 35, 5327-5336.

374

Ladewig, J., Mertens, J., Kesavan, J., Doerr, J., Poppe, D., Glaue, F., Herms, S., Wernet, P., Kogler,

375

G., Muller, F.J., Koch, P., Brustle, O., 2012. Small molecules enable highly efficient neuronal

376

conversion of human fibroblasts. Nat. Methods 9, 575-578. 12

377

Lee, K., Yu, P., Lingampalli, N., Kim, H.J., Tang, R., Murthy, N., 2015. Peptide-enhanced mRNA

378

transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards

379

cardiomyocyte-like cells. Int. J. Nanomedicine 10, 1841-1854.

380

Lee, S., Park, C., Han, J.W., Kim, J.Y., Cho, K., Kim, E.J., Kim, S., Lee, S.J., Oh, S.Y., Tanaka, Y.,

381

Park, I.H., An, H.J., Shin, C.M., Sharma, S., Yoon, Y.S., 2017. Direct reprogramming of human

382

dermal fibroblasts into endothelial cells using ER71/ETV2. Circ. Res. 120, 848-861.

383

Lewis, E.B., 1992. The 1991 Albert Lasker Medical Awards. Clusters of master control genes

384

regulate the development of higher organisms. JAMA 267, 1524-1531.

385

Li, W., Li, K., Wei, W., Ding, S., 2013. Chemical approaches to stem cell biology and therapeutics.

386

Cell Stem Cell 13, 270-283.

387

Li, X., Liu, D., Ma, Y., Du, X., Jing, J., Wang, L., Xie, B., Sun, D., Sun, S., Jin, X., Zhang, X., Zhao,

388

T., Guan, J., Yi, Z., Lai, W., Zheng, P., Huang, Z., Chang, Y., Chai, Z., Xu, J., Deng, H., 2017.

389

Direct reprogramming of fibroblasts via a chemically induced XEN-like state. Cell Stem Cell 21,

390

264-273.e7.

391

Li, X., Zuo, X., Jing, J., Ma, Y., Wang, J., Liu, D., Zhu, J., Du, X., Xiong, L., Du, Y., Xu, J., Xiao,

392

X., Wang, J., Chai, Z., Zhao, Y., Deng, H., 2015. Small-molecule-driven direct reprogramming of

393

mouse fibroblasts into functional neurons. Cell Stem Cell 17, 195-203.

394

Liu, M.L., Zang, T., Zou, Y., Chang, J.C., Gibson, J.R., Huber, K.M., Zhang, C.L., 2013. Small

395

molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons.

396

Nat. Commun. 4, 2183.

397

Liu, X., Li, F., Stubblefield, E.A., Blanchard, B., Richards, T.L., Larson, G.A., He, Y., Huang, Q.,

398

Tan, A.C., Zhang, D., Benke, T.A., Sladek, J.R., Zahniser, N.R., Li, C.Y., 2012. Direct

399

reprogramming of human fibroblasts into dopaminergic neuron-like cells. Cell Res. 22, 321-332.

400

Luo, C., Lee, Q.Y., Wapinski, O., Castanon, R., Nery, J.R., Mall, M., Kareta, M.S., Cullen, S.M.,

401

Goodell, M.A., Chang, H.Y., Wernig, M., Ecker, J.R., 2019. Global DNA methylation remodeling

402

during direct reprogramming of fibroblasts to neurons. eLife 8, e40197.

403

Ma, N.X., Yin, J.C., Chen, G., 2019. Transcriptome analysis of small molecule-mediated

404

astrocyte-to-neuron reprogramming. Front. Cell Dev. Biol. 7, 82.

405

Matsuda, T., Irie, T., Katsurabayashi, S., Hayashi, Y., Nagai, T., Hamazaki, N., Adefuin, A.M.D.,

406

Miura, F., Ito, T., Kimura, H., Shirahige, K., Takeda, T., Iwasaki, K., Imamura, T., Nakashima, K., 13

407

2019. Pioneer factor NeuroD1 rearranges transcriptional and epigenetic profiles to execute

408

microglia-neuron conversion. Neuron 101, 472-485.e7.

409

Mellen, M., Ayata, P., Dewell, S., Kriaucionis, S., Heintz, N., 2012. MeCP2 binds to 5hmC enriched

410

within active genes and accessible chromatin in the nervous system. Cell 151, 1417-1430.

411

Meng, F., Chen, S., Miao, Q., Zhou, K., Lao, Q., Zhang, X., Guo, W., Jiao, J., 2012. Induction of

412

fibroblasts to neurons through adenoviral gene delivery. Cell Res. 22, 436-440.

413

Miskinyte, G., Devaraju, K., Gronning Hansen, M., Monni, E., Tornero, D., Woods, N.B., Bengzon,

414

J., Ahlenius, H., Lindvall, O., Kokaia, Z., 2017. Direct conversion of human fibroblasts to

415

functional excitatory cortical neurons integrating into human neural networks. Stem Cell Res. Ther.

416

8, 207.

417

Niu, W., Zang, T., Zou, Y., Fang, S., Smith, D.K., Bachoo, R., Zhang, C.L., 2013. In vivo

418

reprogramming of astrocytes to neuroblasts in the adult brain. Nat. Cell Biol. 15, 1164-1175.

419

Nizzardo, M., Simone, C., Falcone, M., Riboldi, G., Comi, G.P., Bresolin, N., Corti, S., 2013.

420

Direct reprogramming of adult somatic cells into other lineages: past evidence and future

421

perspectives. Cell Transplant. 22, 921-944.

422

Pang, Z.P., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D.R., Yang, T.Q., Citri, A., Sebastiano,

423

V., Marro, S., Sudhof, T.C., Wernig, M., 2011. Induction of human neuronal cells by defined

424

transcription factors. Nature 476, 220-223.

425

Pfisterer, U., Ek, F., Lang, S., Soneji, S., Olsson, R., Parmar, M., 2016. Small molecules increase

426

direct neural conversion of human fibroblasts. Sci. Rep. 6, 38290.

427

Pfisterer, U., Kirkeby, A., Torper, O., Wood, J., Nelander, J., Dufour, A., Bjorklund, A., Lindvall, O.,

428

Jakobsson, J., Parmar, M., 2011. Direct conversion of human fibroblasts to dopaminergic neurons.

429

Proc. Natl. Acad. Sci. U. S. A. 108, 10343-10348.

430

Polo, J.M., Liu, S., Figueroa, M.E., Kulalert, W., Eminli, S., Tan, K.Y., Apostolou, E., Stadtfeld, M.,

431

Li, Y., Shioda, T., Natesan, S., Wagers, A.J., Melnick, A., Evans, T., Hochedlinger, K., 2010. Cell

432

type of origin influences the molecular and functional properties of mouse induced pluripotent stem

433

cells. Nat. Biotechnol. 28, 848-855.

434

Raposo, A.A., Vasconcelos, F.F., Drechsel, D., Marie, C., Johnston, C., Dolle, D., Bithell, A.,

435

Gillotin, S., van den Berg, D.L., Ettwiller, L., Flicek, P., Crawford, G.E., Parras, C.M., Berninger,

436

B., Buckley, N.J., Guillemot, F., Castro, D.S., 2015. Ascl1 coordinately regulates gene expression 14

437

and the chromatin landscape during neurogenesis. Cell Rep. 10, 1544-1556.

438

Rivetti di Val Cervo, P., Romanov, R.A., Spigolon, G., Masini, D., Martin-Montanez, E., Toledo,

439

E.M., La Manno, G., Feyder, M., Pifl, C., Ng, Y.H., Sanchez, S.P., Linnarsson, S., Wernig, M.,

440

Harkany, T., Fisone, G., Arenas, E., 2017. Induction of functional dopamine neurons from human

441

astrocytes in vitro and mouse astrocytes in a Parkinson's disease model. Nat. Biotechnol. 35,

442

444-452.

443

Schneider, J.W., Gao, Z., Li, S., Farooqi, M., Tang, T.S., Bezprozvanny, I., Frantz, D.E., Hsieh, J.,

444

2008. Small-molecule activation of neuronal cell fate. Nat. Chem. Biol. 4, 408-410.

445

Schugar, R.C., Robbins, P.D., Deasy, B.M., 2008. Small molecules in stem cell self-renewal and

446

differentiation. Gene Ther. 15, 126-135.

447

Sheng, C., Zheng, Q., Wu, J., Xu, Z., Sang, L., Wang, L., Guo, C., Zhu, W., Tong, M., Liu, L., Li,

448

W., Liu, Z.H., Zhao, X.Y., Wang, L., Chen, Z., Zhou, Q., 2012. Generation of dopaminergic neurons

449

directly from mouse fibroblasts and fibroblast-derived neural progenitors. Cell Res. 22, 769-772.

450

Smith, D.K., Yang, J., Liu, M.L., Zhang, C.L., 2016. Small molecules modulate chromatin

451

accessibility to promote NEUROG2-mediated fibroblast-to-neuron reprogramming. Stem Cell Rep.

452

7, 955-969.

453

Smith, Z.D., Meissner, A., 2013. DNA methylation: roles in mammalian development. Nat. Rev.

454

Genet. 14, 204-220.

455

Son, E.Y., Ichida, J.K., Wainger, B.J., Toma, J.S., Rafuse, V.F., Woolf, C.J., Eggan, K., 2011.

456

Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9,

457

205-218.

458

Szulwach, K.E., Li, X., Li, Y., Song, C.X., Wu, H., Dai, Q., Irier, H., Upadhyay, A.K., Gearing, M.,

459

Levey, A.I., Vasanthakumar, A., Godley, L.A., Chang, Q., Cheng, X., He, C., Jin, P., 2011.

460

5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging. Nat. Neurosci.

461

14, 1607-1616.

462

Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and

463

adult fibroblast cultures by defined factors. Cell 126, 663-676.

464

Tanabe, K., Haag, D., Wernig, M., 2015. Direct somatic lineage conversion. Philos. Trans. R. Soc.

465

Lond. B Biol. Sci. 370, 20140368.

466

Thompson, R., Casali, C., Chan, C., 2019. Forskolin and IBMX induce neural transdifferentiation 15

467

of MSCs through downregulation of the NRSF. Sci. Rep. 9, 2969.

468

Tian, C., Li, Y., Huang, Y., Wang, Y., Chen, D., Liu, J., Deng, X., Sun, L., Anderson, K., Qi, X., Li,

469

Y., Mosley, R.L., Chen, X., Huang, J., Zheng, J.C., 2015. Selective generation of dopaminergic

470

precursors from mouse fibroblasts by direct lineage conversion. Sci. Rep. 5, 12622.

471

Torper, O., Pfisterer, U., Wolf, D.A., Pereira, M., Lau, S., Jakobsson, J., Bjorklund, A., Grealish, S.,

472

Parmar, M., 2013. Generation of induced neurons via direct conversion in vivo. Proc. Natl. Acad.

473

Sci. U. S. A. 110, 7038-7043.

474

Treutlein, B., Lee, Q.Y., Camp, J.G., Mall, M., Koh, W., Shariati, S.A., Sim, S., Neff, N.F.,

475

Skotheim, J.M., Wernig, M., Quake, S.R., 2016. Dissecting direct reprogramming from fibroblast to

476

neuron using single-cell RNA-seq. Nature 534, 391-395.

477

Van Pham, P., Vu, N.B., Dao, T.T., Le, H.T., Phi, L.T., Phan, N.K., 2017. Production of endothelial

478

progenitor cells from skin fibroblasts by direct reprogramming for clinical usages. In Vitro Cell.

479

Dev. Biol. Anim. 53, 207-216.

480

Velasco, I., Salazar, P., Giorgetti, A., Ramos-Mejia, V., Castano, J., Romero-Moya, D., Menendez, P.,

481

2014. Concise review: Generation of neurons from somatic cells of healthy individuals and

482

neurological patients through induced pluripotency or direct conversion. Stem Cells 32, 2811-2817.

483

Victor, M.B., Richner, M., Hermanstyne, T.O., Ransdell, J.L., Sobieski, C., Deng, P.Y., Klyachko,

484

V.A., Nerbonne, J.M., Yoo, A.S., 2014. Generation of human striatal neurons by

485

microRNA-dependent direct conversion of fibroblasts. Neuron 84, 311-323.

486

Vierbuchen, T., Ostermeier, A., Pang, Z.P., Kokubu, Y., Sudhof, T.C., Wernig, M., 2010. Direct

487

conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035-1041.

488

Wainger, B.J., Buttermore, E.D., Oliveira, J.T., Mellin, C., Lee, S., Saber, W.A., Wang, A.J., Ichida,

489

J.K., Chiu, I.M., Barrett, L., Huebner, E.A., Bilgin, C., Tsujimoto, N., Brenneis, C., Kapur, K.,

490

Rubin, L.L., Eggan, K., Woolf, C.J., 2015. Modeling pain in vitro using nociceptor neurons

491

reprogrammed from fibroblasts. Nat. Neurosci. 18, 17-24.

492

Wang, P., Zhang, H.L., Li, W., Sha, H., Xu, C., Yao, L., Tang, Q., Tang, H., Chen, L., Zhu, J., 2014.

493

Generation of patient-specific induced neuronal cells using a direct reprogramming strategy. Stem

494

Cells Dev. 23, 16-23.

495

Wapinski, O.L., Vierbuchen, T., Qu, K., Lee, Q.Y., Chanda, S., Fuentes, D.R., Giresi, P.G., Ng, Y.H.,

496

Marro, S., Neff, N.F., Drechsel, D., Martynoga, B., Castro, D.S., Webb, A.E., Sudhof, T.C., Brunet, 16

497

A., Guillemot, F., Chang, H.Y., Wernig, M., 2013. Hierarchical mechanisms for direct

498

reprogramming of fibroblasts to neurons. Cell 155, 621-635.

499

Xiao, D., Liu, X., Zhang, M., Zou, M., Deng, Q., Sun, D., Bian, X., Cai, Y., Guo, Y., Liu, S., Li, S.,

500

Shiang, E., Zhong, H., Cheng, L., Xu, H., Jin, K., Xiang, M., 2018. Direct reprogramming of

501

fibroblasts into neural stem cells by single non-neural progenitor transcription factor Ptf1a. Nat.

502

Commun. 9, 2865.

503

Xie, X., Fu, Y., Liu, J., 2017. Chemical reprogramming and transdifferentiation. Curr. Opin. Genet.

504

Dev. 46, 104-113.

505

Xu, J., Du, Y., Deng, H., 2015. Direct lineage reprogramming: strategies, mechanisms, and

506

applications. Cell Stem Cell 16, 119-134.

507

Xu, Z., Jiang, H., Zhong, P., Yan, Z., Chen, S., Feng, J., 2016. Direct conversion of human

508

fibroblasts to induced serotonergic neurons. Mol. Psychiatry 21, 62-70.

509

Xue, Y., Ouyang, K., Huang, J., Zhou, Y., Ouyang, H., Li, H., Wang, G., Wu, Q., Wei, C., Bi, Y.,

510

Jiang, L., Cai, Z., Sun, H., Zhang, K., Zhang, Y., Chen, J., Fu, X.D., 2013. Direct conversion of

511

fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits. Cell 152, 82-96.

512

Yang, N., Ng, Y., H. Pang, Z. P., Sudhof, T. C., Wernig, M., 2011. Induced neuronal cells: how to

513

make and define a neuron. Cell Stem Cell 9, 517-525.

514

Yin, J.C., Zhang, L., Ma, N.X., Wang, Y., Lee, G., Hou, X.Y., Lei, Z.F., Zhang, F.Y., Dong, F.P., Wu,

515

G.Y., Chen, G., 2019. Chemical conversion of human fetal astrocytes into neurons through

516

modulation of multiple signaling pathways. Stem Cell Rep. 12, 488-501.

517

Yoo, A.S., Sun, A.X., Li, L., Shcheglovitov, A., Portmann, T., Li, Y., Lee-Messer, C., Dolmetsch,

518

R.E., Tsien, R.W., Crabtree, G.R., 2011. MicroRNA-mediated conversion of human fibroblasts to

519

neurons. Nature 476, 228-231.

520

Yoo, J., Lee, E., Kim, H.Y., Youn, D.H., Jung, J., Kim, H., Chang, Y., Lee, W., Shin, J., Baek, S.,

521

Jang, W., Jun, W., Kim, S., Hong, J., Park, H.J., Lengner, C.J., Moh, S.H., Kwon, Y., Kim, J., 2017.

522

Electromagnetized gold nanoparticles mediate direct lineage reprogramming into induced dopamine

523

neurons in vivo for Parkinson's disease therapy. Nat. Nanotechnol. 12, 1006-1014.

524

Yu, C., Liu, K., Tang, S., Ding, S., 2014. Chemical approaches to cell reprogramming. Curr. Opin.

525

Genet. Dev. 28, 50-56.

526

Yu, H., Su, Y., Shin, J., Zhong, C., Guo, J.U., Weng, Y.L., Gao, F., Geschwind, D.H., Coppola, G., 17

527

Ming, G.L., Song, H., 2015. Tet3 regulates synaptic transmission and homeostatic plasticity via

528

DNA oxidation and repair. Nat. Neurosci. 18, 836-843.

529

Zhang, J., Chen, S., Zhang, D., Shi, Z., Li, H., Zhao, T., Hu, B., Zhou, Q., Jiao, J., 2016.

530

Tet3-mediated DNA demethylation contributes to the direct conversion of fibroblast to functional

531

neuron. Cell Rep. 17, 2326-2339.

532

Zhang, L., Lei, Z., Guo, Z., Pei, Z., Chen, Y., Zhang, F., Cai, A., Mok, Y.K., Lee, G., Swaminnathan,

533

V., Wang, F., Bai, Y., Chen, G., 2018. Reversing glial scar back to neural tissue through

534

NeuroD1-mediated astrocyte-to-neuron conversion. bioRxiv doi.org/10.1101/261438.

535

Zhang, L., Yin, J.C., Yeh, H., Ma, N.X., Lee, G., Chen, X.A., Wang, Y., Lin, L., Chen, L., Jin, P.,

536

Wu, G.Y., Chen, G., 2015. Small molecules efficiently reprogram human astroglial cells into

537

functional neurons. Cell Stem Cell 17, 735-747.

538

Zhao, P., Zhu, T., Lu, X., Zhu, J., Li, L., 2015. Neurogenin 2 enhances the generation of

539

patient-specific induced neuronal cells. Brain Res. 1615, 51-60.

540

Zhu, X., Girardo, D., Govek, E.E., John, K., Mellen, M., Tamayo, P., Mesirov, J.P., Hatten, M.E.,

541

2016. Role of Tet1/3 genes and chromatin remodeling genes in cerebellar circuit formation. Neuron

542

89, 100-112.

543

18

Figure legends Fig. 1. Model of direct conversion strategies.

19

Table 1 List of induced neurons (iNs) generated by different methods. Factors in direct conversion

Efficiency (yielda or purityb)

Reference

Ascl1, Brn2, Myt1L

Yield: 19.5%

Vierbuchen et al., 2010

Ascl1, Brn2, Ngn2 (adenovirus)

Yield: 2.9%

Chanda et al., 2014

Ascl1

Yield: 10%

Chanda et al., 2014

Human fibroblasts

Ascl1

N/A

Chanda et al., 2014

Mouse microglia

NeuroD1

Yield: 35%

Matsuda et al., 2019

Sox2

N/Ac

Niu et al., 2013

Yield: 9.1%

Kim et al., 2011b

Yield: 6%

Caiazzo et al., 2011

Yield: 13.93%

Sheng et al., 2012

Donor cells

Target neuronal type

Strategy： ：transcription factors (TFs)

Mouse fibroblasts Unidentified type

Mouse astrocytes

Neuroblasts

Ascl1, Lmx1a, Pitx3, EN1, Nurr1, Foxa2 Ascl1, Nurr1, Lmx1a Mouse fibroblasts Ascl1, Brn2, Lmx1a, Foxa2 or Dopaminergic

Ascl1, Brn2, Lmx1b, Otx2, Nurr1

neurons

Brn2, Sox2, Foxa2

N/A

Tian et al., 2015

Ascl1, Nurr1, LMX1A

N/A

Caiazzo et al., 2011

Ascl1, Ngn2, Pitx3, Nurr1, Sox2

N/A

Liu et al., 2012

N/A

Pfisterer et al., 2011

Human fibroblasts Ascl1,

Myt1L,

Foxa2,

Brn2,

Lmx1A 20

Human fibroblasts Human astrocytes

Brn2, Ascl1, Myt1L, NeuroD1

Yield: 2%–4%

Pang et al., 2011

Glutamatergic

NeuroD1

90% infected cells

Guo et al., 2014

neurons

NeuroD1

92.8% infected cells

Guo et al., 2014

Ngn2

N/A

Heinrich et al., 2010

Ngn1 or Ngn2, Brn3a

Purity: 4.5%

Blanchard et al., 2015

Yield: 5%‒10%

Son et al., 2011

N/A

Xiao et al., 2018

N/A

Son et al., 2011

Ngn1 or Ngn2, Brn3a

Purity: 10%

Blanchard et al., 2015

Ascl1, Isl2, Ngn1, Myt1L, Klf7

Yield: 14%

Wainger et al., 2015

Ascl1,

Yield: >200%

Mouse astrocytes Nociceptor, mechanoreceptor, Mouse fibroblasts

proprioceptor neurons Ascl1, Ngn2, Brn2, Myt1L, Hb9, Motor neurons Lhx3, Isl1 Neural stem cells

Ptf1a Myt1L, Brn2, Ascl1, NeuroD1

Spinal motor neurons Lhx3, Isl1, Ngn2, Hb9 Human fibroblasts

Nociceptor, mechanoreceptor, proprioceptor neurons Nociceptor neurons

Strategy： ：small-molecule compounds + TFs Mouse fibroblasts

Ngn2,

CHIR99021,

Unidentified type

Ladewig et al., 2012 SB431542

Purity: >80% 21

Human fibroblasts

Cholinergic neurons

Ngn2, Forskolin, Dorsomorphin

Purity: 90%

Liu et al., 2013

Purity: 16%

Miskinyte et al., 2017

Purity: 75%

Herdy et al., 2019

CHIR99021, SB431542, Noggin, Cortical

pyramidal

Human fibroblasts

LDN193189, miR-9/9*, miR-124, neurons Brn2, Myt1L, FEZF2 Pyrintegrin, ZM336372, AZ960,

Human fibroblasts

Unidentified type KC7F2, Ascl1, Ngn2 Dopaminergic

NeuroD1,

Ascl1,

Rivetti di Val Cervo et al.,

Lmx1a,

Human astrocytes

Purity: 30.9% neurons

2017

miR218, SB431542, LDN193189

Strategy： ：small-molecule compounds Forskolin,

CHR99021,

Valproic

Mouse fibroblasts

Yield: 90%

Li et al., 2015

Purity: 35%

Hu et al., 2015

Purity: >80%

Dai et al., 2015

acid, I-BET151 CHR99021,Valproic acid, RepSox, Y-27632, Dorsomorphin, GO6983, Unidentified type Forskolin, SP600125 Human fibroblasts SB-431542,

LDN-193189,

CHIR99021,

PD0325901,

Pifithrin-α and Forskolin

22

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

Purity: 67.1%

Zhang et al., 2015

Yield: 71%

Yin et al., 2019

SAG, Purmo

Human astrocytes

SB431542,

LDN193189,

CHIR99021, DAPT Strategy： ：small-molecule compounds + epigenetic modifiers Mouse fibroblasts

Unidentified type

Tet3, Forskolin

Yield: 81.67%

Zhang et al., 2016

Unidentified type

PTB repression

N/A

Xue et al., 2013

miR-124, Brn2, Myt1L

Yield: 1.76%‒3.52%

Ambasudhan et al., 2011

N/A

Yoo et al., 2011

Purity: 63%

Victor et al., 2014

Yield: 78 of cells (10 mm−2)

Yoo et al., 2017

Strategy： ：microRNAs Mouse fibroblasts

Strategy： ：TFs + microRNAs Glutamatergic Human fibroblasts

NeuroD2,

Ascl1,

Myt1L,

neurons miR-124, miR-9/9* Medium

spiny

Myt1L, miR-124, miR-9/9*, Ctip2,

Human fibroblasts neurons

Dlx1, Dlx2

Strategy： ：electromagnetic fields (EMF) + TFs Mouse fibroblast and

AuNPs, EMF, Ascl1, Pitx3, Lmx1a, Unidentified type

astrocytes a

Nurr1

Yield, numbers of iNs divided by numbers of plated cells; b Purity, numbers of iNs divided by numbers of DAPI; c N/A, not available. 23

Figure1. Model of direct conversion strategies