Phenotypic and functional comparison of rat enteric neural crest-derived cells during fetal and earlypostnatal stages

Dong-Hao Tian , Chuan-Hui Qin , Wen-Yao Xu , Wei-Kang Pan Yu-Ying ZhaoBai-Jun Zheng Xin-Lin Chen, Yong Liu, Ya Gao , Hui Yu ,

Abstract In our previous study, we showed that with increasing time in culture, the growth characteristics of enteric neural crest-derived cells (ENCCs)change, and that the proliferation, migration and neural differentiation potential of these cells in vitro notably diminish. However, there are no studies on the developmental differences in these characteristics between fetal and early-postnatal stages in vitro or in vivo. In this study,we isolated fetal (embryonic day 14.5) and postnatal (postnatal day 2) ENCCs from the intestines of rats. Fetal ENCCs had greater maximum cross-sectional area of the neurospheres, stronger migration ability, and reduced apoptosis, compared with postnatal ENCCs. However,fetal and postnatal ENCCs had a similar differentiation ability. Fetal and postnatal ENCCs both survived after transplant into a rat model of Hirschsprung’s disease. In these rats with Hirschsprung’s disease, the number of ganglionic cells in the myenteric plexus was higher and the distal intestinal pressure change was greater in animals treated with fetal ENCCs compared with those treated with postnatal ENCCs. These findings suggest that, compared with postnatal ENCCs, fetal ENCCs exhibit higher survival and proliferation and migration abilities, and are therefore a more appropriate seed cell for the treatment of Hirschsprung’s disease. This study was approved by the Animal Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University (approval No. 2016086) on March 3, 2016.

Key Words: cell transplantation; enteric nervous system; enteric neural crest-derived cells; Hirschsprung’s disease; stem cells

Introduction

The enteric nervous system (ENS) is derived from enteric neural crest-derived cells (ENCCs) and forms an intricate network that performs complex functions such as transport,digestion, secretion and preservation of intestinal barrier integrity, in addition to protecting against pathogens in the gastrointestinal tract (Nagy and Goldstein, 2017). ENCCs can be isolated from healthy and even aganglionic bowels, exhibit self-renewal and migratory abilities, and can differentiate into various cell types (Almond et al., 2007; Heuckeroth, 2016;Cheng et al., 2017).

Perturbation of ENS development can lead to enteric neuropathic diseases such as Hirschsprung’s disease (HSCR),one of the most common congenital diseases in newborns and infants. At present, surgery is the main treatment for HSCR,but while it alleviates the symptoms, it is far from curative.Moreover, as many as one-third of children diagnosed of HSCR experience post-operative complications such as enterocolitis,which severely affects their growth and development, and can even endanger their lives (Heuckeroth, 2016). Therefore, it is urgent to optimize existing treatment modalities and explore new alternative strategies.

Previous studies demonstrated the potential of ENCCs as a cell-replacement therapy for HSCR (Pan et al., 2011; Hotta et al., 2013; Fattahi et al., 2016; Yu et al., 2017). ENCCs, which receive signals to differentiate into the appropriate subtypes of neurons throughout development (Avetisyan et al., 2015;Burns et al., 2016), may be suitable as seed cells. During development, ENCCs are spatiotemporally regulated by cues from the surrounding environment (Lake and Heuckeroth,2013), which may regulate ENS development. Our previous study revealed that ENCC growth characteristics change with increasing time inin vitroculture (Yu et al., 2016b),particularly decreases in the proliferative, migratory and neuro-differentiative potential of postnatal ENCCs. However,differences between fetal and postnatal ENCCs have not yet been systematically analyzed (Anitha et al., 2008; Stamp,2017). Therefore, in this study, we compare the biological characteristics of fetal and postnatal ENCCs.

Materials and Methods

Experimental animals

Pregnant (embryonic day 14.5) and newborn (postnatal day 2) Sprague-Dawley rats were used for collecting the fetal and postnatal ENCCs, respectively. Female Sprague-Dawley rats,weighing approximately 250 g, and housed under a 12-hour light/dark cycle, were used for the HSCR model. All rats had ad libitum access to food and water, and were fasted for 12 hours before abdominal surgery. All animals were supplied by the Experimental Animals Center, Xi’an Jiaotong University(SCXK (Shaan) 2018-001) and handled in accordance with the guidelines outlined by the Animal Care and Ethics Committee of Xi’an Jiaotong University. All procedures were approved by the Animal Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University (approval No. 2016086) on March 3, 2016. All experiments were designed and reported according to the Animal Research: Reporting ofIn Vivo(ARRIVE) Experiments guidelines.

Cell culture

Fetal and postnatal ENCCs were extracted from fetal and postnatal rats, respectively. ENCC culture procedures were performed under aseptic conditions. Pregnant rats were sacrificed by intraperitoneal injection of 0.3% pentobarbital sodium solution (1 mL/100 g, Cat# 100203; Guo An Reagent Biotechnology, Xi’an, China) followed by quick cervical dislocation. Fetal and postnatal rats were rinsed for a few seconds with alcohol, and then rapid laparotomy was conducted to extract the intestinal tissues. The fetal and postnatal ENCCs were extracted from the entire digestive tract and the muscular layer of the digestive tract, respectively.The procedure for cell culture of the postnatal ENCCs was as previously described (Yu et al., 2015). Briefly, the outer layers of the gut were dissected under aseptic conditions under an anatomical microscope (SZX16; Olympus, Tokyo, Japan). After dissociation by trituration and filtration through a 350-mesh strainer, the single-cell suspensions were plated in Dulbecco’s modified Eagle medium (DMEM)/F12 (1:1; Cat# SH30023.01B;Life Sciences, Woburn, MA, USA) supplemented with N2(0.5%; Cat# 17502048; Gibco, Grand Island, NY, USA), B27(1.0%; Cat# 12587-010; Gibco), recombinant basic fibroblast growth factor (10 ng/mL; Cat# PHG0021; Gibco), recombinant epidermal growth factor (10 ng/mL; Cat# PHG0311; Gibco),2-mercaptoethanol (50 µM; Cat# M3148; Gibco), and penicillin and streptomycin (100 U/mL, Cat# 15140148; Gibco). ENCCs were purified by fluorescence-activated cell sorting (Becton Dickinson, San Jose, CA, USA) using a p75NTR antibody (1:500,rats; Cat# ab104576, RRID AB_10899157; Abcam, Cambridge,MA, USA). These ENCCs were seeded into culture flasks at a density of 5.0 × 105cells/mL and cultured for 5 days for subculture and subsequent experiments. Cells were routinely counted by trypan blue staining with a hemocytometer under a light microscope (CKX41; Olympus).

Mean maximum cross-sectional area of neurospheres

To analyze cell proliferation, 5 mL of the cell suspension was re-plated at a density of 5.0 × 105cells/mL, and the secondary neurospheres derived from fetal and postnatal ENCCs at culture day 5 were collected, resuspended in a 5-mL volume of medium, and randomly selected to measure their mean maximum cross-sectional area (MCSA) in 10 randomly chosen fields at 20× magnification. Using the photographs obtained with a light microscope equipped with a DP70 digital camera and Manager software (Olympus America Inc., Center Valley,PA, USA), the MCSA was calculated as the longest size of the neurospheres multiplied by the vertical diameter.

Immunohistochemical analysis of ENCC differentiation

Neurospheres derived from fetal and postnatal ENCCs were dissociated into single-cell suspensions using trypsin/ethylenediaminetetraacetic acid (Life Sciences; Cat#SH30042.01) and re-plated onto polylysine-coated plates at a density of 5.0 × 105cells/mL for differentiation under aseptic conditions. After culture for 3 days in DMEM/F12(1:1) supplemented with N2, B27 and 10% fetal calf serum(Cat# 10099-141; Gibco) in the absence of basic fibroblast growth factor and epidermal growth factor, the re-plated cells were subjected to conventional immunocytochemistry using primary antibodies against p75NTR (ENCC marker; 1:300;rat; Cat# ab104576, RRID AB_10899157; Abcam), peripherin(neuronal marker; 1:2,000; rat; Cat# ab4666, RRID AB_449340;Abcam) and glial fibrillary acidic protein (GFAP; glial marker;1:100; rat; Cat# MA5-12023, RRID AB_10984338; Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4°C, as described previously (Yu et al., 2015). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Cat# 10236276001;Roche, Basel, Switzerland). After acquiring photographs at 20×magnification with a fluorescence microscope equipped with a DP70 digital camera (Olympus America Inc.), p75-, peripherin-,GFAP- and DAPI-positive cells were counted in five random fields. After adding fetal calf serum (1.0%; Cat# 10099-141;Gibco), cell differentiation of fetal and postnatal ENCCs was assessed and compared at day 3 in differentiating medium as the ratio of peripherin- and DAPI-positive cells.

Apoptosis analysis by flow cytometry

Single-cell suspensions of fetal and postnatal ENCCs at day 5 were collected by centrifugation to assess cell apoptosis by flow cytometry using an Annexin V-fluorescein isothiocyanate/propidium iodide Apoptosis Detection Kit (Cat# 40302ES20,Yeasen, Shanghai, China) as described in our previous studies(Yu et al., 2015). Briefly, 1 × 105cells suspended in 500 µL of binding buffer were mixed with 5 µL Annexin V-fluorescein isothiocyanate and 5 µL propidium iodide and incubated for 10 minutes at room temperature. Apoptosis was measured with FACSCalibur and CELLQuest software version 2.0 (Becton Dickinson) within 1 hour according to the manufacturer’s instructions.

Migration analysis using Hanging Cell Culture Inserts

Cell migration was determined using Millicell Hanging Cell Culture Inserts (8.0 µm pore size, PET track-etched membrane, BD Biosciences, San Jose, CA, USA) in a 24-well plate as described previously (Yu et al., 2015). Briefly, 500 µL of single-cell suspension (1.0 × 105cells/mL) was added to the top chamber of the Millicell Hanging Cell Culture Insert in serum-free DMEM/F12 medium supplemented with N2,B27, basic fibroblast growth factor, epidermal growth factor,2-mercaptoethanol, and penicillin and streptomycin, while the bottom chamber was filled with 750 µL of DMEM/F12 medium containing 1.0% fetal calf serum, as a chemoattractant.ENCCs that had migrated through the filter after 72 hours of incubation were fixed with methanol, stained with hematoxylin and eosin, and counted under a fluorescence microscope (BX51; Olympus) in five random fields.

Production of the rat HSCR model

All procedures used to produce the HSCR model have been previously described (Yu et al., 2016a). Briefly,after anesthetization by intraperitoneal injection of 0.3%pentobarbital sodium solution (1 mL/100 g), 1-cm segments of the descending colon were excised, wrapped tightly with filter paper, and rinsed thoroughly with 0.1% benzalkonium chloride (BAC; Cat# 76857, Sigma-Aldrich, St. Louis, MO, USA)for 30 minutes.

ENCC transfection and transplantation

Animal grouping and cell transplantation were conducted in a randomized manner. Protocols for labeling ENCCs have been described previously (Yu et al., 2015). Single-cell suspensions of fetal and postnatal ENCCs were sub-cultured. Lentivirus (Cat#HYXH-lenti-210026; NeuronBiotech, Beijing, China), which was produced from the plasmid vector pLOV.CMV.eGFP.2A.EF1a.PuroR (Cat# HYXH-lenti-210026; NeuronBiotech) and packaged in human embryonic kidney 293T cells (Cat# HYXHlenti-210026; NeuronBiotech), was applied at a multiplicity of infection of 25 for 24 hours. The ENCCs were purified by fluorescence-activated cell sorting using a p75NTR antibody before transplantation. Rats with a poor appetite and insufficient activity were excluded from the study. All rats (n=72) were randomly divided into the following four groups: fetal ENCCs, postnatal ENCCs, control and no-transplantation (no cell transplantation) (n= 18 each). Cell transplantation at a density of 1 × 106ENCCs in 20 µL medium was performed 1 week after model production. Protocols for ENCC transplantation have been described previously (Yu et al., 2017; Zhao et al.,2020). Briefly, the BAC-treated colons were exteriorized, and the muscular layer was injected using a capillary micropipette(Syltech, Hangzhou, China) at the 0, 3, 6 and 9 o’clock positions in a clockwise pattern. The injected sites were marked with India ink solution (Cat# DM0103; Huayueyang, Beijing, China).After the transplantation procedure, the treated segments were gently washed with lukewarm saline, followed by closure of the abdomen. All rats were housed with access to standard diet and tap waterad libitum.

Intraluminal pressure measurement

We applied immediate stimulation to the proximal bowel to simultaneously measure the intraluminal pressure change in the colon, as a functional assay, at day 84 after transplantation.All animals were housed under a 12-hour light/dark cycle with ad libitum access to food and water, and were fasted for 12 hours before the measurements. Thisin vivoprocedure was performed under anesthesia with 0.3% pentobarbital sodium solution (1 mL/100 g, intraperitoneal injection). The detailed procedures have been described previously (Pan et al., 2011).Briefly, a pressure balloon connected to a pressure transducer(AD Instruments, Sydney, Australia) was inserted 2 cm through the anus. Another stimulating balloon was inserted and proximally placed at 5 cm from the pressure balloon.

Pathological analysis

At days 28, 56 and 84 after transplantation, pathological analysis was performed as described previously (Pan et al.,2011). Twenty-five 5-µm-thick transverse sections were randomly selected for hematoxylin and eosin staining and immunohistochemical analyses. The sections were blocked and incubated with antibodies to p75NTR (1:300; rat; Cat#ab104576, RRID AB_10899157; Abcam), peripherin (1:2000;rat; Cat# ab4666, RRID AB_449340; Abcam) and GFAP (1:100;rat; Cat# MA5-12023, RRID AB_10984338; Thermo Fisher Scientific) overnight at 4°C. p75NTR-, peripherin-, GFAP- and DAPI-positive cells were counted by fluorescence microscopy(Olympus, BX51) and laser scanning confocal microscopy (Cat#TCS-SP2; Leica, Frankfurt, Germany). Ganglionic cells were defined as cells positive for the neuronal marker peripherin and for DAPI under a DP70 digital camera (Olympus America).

Statistical analysis

Statistical analysis was conducted using SPSS 13.0 (SPSS,Chicago, IL, USA; RRID:SCR_002865). Numerical data are presented as the mean ± standard error of mean (SEM).Depending on the result of distribution and variances, a twotailed Student’st-test was applied to compare differences in the MCSA of the neurospheres, numbers of migrating and apoptotic ENCCs, and peripherin-positive cells per ganglia between the fetal and postnatal ENCC groups. The mean number of ganglionic cells per ganglia and intraluminal pressure change in different animals were assessed by oneway analysis of variance followed by Bonferronipost hoctest.Significance was set atP＜ 0.05.

Results

Proliferative ability of fetal ENCCs is higher than that of postnatal ENCCs

Both postnatal and fetal ENCCs proliferated rapidly and formed neurosphere-like bodies on day 2 (Figure 1A and B)and free-floating neurospheres on day 5 (Figure 1C and D).However, the MCSA of neurospheres derived from fetal ENCCs was larger than those from postnatal ENCCs (P= 0.0045;Figure 1E).

Differentiation potency of fetal ENCCs is similar to that of postnatal ENCCs

During culture in differentiation medium, neurospheres derived from postnatal ENCCs adhered gradually and showed neurite outgrowth and radial migration at day 7 (Figure 2A). Most postnatal ENCCs extended axon-like projections (Figure 2B) and differentiated into neuron- or glial-like cells (Figure 2C and D).Given the similar differentiation potential of fetal and postnatal ENCCs, we did not evaluate fetal ENCC differentiation in this study. Further analysis revealed no significant difference in the number of peripherin/DAPI-positive cells per field between fetal and postnatal ENCCs (P= 0.32; Figure 2E). These results suggest that the neuronal differentiation potential of ENCCs is similar in the embryonic to postnatal stages.

Fetal ENCCs have better migration ability than postnatal ENCCs

Cell migration was evaluated using Hanging Cell Culture Inserts. The fetal (Figure 2F) and postnatal ENCCs (Figure 2G)had migrated through the membrane to the lower surface.The number of migrating fetal ENCCs was significantly greater than the number of migrating postnatal ENCCs (P＜ 0.0001;Figure 2H). These results suggest that fetal and postnatal ENCCs have different migratory abilities.

The number of apoptotic fetal ENCCs is less than the number of postnatal ENCCs

To analyze the apoptosis of fetal and postnatal ENCCs,we conducted flow cytometry and Annexin V-fluorescein isothiocyanate/propidium iodide staining. The number of apoptotic fetal ENCCs was significantly less than the number of apoptotic postnatal ENCCs (P= 0.0002; Figure 2I-K).

The number of ganglionic cells in the myenteric plexus is greater in HSCR rats treated with fetal ENCCs compared with those treated with postnatal ENCCs

Rats in all groups survived during the experimental period. We compared hematoxylin and eosin staining between the control(Figure 3A) and the other intervention groups (Figure 3B),and found constricted segments as well as dilatation and stool retention proximal to the treated bowel (Figure 3C). On day 84,autopsy of rats in the postnatal ENCCs group revealed dilatation of the colonic gut proximal to the treated bowel and adhesion between the BAC-treated intestinal segment and surrounding tissues (Figure 3C). Quantitative analysis revealed more enhanced green fluorescent protein/peripherin double-positive cells per ganglia in the fetal ENCCs group compared with the postnatal ENCCs group on days 56 and 84 (Figure 4A-E).

Distal intestinal pressure change is greater in HSCR rats treated with fetal ENCCs compared with those treated with postnatal ENCCs

In the control group, rats displayed more reflexive contractions or pressure changes when the proximal colon was stimulated.In comparison, rats in the other groups showed less reflexive contractions and decreased intraluminal pressure changes. The pressure in the fetal ENCCs group was significantly higher than that in the postnatal ENCCs group (P= 0.0056; Figure 4F).

Figure 1 ｜Differentiation and MCSA of postnatal and fetal ENCCs on days 2 and 5.

Figure 2 ｜Differentiation, apoptosis and migration analysis of the postnatal and fetal ENCCs in vitro.

Figure 3 ｜The morphological characteristics of the Hirschsprung’s disease rat model.

Figure 4 ｜Effects of fetal and postnatal ENCC transplantation on ganglionic cell numbers and distal intestinal pressure change in the Hirschsprung’s disease rat model.

Discussion

In vitroexperiments have demonstrated that fetal and postnatal ENCCs can be isolated from fetal and postnatal enteric tissues, respectively. These cells can proliferate rapidly into neurospheres, migrate through membranes,and differentiate into neurons and glia. Fetal ENCCs show obvious advantages in proliferation, migration and apoptosis,but not differentiation, compared with postnatal ENCCs.Transplantation experiments with fetal and postnatal ENCCs confirmed the advantages of fetal ENCCs over postnatal ENCCs. These studies uncovered the phenotypic and functional differences between fetal and postnatal ENCCs during ENS development. These differences suggest that fetal ENCCs may be more appropriate than postnatal ENCCs for reconstructing the injured ENS. Future cell transplantation studies should further clarify the developmental differences between fetal and postnatal ENCCs.

Because of refractory complications after surgery, cell transplantation has been considered for treating HSCR, with significant advancements achieved in the past two decades.Some tissue-derived stem cells/progenitors (central nervous system, ENS, intestinal mucosa, and appendix) (Bixby et al.,2002; Sieber-Blum et al., 2004; Findlay et al., 2014; Fattahi et al., 2016) exhibit proliferation, migration and directed differentiation into neurons and glia, which may improve intestinal functions upon transplantation into embryonic/neonatal/adult animal colons and even the diseased bowels of patients suffering from HSCR. However, the compensatory effects do not cure HSCR. Therefore, we conjectured that intrinsic deficiencies in the transplanted cells themselves or insufficient compatibility between transplanted cells and their environment could explain the lack of adequate compensation.Additionally, some studies showed positive effects using stem cells/progenitors by activation of Wnt signaling or the addition of growth factors (McKeown et al., 2017; Zhang et al., 2017)to repair the ENS. It is necessary to compare the proliferation,migration and differentiation abilities of neural stem cells/progenitors before cell transplantation.

The following sources are available for cell therapy: ENS,central nervous system, and other somatic tissues, as well as embryonic and induced pluripotent stem cells. The advantages, disadvantages, and properties of these stem cells have been widely investigated (Findlay et al., 2014;Gonsalvez et al., 2015; Fatima et al., 2019). Findlay et al.(2014) compared ENS- with central nervous system-derived stem cells/progenitors and concluded that ENS-derived stem cells had superior migration, proliferation, and neuronal differentiation abilities. Additionally, ENCCs can be stimulated by inducible factors and signals in the developing gut to colonize, migrate and differentiate into the appropriate cell types to form a functional ENS. Thus, ENCCs can be used as seed cells for transplantation. We isolated ENCCs from the fetal/postnatal gut, and these cells rapidly proliferated into neurosphere-like bodies, easily differentiated into neuron- and glial-like cells by induction, and improved intestinal functions upon transplantation. Given the developmental complexities and hurdles to cell-based therapy, including immune rejection,limits in differentiation potential and capacities for selfrenewal, proliferation and migration, difficulties in isolation,purification and transplantation, and long-term safety after transplantation (Zhang et al., 2017), there is currently no ideal cell source for transplantation.

In this study, the proliferative potential of ENCCs showed a decreasing trend over time. This finding is partially supported by the findings of Mosher et al. (2007), who examined ENCC cycle dynamics in the embryonic gut. They showed that the cell cycle length of ENCCs increased significantly between E10.5 and E12.5, while no further increases occurred at E16.5. The proliferation of ENCCs depends on frequent cell division and the proportion of proliferative cells (Mosher et al., 2007), and their proliferation is tightly regulated in the developing gut. We hypothesized that most fetal ENCCs had a strong proliferative ability for constructing the ENS, while the postnatal ENS contained the most cell components, consistent with the lower proliferation of postnatal ENCCs. Therefore,the spatiotemporal patterns of ENCC proliferation should be evaluated in subsequent transplantation studies.

Enteric changes may be one cause of migrative disorders after ENCCs transplantation. Iwashita et al. (2003) showed that Ednrbsl/sl ENCCs could engraft and undergo neurogenesis even in the aganglionic region of the Ednrbsl/sl gut. These observations indicated that ENCC migration is inducible, and we predicted that inadequate migration of ENCCs was an important factor affecting the outcomes of transplanted cells in preclinical studies. Future transplantation experiments should be conducted to investigate the spatial and temporal differences in ENCCs (Goldstein et al., 2013). Iwashita et al.(2003) found intrinsic differences among spatially distinct ENCCs in migratory properties. In this study, we compared the migration abilities of ENCCs in the fetal and postnatal periods and detected temporal differences. Glial cell linederived neurotrophic factor/RET/GFRɑ1 signaling pathways are important for ENCC migration and ENS development, and the absence of Ret expression may account for the migration defect of sciatic nerve ENCCs (Iwashita et al., 2003; Goldstein et al., 2013). Further research on the relationship between ENCCs and the transplant environment are needed.

In this study, we used serum to induce the differentiation of ENCCs and examined the differentiation abilities of fetal ENCCs and postnatal ENCCs. The percentage of neurons was approximately equal among the two groups of cells. However,some questions remain, such as whether serum has an inhibitory effect on cell differentiation of fetal and postnatal ENCCs and whether the absence ofin vivostimuli or the presence ofin vitrosupplements affects their differentiation.

ENCC proliferation, migration and differentiation are not sequential, but occur simultaneously during ENS development.For example, sufficient proliferation can promote migration at the beginning of differentiation as well as the cessation of proliferation. Therefore, the complex associations that exist among these processes should be further examined.

Apoptosis of ENCCs occurs during ENS development, which has specific and stage-specific characteristics, particularly high apoptosis during migration and low apoptosis of ENCCs and their differentiated cells upon colonization, with increased apoptosis of neurons after the perinatal stage. These characteristics of the ENS are partially different from those of the central nervous system and other peripheral nervous systems, perhaps because the enteric microenvironment is rich in trophic factors such as glial cell line-derived neurotrophic factor and anti-apoptotic factors that promote the survival of precursors and their differentiated progeny in the fetal period. In this study, we observed ENCC apoptosis in both the embryonic and postnatal periods, and the proportion of apoptotic ENCCs in the former was lower than in the latter.Therefore, it is necessary to regulate apoptosis to maintain the number of critical ENCCs to ensure complete ENS formation.Further study is needed to develop methods for reducing the apoptosis of ENCCs, as apoptosis can lead to neurointestinal diseases such as HSCR.

The limitations of this study need to be pointed out. We cannot exclude an influence of remaining neurons, nontransfected cells, endogenous ENCCs, unsuccessfully transduced ENCCs or sex differences as biological variables in the transplanted animals. Moreover, some appropriate alternative such as transgenic or knockout mouse model seem to be better. Additionally, the sample size in this study was small. Furthermore, ectopic or neuronal transdifferentiation may have affected the results of this study. We only observed and analyzed ENCC developmental potentials during ENS formation from the perspective of cell biology without considering ethics, immune rejection, or surgical difficulties.

Taken together, our present findings provide insight into the phenotypic and functional differences among ENCCs during ENS development, and provide an important reference point for future studies. Further study is needed to elucidate the mechanisms involved in the developmental regulation of the ENS and to develop strategies for cell optimization. Studies are also required to clarify the mechanisms of the decreasing developmental potential of ENCCs, which may help towards clinical translation.

Author contributions:Study design: YG, HY; experimental implementation: WYX, WKP, YYZ, DHT; data analysis: CHQ, BJZ, XLC, YL;manuscript writing: HY, DHT. All authors participated in the manuscript writing and approved the final version of the manuscript.

Conflicts of interest:The authors declare that they have no conflicts of interest.

Financial support:This study was supported by the National Natural Science Foundation of China, Nos. 82071692 (to HY), 81741096 (to HY), 81770513 (to YG), 81270435 (to WKP); Shaanxi Youth Science and Technology Project of China, No. 2019KJXX-044 (to HY); the National Natural Science Foundation of Shaanxi Province of China, No. 2020JM-407(to HY); and Fund Project of the Second Affiliated Hospital, Xi’an Jiaotong University No. RC(XM)201703 (to HY). The funders had no roles in the study design, conduction of experiment, data collection and analysis, decision to publish, or preparation of the manuscript.

Institutional review board statement:The study was approved by the Animal Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University (approval No. 2016086) on March 3, 2016.

Copyright license agreement:The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement:Datasets analyzed during the current study are available from the corresponding author on reasonable request.

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Peer review:Externally peer reviewed.

Open access statement:This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix,tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewers:Willian Orlando Castillo, Universidade de Sao Paulo, Brazil; Ghulam Md Ashraf, King Abdulaziz University, Saudi Arabia;Sanusi Mohammad Bello, King Faisal University, Saudi Arabia.

Additional file:Open peer review report 1.