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Small molecules combined with collagen hydrogel direct neurogenesis and migration of neural stem cells after spinal cord injury

 Yaming Yang, Yongheng Fan, Haipeng Zhang, Qi Zhang, Yannan Zhao, Zhifeng Xiao, Wenbin Liu, Bing Chen, Gao Lin, Zheng Sun, Xiaoyu Xue, Muya Shu, Jianwu Dai

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Small molecules combined with collagen scaffold direct neurogenesis and migration of neural stem cells after spinal cord injury

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Small molecules combined with collagen hydrogel direct neurogenesis and migration of neural stem cells after spinal cord injury

Yaming Yang1,2, Yongheng Fan1,2, Haipeng Zhang1,2, Qi Zhang1, Yannan Zhao1, Zhifeng Xiao1, Wenbin Liu1, Bing Chen1, Gao Lin1,2, Zheng Sun1,2, Xiaoyu Xue1,2, Muya Shu1,2, Jianwu Dai1,2,*

1State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; 2University of Chinese Academy of Sciences, Beijing 100049, China;


Complete spinal cord injury (SCI) leads to cell death, interruption of axonal connections and permanent functional impairments. In the development of SCI treatments, cell transplantation combined with biomaterial-growth factor-based therapies have been widely studied. Another avenue worth exploring is the generation of neurons from endogenous neural stem cells (NSCs) or reactive astrocytes activated by SCI. Here, we screened a combination of four small molecules, LDN193189, SB431542, CHIR99021 and P7C3-A20, that can increase neuronal differentiation of mouse and rat spinal cord NSCs. Moreover, the small molecules loaded in an injectable collagen hydrogel induced neurogenesis and inhibited astrogliogenesis of endogenous NSCs in the injury site, which usually differentiate into astrocytes under pathological conditions. Meanwhile, induced neurons migrated into the non-neural lesion core, and genetic fate mapping showed that neurons mainly originated from NSCs in the parenchyma, but not from the central canal of the spinal cord. The neuronal regeneration in the lesion sites resulted in some recoveryof locomotion. Our findings indicate that small molecules combined and collagen hydrogel is a potential therapeutic strategy for SCI by inducing endogenous in situ NSCs to form neurons and restore damaged functions.

Keywords: complete spinal cord injury, small molecules, collagen hydrogel, neural stem cells, neurogenesis, in vivo, migration


Complete spinal cord injury (SCI), which is typically caused by external insults, including traffic accidents, sports-related injuries and violent incidents, can lead to severe functional impairments of locomotion and sensation, and is incurable with current therapies [1]. Over the last few decades, many studies have investigated traumatic SCI with respect to fundamental cellular processes and therapeutic evaluation of treatments. However, models of traumatic SCI often have non-uniform injuries that can lead to differences in individual responses to treatments. Furthermore, it is difficult to evaluate whether axons detected in lesion sites represent regenerating axons or sprouting axons [2]. Anatomically complete SCI results in larger non-neural areas in the lesion core, and this model avoids the problem of sprouting/regeneration triggered in some traumatic SCI models and, therefore, reflects genuine axonal regeneration [3,4].

Neural stem cells (NSCs) are a group of multipotent and self-renewing cells that be able to produce neural lineage cells, including neurons, astrocytes, and oligodendrocytes. NSCs are also present in the adult spinal cord and can be proliferated in vitro [5,6]. After injury, NSCs can be directly activated from the quiescent state and are, therefore, promising candidates to produce neuronal progeny for treating SCI [7,8]. Compared with cell transplantation, the approach of recruiting reactive endogenous NSCs to generate neurons avoids the difficulties of cell production and concerns of graft immunogenicity and rejection. However, activated NSCs show limited migration to neighboring injury sites and usually differentiate into astrocytes under pathological conditions [9]. Consequently, inducing reactive endogenous NSCs to differentiate into neurons for tissue repair and neuroregeneration is highly desirable [10].

Approaches have been established for the treatment of neurodegenerative diseases that direct reactive NSCs towards a neuronal fate by modulating cell activation, differentiation or reprogramming [7]. One strategy that has produced encouraging results is the employment of small molecules to stimulate differentiation of pluripotent stem cells or NSCs into neurons [11–13]. Small molecules have advantageous features that confer the ability to regulate specific cellular processes and to facilitate investigation of basic cell biology. Moreover, different combinations of small molecules can reprogram astrocytes or fibroblasts into functional neurons without introduction of any exogenous genes [14–16]. Although small molecules hold great potential in regenerative medicine and their practical application has been improved in vitro, it is uncertain whether small molecules can modulate the differentiation of endogenous NSCs into neurons in the complex environment that is hostile to neurogenesis after SCI.

Here, we identified a combination of small molecules that can significantly increase the differentiation rate of NSCs into neurons in vitro as well as in the SCI injury site with assistance from an injectable collagen hydrogel. Meanwhile, genetic fate mapping showed that neurons generated from NSCs can gradually migrate into the non-neural lesion core. Our data also indicate that neurons mainly originated from NSCs in the parenchyma, but not from the central canal of the spinal cord. In brief, our data indicate that chemical compounds provide a promising strategy for treating SCI by employing the regenerative potential of endogenous NSCs to generate new neurons and restore damaged functions.


Improving neuronal differentiation of spinal cord NSCs using small molecules

To investigate whether small molecules can induce neurogenesis from mouse spinal cord NSCs, we first selected SMAD signal inhibitors (LDN193189, 0.25 μM and SB431542, 5 μM) [17] and a WNT signal agonist (CHIR99021, 3 μM) [18], which have been widely used to inhibit a glial fate and to induce a neuronal fate. We also selected molecules that play essential roles in the direct reprogramming of fibroblasts or astrocytes into neurons and in the induction to neurons from NSCs: DAPT (a γ-secretase inhibitor, 1 μM) [19], P7C3-A20 (a proneurogenic and neuroprotective chemical, 3 μM) [20], ISX9 (a neurogenesis modulator, 5 μM) [16], purmorphamine (a Smoothened agonist, 1 μM) [21], dorsomorphin (an AMPK inhibitor, 1 μM) [15], and SU5402 (an inhibitor of fibroblast growth factor signaling, 1 μM) [22].

We isolated spinal cord NSCs (sc-NSCs) from postnatal mice (Figure 1A) and cultured them for at least two passages. Then we determined the identity of NSCs by immunostaining with Nestin, SOX2, GFAP and TUJ1. The majority of NSCs were immunopositive for Nestin (91.8±2.1%) and SOX2 (97.3±1.3%), and about 20% of the cells were also positive for GFAP (Figure 1B and C). No more than 3% of cells spontaneously differentiated toward neurons (Figure 1C). To evaluate the effect of small molecules on neuronal differentiation, we digested the neural spheres into single cells, cultured them in a monolayer and treated them with small molecules. We included LDN193189, SB431542 and CHIR99021 (LSC) in a basal induction medium, and individually supplemented the medium with DAPT (D), P7C3-A20 (P), ISX9 (I), purmorphamine (Pu), dorsomorphin (Do) or SU5402 (Su). NSCs were then exposed to the different chemical cocktails and the effect on neuronal differentiation was evaluated after 7 days. TUJ1 immunostaining indicated that the addition of small moleculesincreased the differentiation capacity of NSCs towards neurons. Compared with control medium without chemical compounds, LSC-P had the most significant positive effect on neuronal differentiation, followed by LSC-D and LSC-Pu, while LSC-Su, LSC-Do, and LSC-I had almost the same effect as LSC (Figure 1D). Hence, to further increase efficiency of neuronal differentiation, we performed another round of screening in which LSC-P was supplemented with one additional small molecule (Table S2). Addition of RepSox (an inhibitor of TGFβ, 0.5 μM), RG108 (an inhibitor of DNA methyltransferase, 10 μM), VPA (an inhibitor of histone deacetylase, 0.5 mM), or forskolin (an activator of cAMP, 10 μM) produced slight positive effects on neuronal differentiation, but at the expense of increased cell death (data not show). We therefore used LSC-P in the subsequent experiments.

To investigate the effect of LSC-P on the neural fate of sc-NSCs, we first detected changes to astroglial differentiation. Immunostaining indicated that LSC-P inhibited expression of GFAP in sc-NSCs, and astroglial differentiation was down-regulated from 65.8±1.9% to 30.5±1.3% (Figures 1E and F). We also examined the neuronal differentiation of sc-NSCs using three well established neuronal markers, TUJ1, MAP2 and NeuN (Figures 1G–J). Compared with control groups, LSC-P greatly induced expression of TUJ1 from 24.6±0.5% of cells to 62.4±0.8% of cells (Figure 1K). Expression of the mature neuronal markers, MAP2 and NeuN, was enhanced to 53.2±0.6% and 51.4±1.2% of cells, respectively (Figure 1K). These data indicated that LSC-P efficiently inhibited astroglial differentiation and induced neurogenesis from sc-NSCs.

We also performed whole-cell patch-clamp recording to test electrophysiological activities of the induced neurons generated from sc-NSCs (Figure S1A). The majority of detected neurons showed expected electrophysiological features. The neurons were capable of eliciting sodium and potassium currents in response to a series of voltage pulses (Figure S1B). Furthermore, characteristic traces of membrane action potentialwere detected when injecting a series of currents into neurons (Figure S1C). Additionally, spontaneous action potentials were examined in mature neurons (Figure S1D). These data collectively indicated that neurons induced from sc-NSCs by the cocktail of small were functionally mature.

Small molecules combined with collagen hydrogel increase neuronal differentiation of NSCs and induce their migration into the injury site after spinal cord injury

To expose cells in the lesion site to small molecules for longer periods of time, LSC-P was applied with an injectable collagen hydrogel that has excellent biocompatibility, adhesiveness and biodegradability [23,24] (Figure 2A). The in vitro release of LSC-P from collagen hydrogel was investigated by liquid chromatography mass spectrometry (LC-MS) for the first 6 days (Figure S2A). SB431542, LDN193189, P7C3-A20 and CHIR99021 displayed 76.87%, 84.75%, 83.77% and 79.47% accumulated release after 6 days, respectively (Figure 2B and Table S3). Meanwhile, to explore the in vivo release of small molecules from collagen hydrogel, we also detected the retention of LSC-P in collagen hydrogel in lesion sites of the spinal cord after the first 6 days. Our data showed that small molecules could still be detected at day 6 (Figure 2C). However, without the collagen hydrogel, small molecules were no longer detected in lesion sites even at day 2 (data not shown in the figure). These data indicated that injection with collagen hydrogel could extend the exposure of cells in injury sites to LSC-P after SCI.

To explore whether small molecules can induce neurogenesis from in situ sc-NSCs after SCI, we crossbred mice with CreERT2 inserted into the Nestin locus with Rosa26-tdTomato (tdT) reporter mice to target endogenous NSCs [25] (Figure S2B). To exclude ectopic labeling of neuronal cells, we chose the line that displayed the highest specificity for NSCs [26]. Recombination was induced in adult transgenic animals by five daily injections of tamoxifen followed by a 7 day clearing time (Figure S2C). We then detected the distribution of sc-NSCs in NestinCreERT2-tdT mice. In transverse and longitudinal sections of the spinal cord, sc-NSCs were detected in the parenchyma and the central canal (Figure S2D).

Spinal cords were surgically injured by creating 2 mm long gap at level T8, and then the mixture of small molecules and collagen hydrogel was immediately injected into the lesion site (Figure S2E). Mice were sacrificed on day 14 after injury, and longitudinal sections of each injury site were obtained and observed. Unexpectedly, compared with control groups treated with collagen hydrogel alone or collagen hydrogel with normal saline, cells originating from Nestin-positive cells in the group treated with LSC-P could efficiently migrate into the injury site (Figure 2D).

We then evaluated differentiation of sc-NSCs toward neural lineage cells in the lesion zone. Approximately 80±2.9% of tdT+ cells in control groups showed positive immunostaining for GFAP (Figures 2E, G), whereas, in experimental groups, no more than 10% of tdT+ cells displayed immunopositive signals for GFAP (Figures 2F, G). GFAP immunopositive signals were not detected in the lesion core in experimental or control groups. We then investigated fate changes of tdT+ cells to neurons with different treatments after SCI. TUJ1 immunostaining produced the opposite trends to GFAP immunostaining in different groups. In control groups, no more than 4% of tdT+ cells were immunopositive for TUJ1 (Figures 2H, J), which was consistent with previous studies showing that NSCs usually differentiate into astrocytes but not neurons under pathological conditions after SCI. However, about 70% of tdT+ cells were immunostained for TUJ1 in lesion sites (Figures 2I and J). Unfortunately, we did not observe positive signals in lesion cores for the mature neuronal markers, MAP2 or NeuN. These results indicated that LSC-P induced migration of sc-NSCs to lesion cores and promoted their differentiation into neurons but not astrocytes after SCI. It is worth noting that application of LSC-P did not affect the formation of glia scar in the lesion site (Figure S2F). Considering that the environment is hostile to the survival and maturation of induced neurons differentiated from sc-NSCs, we decided to supplement trophic factors into injury sites. As shown in Figure 3A, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and glial cell line-derived neurotrophic factor (GDNF) were injected into injury sites on day 10 after SCI (Figure S3A), and then maturity of the induced neurons was evaluated. We found that neurofilaments of induced neurons were longer when treated with trophic factors compared with those in the non-treated groups (Figure 3B and C). Next, we explored neurogenesis in different tissue compartments of the injury site that have been previously defined based on unique cell morphology [8, 27]. TUJ1 immunostaining indicated that LSC-P does not have an obvious impact on neuronal differentiation of NSCs in uninjured tissue (1 cm away from lesion sites) or in spared but reactive neural tissue, whereas positive RFP signals were detected in the astrocyte scar border (43.8±1.5%) and injury site (64.7±3.9%), including the lesion edge and lesion core (Figures 3D and E). Noticeably, TUJ1+ neurons in the uninjured zone had a round morphology, while those in the lesion zone were thinner and longer. Meanwhile, NeuN-positive cells in the lesion edge in control groups were much less numerous than in LSC-P treated groups (Figure 3F). It was worth noting that P7C3-A20 alone also have neuroprotective effect in the lesion edge (Figure S3B). We then investigated the maturity of induced neurons, and some tdT+ cells were immunopositive for NeuN, which indicated that small molecules and trophic factors promoted the survival of neurons after injury. Similar to the situation for TUJ1, NeuN+ tdT+ cells were only detected in the astrocyte scar border and lesion zone, and the percentage was no more than 30% (Figures 3G and H). Moreover, the nuclei of NeuN+ cells in injury sites were smaller than those in normal neural tissues. A single nutritional supplementation only maintained maturation of neurons for a period of time, and the survival rate of induced neurons progressively declined over time (Figure 3I and J). Together, these data indicated that the NSC-derivedinduced neurons in injury sites can mature to a certain extent with the assistance of neurotrophic factors.


Neurogenesis at the injury site improved electrophysiological and locomotor recovery

To investigate the specific subtypes of induced neurons in the injury site, we immunolocalized vesicular glutamate transporter 1 (VGlut1), γ-aminobutyric acid (GABA), 5-hydroxytrptamine (5-HT), and choline acetyltransferase (ChAT) (Figures 4A–C). The data indicated that 18.3±2.0% of tdT+ cells co-expressed ChAT, with no more than 6% of tdT+ cells positive for 5-HT or VGlut1. No GABA signals were observed at injury sites (Figures 4D). We then performed immunostaining for synapsin 1 (SYN1) to assess the formation of synapses by induced neurons in the lesion zone. We found that only a small population of tdT+ cells (cell number, 65±5) in the lesion zone were positive for SYN1 in the collagen+SM+GBN group (Figures 4E and F). In contrast, SYN1 staining was rarely observed in the lesion edge or core in control groups (Figure 4F). We then examined whether LSC-P influenced the microenvironment of the lesion zone. First, the axon growth-supporting substrate, laminin, was detected and its expression was much higher in LSC-P groups than in control groups (Figures S4A and C). Then we investigated levels of chondroitin sulfate proteoglycans (CSPGs), which is a primary indicator of neuronal regeneration in lesion sites. The CSPGs immunostaining indicated that it was obviously down-regulated in injury sites by LSC-P compared with control groups (Figures S4B and D). These data showed that LSC-P treatment after SCI improved the lesion site microenvironment for neuronal regeneration.

We next performed electrophysiological evaluation and behavioral assessments to verify functional motor regeneration. The electrophysiological analyses indicated that the amplitude in all SCI groups, with or without treatment, was obviously lower than that in uninjured mice (Figures 4G and H). However, among the five SCI groups, amplitude was moderately restored in the collagen+SM+GBN group compared with the other four groups. Meanwhile, the latency response was also partially recovered in the collagen+SM+GBN group, but it was insignificant compared with that in healthy mice (p<0.01, Figure 4I). We also explored locomotor recovery of hind limbs according to the Basso Mouse Scale (BMS) during a twelve-week observation period. Mice in the collagen+SM+GBN group displayed substantial improvement compared with the other four control groups from the sixth week post-surgery until the animals were sacrificed (p<0.01, Figure 4J) and reached a plateau at eight weeks (BMS score 2.2, plantar placing of the paw with weight support, occasionally dorsal stepping but no plantar stepping). These findings showed that a combination of LSC-P, collagen hydrogel and trophic factors had beneficial effects on locomotor outcome after complete SCI.

 Origins of TUJ1-positive cells in the lesion core

The TUJ1 immunostaining in figure 3B shows more TUJ1-positive cells than tdT+ cells in NestinCreERT2-tdT mice, indicating that a fraction of the TUJ1+ cells may be derived from cells other than recombinant or non-recombinant NestinCreERT2-tdT cells. To further dissect the origins of TUJ1+ cells in the lesion core, we first verified the response of astrocytes and oligodendrocyte progenitors to small molecules, which are presumed to be reactivated after SCI [28]. We then used GFAPCreERT2-tdT mice [29] and NG2CreERT2-tdT mice [30] to validate our hypothesis by linage tracing (Figure S4E). We detected no tdT+ cells in the lesion core in control or experimental groups, but a small proportion of LSC-P-induced tdT+ cells was immunopositive for TUJ1 (Figures 5A and B), indicating that small molecules can change the fate of reactive astrocytes (15.2±0.7%) or oligodendrocyte progenitors (17.0±2.7%) towards neurons (Figure 5E). After excluding neurogenesis as the origin of reactive astrocyte or oligodendrocyte progenitors in thelesion core, we investigated whether part of the TUJ1 immunoreactivity was from intrinsic neurons. Then the neurons were labeled by biotinylated dextran amine (BDA) tract-tracer between one and two segments rostral to SCI sites [31] (Figure S3A), and surprisingly, labeled axons were found 1 mm anterior to the lesion core (25.2±2.2%) (Figures 5C and F).

NSCs in the adult spinal cord are distributed in the parenchyma and central canal. Immunopositivity for the NSCs markers, Vimentin and CD133, together with tdTomato, verified the identity of NSCs in the central canal (Figure S4F). To determine which NSCs could migrate into the lesion core following LSC-P treatment, we used another transgenic mouse, Foxj1 CreERT2-tdT [32], which expresses RFP only in the central canal of the spinal cord. Then, we performed SCI, and no positive tdT+ signals were found in the lesion core in either group (Figures 5D, F), and no tdT+ cells were immunopositive for TUJ1 in the lesion edge (Figure 5E). These data indicated that TUJ1-positive cells in the lesion core mainly originated from Nestin-positive cells of the parenchyma and regenerated axons. Meanwhile, LSC-P was also able to induce reactive astrocyte or oligodendrocyte progenitors to differentiate into neurons in the lesion edge to a certain extent.

LSC-P induced differentiation of NSCs into neurons before their migration into the lesion core

The induced neurons in the lesion core raised the question of whether migration precedes differentiation or vice versa. To observe dynamic changes at early stages, we obtained samples from control groups andsmall molecule groups at day 3, 5, 7, and 10 after SCI. We then performed DCX immunostaining, and DCX-positive signals were apparent at day 3 and were concentrated in the lesion edge after LSC-P treatment. At this time point, some of the tdT+ cells were positive for DCX, but no tdT+ cells were visible in the injury site except for those at theedge (Figure S5A). Two days later, some DCX+ tdT+ cells were detectable within 0.5 mm of the lesion edge, and they had migrated farther towards the lesion core by day 7 (Figures S5B and C). At day 10, some of the DCX+ tdT+ cells had moved into the lesion core from either side of the injury zone (Figure S5D). However, in control groups, almost no tdT+ cells were labeled with DCX at early days (data not shown), or at day 7 (Figure S5E). This indicated that after SCI, activated NSCs first differentiated into neurons before migrating to the lesion core.

 Evaluation of LSC-P compounds in the treatment of spinal cord injury

We explored the specific roles of the LSC-P chemicals according to three considerations: induction of neurons, inhibition of astrocytes, and migration of Nestin-positive cells. LSC-P was divided into three groups based on the mechanism of action of different compounds: LDN193189+SB431542, CHIR99021 and P7C3-A20. Immunostaining showed that the number of TUJ1+ cells induced by CHIR99021 (21.7±2.0%), LDN193189+SB431542 (28.9±1.8%) and P7C3-A20 (35.0±1.3%) gradually increased in the lesion zone (Figures 6A and B). We also found that CHIR99021 (332±27 tdT+ cells) plays an important role in the migration of Nestin+ cells in the lesion core, followed by P7C3-A20 (233±15 tdT+ cells) and LDN193189+SB431542 (144±17 tdT+ cells) (Figure 6C). Next, we assessed each of the small molecules for inhibition of astrocytes by detecting GFAP. LDN193189+SB431542 (33.9±2.2%) clearly decreased GFAP expression as predicated, and this effect was less for P7C3-A20 (42.7±2.8%) and CHIR99021 (55±4.1%) (Figures 6D and E). Quantitative analysis revealed that the small molecules in LSC-P play different but important roles in the induction of neurogenesis after SCI.

Neurogenesis induced by LSC-P in rats in vitro and in vivo

Finally, we examined whether neurogenesis induced in mice by LSC-P could be extended to rats, in which injury site pathophysiology is thought to be similar to that of humans. First, we isolated sc-NSCs from postnatal rats and cultured them for 10 days to form spheres. Next, these cells were exposed to LSC-P medium, and the efficiency of neuronal differentiation was estimated by immunostaining with TUJ1, MAP2 and NeuN. LSC-P increased the expression of the neuronal markers compared with control groups (TUJ1, from 23.8±1.9% to 54.8±2.4%, MAP2, from 18.6±1.0% to 44.5±2.5% and NeuN, from 16.9±1.5% to 41.3±0.9%) (Figures 7A–E). Furthermore, we assessed whether LSC-P could stimulate neurogenesis in the injury site after SCI. Rats were divided into two groups (collagen hydrogel+normal saline and collagen hydrogel+small molecules), and neurogenesis was evaluated at day 21 post-injury. TUJ1 immunostaining indicated that neurogenesis in the lesion sites was greatly induced by LSC-P (cell number, 661±20) compared with the control group (cell number, 53±6) (Figures 7F, H). Meanwhile, LSC-P also increased expression of laminin, which provides a more conducive environment for neuronal survival (Figures 7G, I). Our findings showed that the combined chemicals of LSC-P were able to induce neurogenesis and stimulate the migration of regenerated neurons in rats after SCI. 


In this study, we identified four small molecules that regulate signaling pathways involved in neuronal induction in the injury site after spinal cord injury. Furthermore, the induced neurons could migrate into the lesion core and mature with additional supplementation of neurotrophic factors. We also showed that LSC-P induced axonal regeneration that was labeled by BDA, but genetic fate mapping showed that LSC-P failed to activate neurons generated from reactive astrocytes and oligodendrocyte progenitors in the lesion core. Finally,  the neurogenesis stimulated in the injury siteresulted in a degree of movement recovery. Our findings indicate that small molecules provide an alternative approach for treating SCI by promoting neurogenesis from endogenous NSCs at the injury site.

Regenerative medicine mainly focuses on replacement or regeneration of organs, tissues or cells, to repair normal function. The application of small molecules in regenerative medicine is at an early stage but their use is increasing, especially for neuroregeneration [33]. Many studies have indicated that the stepwise use of small molecules can facilitate the maintenance and proliferation of pluripotent stem cells, as well as efficiently guiding and accelerating their fate towards specific neural progenitors or neurons [34]. Meanwhile, small molecules are capable of directly reprogramming fibroblasts and astrocytes into neurons or NSCs in vitro without any exogenous gene integration [14, 15]. However, it is uncertain whether small molecules have positive effects for neuroregeneration in situ, although they confer a low risk of tumor formation compared with transplanted neural cells derived from pluripotent stem cells. Adult NSCs and reactive astrocytes are the best candidates for in situ neurogenesis at injury sites [7, 8]; therefore, in our primary screen, we mainly selected small molecules known to inhibit astrocytes and promote neuronal induction. However, inefficiency of small molecule combinations may result from species differences or differences in central nervous system areas studied [14]. The study also suggested that their protocol is more appropriate for astrocytes originating from human brains. Adult neurogenesis in the mouse hippocampus was investigated by combination of compounds that was simplified from nine to four and injected in vivo [19]. However, the effect of the four compounds was moderate on NSC neurogenesis in the spinal cord, which may have an intrinsically lower capacity for neuronal differentiation compared with the brain. We found that addition of P7C3-A20 clearly increased the efficiency of sc-NSC neuronal differentiation in vitro, and was also effective on adult NSCs in the injury site after SCI. Our findingsindicate that small molecules may be a preferred choice for neuronal regeneration by recruiting resident adult NSCs in lesion sites.

LSC-P induced neurogenesis in the lesion core after SCI, but the recovery of electrophysiological activities and locomotor function was not obvious. We suggest four reasons to explain these results. First, although the number of induced neurons in the lesion zone appeared substantial, it was still far below the normal innervation density. Second, positive signals for the mature markers, MAP2, NeuN and SYN, were limited in the induced neurons of the initial experiments. With the addition of neurotrophic factors, a small proportion of induced neurons were immunopositive for MAP2 and NeuN. However, a single nutritional supplementation can only maintain maturation of neurons for a period of time, and multiple injections of growth factors to the injury site can cause secondary damage to induced neurons. Third, apart from neuronal rehabilitation and neurotrophic factors, remyelination of induced neurons or regenerated neurons is also required to shape reformation of neural circuitry [35], but this is difficult to achieve in the lesion core without the participation of oligodendrocytes and other necessary components. Last but not least, the recovery of behavior requires the restoration of neural circuitry to pre-injury patterns and an accurate orchestration of sequential genetic and epigenetic events [36], which are impossible to complete under pathological conditions. The induced neurons or regenerated axons may, therefore, be mistargeted and misdirected along incorrect routes, resulting in misplaced connections. Similarly, although robust axon regrowth can occur in the lesion sites after SCI, improvement in locomotor function and electrophysiological activity is not guaranteed [31]. This suggests that functional recovery results from improvements in the multiple factors mentioned above, and that much still needs to be discovered to enable spinal cord injury repair.


Our findings show that application of compounds, LDN193189, SB431542, CHIR99021 and P7C3-A20 efficiently induced NSCs to differentiate into neurons and inhibited their previous destiny towards astrocytes under pathological conditions. These results indicate that small molecules provide a potential therapeutic strategy for treating SCI by recruiting regenerative potential through modulating the fate of resident NSCs in injury sites. Furthermore, the successful neurogenesis induced in the injured spinal cord indicates that small molecules can be applied to the treatment of other neurological diseases.

Author contribution

 Y.Y. and J.D. conceived and designed the experiments. Y.Y., Y.F., H.Z., W.L., Z.S. L.G., Q.Z., Y.Z., Z.X., B.C., X.X. and M.S. performed the experiments and analyzed the data. Y.Y. and J.D. wrote the manuscript.


 This work was supported by grants from the National Natural Science Foundation of China (No. 81891000), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16020100, XDA16040000), the National Key R&D Program of China (2017YFA0104701, 2017YFA0104704, 2016YFC1101501, 2016YFC1101502) and the National High Technology Research and Development Program of China (863 project, No.2015AA020312).

Materials and Methods

Ethics statement: Animal experiments were approved by the Animal Care and Use Committee of the Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences.

Animals: Female C57BL/6 mice and female Sprague-Dawley rats were 8–10 weeks old and were purchased from Beijing Vital River Laboratory Animal Technology. All mice and rats were housed in conditions of controlled temperature and humidity with a 12 h light/dark cycle.

 Nestin-CreERT2 (# 016261), GFAP-CreERT2 (# 012849), Foxj1 (# 027012)-CreERT2, NG2 (# 008538)-CreERT2, and Rosa26-CAG-LSL-tdTomato (#

007909) mice were purchased from the Jackson Laboratory. Recombination was initiated by five daily intraperitoneal injections of tamoxifen (Sigma). The recombined mice were used in experiments at least 7 days after tamoxifen injection to allow tamoxifen clearance.

NSCs culture and differentiation: NSCs were isolated from spinal cords of newborn C57BL/6 mice. After carefully striping away the meninges, spinal cord tissue was cut into 1 mm3 pieces, and digested in TrypLE Express Enzyme (Gibco) for 15 min at 37°C. The trypsin was then diluted with PBS and cells were collected by centrifugation at 500 g for 5 min. Next, cells were suspended in serum-free DMEM/F12 medium containing 20 ng mL−1 bFGF, 20 ng mL−1 EGF, and 2% B27. Half the medium was changed for fresh medium every three days. Ten days later, the newly formed neurospheres were digested into single NSCs and cultured in adhesion medium containing 10% FBS. Single NSCs were cultured on poly-D-lysine-coated coverslips in 24-well plates at 1 × 105 cells/well. The medium was then replaced with neuronal induction medium (NIM) consisting of DMEM/F12 medium, 2% B27 and small molecule drugs (3 μM CHIR99021, 5 μM SB431542, 0.25 μM LDN193189, 3 μM P7C3, 1 μM DAPT, 5 μM ISX9, 0.5 μMpurmorphamine, 1 μM dorsomorphin, 1 μM SU5402, 0.5 μM Repsox, 10 μM RG108, 0.5 mM VPA, 10 μM forskolin). Half the medium was changed for fresh medium every 2 days. The culture of rat NSCs was similar to that of mice NSCs.

Spinal cord injury: Surgical procedures were performed on mice and rats as previously described with some alterations [37]. Briefly, 2 mm-long T7–T8 spinal cord sections were removed after intraperitoneal anesthesia with sodium pentobarbital (35 mg/kg). The injury site was irrigated with normal saline and bleeding was controlled with cotton-tipped applicators. The musculature and skin were then sutured and experimental treatments applied [37]. Rats weighing 200–220 g were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg) until responsiveness to tail and paw pinch were completely absent. Then the lower thoracic area of the back was shaved and the skin cleaned with Betadine. The skin and muscle were opened to expose vertebrae T8–T10 using a #15 blade. Then a laminectomy was performed at T9 to expose the spinal cord and a 2 mm long spinal cord section was cut and removed. The transection site was irrigated with normal saline, and bleeding was controlled using Merocel. Then the muscle layers and the skin were sutured.

Manual bladder voiding for mice and rats was performed twice daily after SCI in the first 14 days. Then, it reduced to once daily, and two months later, once every two days until reflex voiding recovered. All of the mice and rats used for SCI were female. The numbers of animals used in the experiments of spinal cord injury were shown in Table S1. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health.

Preparation of injectable collagen hydrogel: Collagen hydrogel were prepared for injection from bovine collagen as described previously [38]. Freshly harvested bovine aponeurosis was incubated with 1% tri(n-butyl) phosphate for 48 h. The resulting collagen was then incubated in 1% trypsin for 1 h to remove debris, washed in deionized water and then dissolved in 0.5 M acetic acid for 24 h. The collagen was then dialyzed in deionized water for 10 days, lyophilized and dissolved in normal saline to give a 30 mg/ml collagen hydrogel for injection.

Small molecule treatment in vivo: The final concentration of small molecules for in vivo treatment was double that used in vitro (2×) (6 μM CHIR99021, 10 μM SB431542, 0.5 μM LDN193189, and 6 μM P7C3). First, 4× small molecule cocktails and the equivalent volume of injectable collagen hydrogel were prepared and each was transferred into a 1 ml syringe (BD, 309628), which were connected by a three way plastic tube (BD,394605). After sufficient mixing (being careful to avoid the introduction of air bubbles), the 2× small molecule-collagen hydrogel mixture was applied to each spinal cord injury site when bleeding was controlled. A control group was similarly prepared but the 4× small cocktail was replaced with 2× normal saline.

Whole cell patch clamp: The detection of induced neurons was performed at day 14 after small molecule treatment. Neurons cultured on poly-D-lysine-coated coverslips were used for electrophysiological analysis. The whole-cell recording was done in voltage-or current-clamp mode. The sodium/potassium currents, action potentials, and spontaneous synaptic currents were measured using an Axopatch 200B or MultiClamp 700A amplifier (Molecular Devices). Details of the parameters used were as previously described [39].

Motor evoked potential (MEP) detection: MEP was assessed by scalp stimulation to evaluate muscle responses. For electrophysiological analyses, all mice received the same anesthesia treatment. The operational procedures were performed as previously described [37].

Behavioral assessment: We used the Basso Mouse Scale (BMS) to estimate functional recovery in mice as previously described [37]. Our observations mainly focused on ankle movement of the hind limbs, the position of the soles of the feet, the state of the toes during walking, and the stability of the body. Ankle motor status was rated as: no, slight, and extensive. The placement of the sole of the foot included landing of the dorsal foot and landing of the sole. Plantar stepping was divided into incidental and recurrent. The position of toes mainly included: digits of the paw being parallel to the body, turned out away from the body, or turned inward toward the midline. The assessment was performed by two observers who were blind to the experimental conditions. They recorded a BMS score for each mouse at week 2, 4, 6, 8, 10, and 12 after surgery based on spontaneous or voluntary hind limb movements.

Immunostaining: Immunohistochemistry was performed as described previously. T6– T10 spinal cord samples were collected, fixed in 4% formaldehyde for 24 h and then cryoprotected in 30% sucrose for 24 h at 4°C. The tissues were embedded in optimal cutting temperature compound (OCT) and cut into 20-µm-thick sections (CM1950, Leica) as previously described. [21]. Images were obtained under a Leica SP8 confocal microscope.

The following primary antibodies were incubated overnight at 4°C: chicken anti-GFAP (1:1000, ab4674, abcam), rabbit anti-NeuN (1:200, ab17748, abcam), rabbit anti-Tuj1 (1:500, GTX130245, GeneTex), mouse anti-Tuj1 (1:500, ab78078, abcam), rat anti-RFP (1:500, 5F8, chromoTek), chicken anti-MAP2 (1:500, ab5392, abcam), goat anti-DCX (1:500, ab113435, abcam), rabbit anti-Laminin (1:500, ab11575, abcam), mouse anti-CSPG (1:500, C8035, Millipore), rabbit anti-Synapsin I (1:500, ab64581, abcam), goat anti-ChAT (1:100, AB144P, Millipore), rabbit anti-5-HT (1:1000, AB572263, Immunostar), ), rabbit anti-GABA (1:200, ab75838, abcam). Fluorescent secondary antibodies from different host sources were labeled with Alexa Fluor 488, 594, or 633 (all 1:500, Invitrogen) and were incubated for an hour at room temperature.

BDA and neurotrophic factor injection: Tract-tracing of axons was performed by BDA (Dextran, Alexa Fluor™ 488, 10000 MW, D22910, Thermo Fisher) injection using a MICRO2T SMARTouchTM microinjection controller (WPI). BDA was prepared at 10% w/v in saline, and 2 × 0.5 μl was injected into neuronal axons between one and two segments rostral to the lesion site 7 days post injury. BDA labeling was evaluated 14 days post injection.

Neurotrophic factors were administered 10 days post injury via a second surgical procedure under anesthesia. The injury site was exposed after opening the skin and muscle on the back. The growth factor mixture containing BDNF (200 ng/ml, Peprotech), NT3 (200 ng/ml, Peprotech), and GDNF (100 ng/ml, Peprotech) was immediately injected into four sites (two on each side of the lesion core; 0.25 μl into each site) using a glass micropipette, as previously described [31].

 Release of small molecules from collagen hydrogel in vitro and in vivo First, 100 μl of 2× concentrations of small molecules in normal saline were mixed well with an equal volume of collagen hydrogel in a well of 24-well plate. Then, 100 μl of normal saline was added to the surface of the mixture at a temperature of 37°C. Next, 100 μl of liquid on the surface of the mixture was collected on day 1, 2, 3, 4, 5, and 6 for LC-MS analysis (Shimadzu, LC30AD) [40, 41]. One hundred microliters of fresh normal saline was added to the mixture surface after each collection, n=3 at each time point. The release of small molecules from the collagen hydrogel was calculated by detected weight/original weight.

C57/BL6 mice were randomly assigned into two groups for detection of small molecule release. One group received a small molecule injection and the other received a small molecule and collagen hydrogel injection (15 μl) into the spinal cord injury site (to ensure sensitivity and accuracy of the release amount with small volume in vivo, the final concentration of small molecules for in vivo treatment was 15×). Tissues from the injury site (about 2 mm long) were collected at day 1, 2, 3, 4, 5, and 6 (n=4 mice at each time point). Tissues were disrupted and 200 μl HBSS added. Then small molecules were released from the tissues using ultrasound for 20 min. Suspensions were collected and analyzed by LC-MS, as previously described [41].

Efficiency evaluation: For evaluating in vitro efficiency, we randomly selected 10–20 fields of view (20×) from each sample using a Leica SP8 microscope, and counted the number of Nestin+, SOX2+, GFAP+, TUJ1+, MAP2+, and NeuN+ cells. The percentage represents positive cells among total cells stained by DAPI. Experiments was repeated three times in vitro. For evaluating in vivo efficiency, 10 fields of view (20×) from specific regions of each sample were randomly selected using a Leica SP8 microscope,and the numbers of Tdtomato+, GFAP+, TUJ1+, MAP2+, NeuN+, and SYN+cells were counted. The percentage represents positive cells among Tdtomato positive cells. We examined at least four slices from each sample (selected every five slices). And at least 20 images (20×) were obtained from each slice.

 Statistical analysis: Statistical analyses were performed using Student’s t test. Quantitative data are presented as the mean ± SEM of at least three independent experiments. Probabilities of p<0.05 were considered as significant (in figures, *p 0.05,

**p 0.01, and ***p 0.001).



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