Enhanced neuronic differentiation of neural stem cells by SHH gene-engineered EMSCs co-culture in vitro

doi:10.21203/rs.3.rs-2618923/v1

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Enhanced neuronic differentiation of neural stem cells by SHH gene-engineered EMSCs co-culture in vitro

https://doi.org/10.21203/rs.3.rs-2618923/v1

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Spinal cord injury is a severe central nervous system disease. Neural crest-derived ectoderm mesenchymal stem cells (EMSCs) have become an important tool in treating SCI owing to their excellent neuronal differentiation efficiency and promising paracrine effects. Sonic hedgehog (shh) was proven to promote both the proliferation and chemotaxis of NSCs and induce their differentiation into mature neurons and the formation of synapses. In this study, we examined the neuronal differentiation induced by SHH-engineered EMSCs in a co-culture system. The levels of the neuronal markers, tubulin beta 3 class III and growth-associated protein 43, were higher in NSCs in the SHH-EMSC co-culture group, and those of the astrocytic marker glial fibrillary acidic protein (GFAP) were lower, compared with the mono and EMSCs-NSCs groups The expression of GAP43, GFAP, MBP, and NF200 was also confirmed by western blot analysis. Most importantly, the upregulation of con43, con47, smoothen, and integrin might reveal the construction of the gap junction during the overexpression of SHH in EMSCs. Overall, our findings indicate that SHH-EMSCs might facilitate the neuronal differentiation of NSCs via the gap junction formation.

EMSCs

SHH

Neural differentiation

Gap junction

Spinal cord injury (SCI) is a severe neurodegenerative disease that leads to a heavy economic burden and decreased quality of life[1]. One of the essential features of spinal cord injury is the loss of a large number of neurons. This irreversible pathological process encourages us to replenish the missing neurons to rebuild the central nervous system (CNS). Neural stem cells (NSCs) are conceived as promising seed cells for the regeneration of the CNS owing to their remarkable differentiation ability into neurons, astrocytes, and oligodendrocytes[2]. However, the therapeutic applications of NSCs still face inevitable challenges, such as limited sources, immunorejection, uncontrollable differentiation, and inflammatory reaction[3]. Thus, the transplantation of neurons derived from NSCs is an alternative strategy for SCI restoration.

Sonic hedgehog (shh) was proven to promote both the proliferation and chemotaxis of NSCs and induce their differentiation into mature neurons and the formation of synapses[4–6]. SHH promotes neural stem cell proliferation through the Patched-Smoothened-Gli classical signaling pathway in a Gli transcriptional activity-dependent manner. In addition, SHH also regulates the non-classical pathway named BOC-SFKs (Src family kinases, such as Src) to facilitate the assembly of the endoskeleton of neuronal protrusions and promote the extension of neuronal protrusions to areas of high SHH concentration, and encourages synapse formation between neurons. Hao Yang et al. reported that SHH effectively improves Oct-4 mediated reprogramming of astrocytes into NSCs via PI3K/Cdk2 signaling pathway in vitro and vivo[7]. Moreover, SHH can strikingly improve the motor function of the cortical contusion injury model[8]. The combination of retinoic acid (RA) and SHH also facilitate the motor neuron-like cells (MNLC) transdifferentiation of NSCs from adipose-derived stem cells[9]. The SHH pathway was activated by taurine in cochlear NSCs' transplantation to replace degenerated spiral ganglion neurons[10].

Nasal mucosa-derived ectoderm mesenchymal stem cell (EMSC)is a newly explored cell line that shows excellent regenerative activities in many areas. Zhijian Zhang et al. isolated EMSCs from nasal olfactory and respiratory mucosa and proved their preference for neural directional differentiation in three-dimensional scaffolds[11]. Moreover, EMSCs, similar to other MSCs, possess great paracrine effects that determine the fate of other cells such as oligodendrocyte precursor cells[12] and NSCs[13]. Strikingly, Wenwen Deng et al. illustrated that the enhanced neuronal differentiation and inhibited astrocytic differentiation of NSCs when co-culturing with EMSCs is due to the assembly of a cell-cell contact all-in-one lipid raft niche via the connexin43/YAP/143-3 pathway[14]. Based on this, EMSCs have been extensively applied in neural regeneration[15]. Besides, the promising paracrine effects of EMSCs also alleviate LPS-induced acute liver injury by IL-10 secretion[16]. Furthermore, the minimal trauma and handy sampling of EMSCs, as well as the said promising activities, would give new hope for clinical application.

In this study, we genetically engineered EMSCs that overexpress SHH, thus enhancing the neuronal entiation of NSCs. The results of the current study highlight novel roles for SHH within EMSCs, and bring a bright future for SHH-EMSCs in SCI therapy.

2.1 NSCs isolation

The animal studies were approved by the Animal Ethical Experimentation Committee of Jiangsu University (Jiangsu, China). Two cell types, which are primary NSCs and EMSCs, were used in this work. Neonatal rat (24 ~ 48h) SD rats were sacrificed by cervical dislocation. Primary NSCs were cultured using the neurosphere method. Briefly, cells were dissociated by mechanical shearing followed by filtration through a 70-micron mesh (Gibco, Grand Island, NY, USA). Next, cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Sigma-Aldrich, St. Louis, MO, USA) containing 20 ng/ml basic fibroblast growth factor, 20 ng/ml epidermal growth factor (both PeproTech, Inc.), 2% N2 supplement (Sigma-Aldrich), 2% B27 supplement (Sigma-Aldrich), and 2 mM l-glutamine (Sigma-Aldrich) and passaged (1:1) every week. All cultures were maintained at 37 ℃ in a humidified atmosphere of 5% CO₂. NSCs at passage three were identified by immunofluorescence analysis.

2.2 EMSCs preparation

EMSCs were isolated from rats, according to the reported methods[11]. Briefly, rats were sacrificed using pentobarbital sodium (200 mg/kg). Subsequently, nasal septum mucosa tissue samples were collected from the lower third of the rat nasal septum. These mucous membranes were gently minced into pieces (0.5 mm3) and cultured in DMEM/F12 containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). The isolated mucosa was minced and incubated in a 0.25% trypsin solution at 37°C for 25 min. The trypsin was neutralized using fresh DMEM/F12, and then the obtained tissues were centrifuged at 1000 g for 5 min and washed twice with phosphate-buffered saline (PBS). The cultures were maintained in an atmosphere of 5% CO₂ at 37°C. The media were replaced every 3 days, and the cells were passaged every week.

2.3 SHH-GFP recombinant adenovirus transfection

The following primers were designed to synthesize the SHH gene: SHH-GFP, forward 5′-AAAGAATTCGCCACCATGCT-GCTGCTGCTGGCCAGATGT-3′; recombinant rat (rSHH), reverse 5′-AAAGAAT-TCAGATTTGGCCGCCACGGAGTTC-3′. The recombinant SHH adenovirus shuttle plasmid was constructed by SinoGenoMax Co., Ltd. (Beijing, China) and confirmed by sequencing, as described in our previous study[13]. SHH-containing adenovirus was added to the cells in the appropriate volume of EMSC culture medium. EMSCs (2 × 10⁵) at passage three were infected with Ad-SHH-GFP at a multiplicity of infection (MOI) of 70 pfu/cell following standard infection procedures. The SHH-EMSCs were identified by detecting the expression of nestin and vimentin, CD133, and connexin 43 (con 43) via immunofluorescence analysis.

2.4 Co‑culturing NSCs with EMSCs

To investigate the effect of SHH-EMSC on the differentiation of NSCs, an NSC/SHH-EMSC co-culture system was established. Specifically, EMSCs were seeded in 48-well plates at 15,000 cells/well density. These cells were maintained in DMEM/F12 supplemented with 5% FBS in 5% CO₂ at 37°C, and the medium was changed every 48 hours. After four days, neurospheres were collected and then seeded on SHH-EMSCs at a concentration of 10 neurospheres/well. The NSCs/SHH-EMSCs contact co-culture system was incubated in the DMEM/F12 medium containing 5% FBS, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin. The medium was changed every 3 days. Meanwhile, the mono-cultured NSCs and the co-culture of NSCs and EMSCs were performed as control groups. Following co-culture for 14 days, the cells obtained from the differentiated NSCs were fixed and labeled with the neuronal-specific markers, tubulin beta 3 class III (Tuj1), growth-associated protein 43 (gap43), and glial fibrillary acidic protein (GFAP). Meanwhile, the protein samples were extracted for western blot analysis.

2.5 Western blot analysis

Total protein samples were harvested using RIPA lysis buffer (Beyotime Tech, China) with a protease inhibitor cocktail (Merck Millipore, USA), and the protein concentration was determined with a bicinchoninic acid protein assay kit (Sigma-Aldrich, St. Louis, MO, USA). An equal amount of each protein sample was transferred onto polyvinylidene difluoride membranes (Millipore EMD, Darmstadt, Germany). Then, the membranes were blocked in 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) in TBST (20 mM Tris/HCl, 137 mM NaCl, 0.4% Tween 20, pH 7.6) for 1 h, and incubated overnight at 4°C with primary anti-bodies including rabbit monoclonal antibody anti-TUBB3 (1:1000; sc-53140, Santa Cruz Biotechnology, Inc.), goat polyclonal antibody anti-GAP-43 (l:1000; cat. no. ab16053; Abcam), rabbit monoclonal antibody anti-laminin (1:500; cat. no. BM4921; Wuhan Boster Biological Technology, Ltd.), goat polyclonal antibody anti-Shh (1:1000; sc-365112, Santa Cruz Biotechnology, Inc.), anti-FN (1:1000; cat. no. BA1772; Wuhan Boster Biological Technology, Ltd.), rabbit monoclonal antibody anti-SHH (1:1000; sc-48387, Santa Cruz Biotechnology, Inc.), monoclonal antibody glial fibrillary acidic protein (1:1000; sc-33673, Santa Cruz Biotechnology, Inc.), and goat polyclonal antibody anti-β-actin (1:1000; sc-56459, Santa Cruz Biotechnology, Inc.), followed by incubation with horseradish peroxidase- (HRP-) conjugated rabbit anti-goat IgG/goat anti-rabbit IgG secondary antibody 1:4000 (Millipore EMD, Darmstadt, Germany) for 2 h at room temperature. Immunoreactive proteins were visualized using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). All different molecular weights of protein fragments were chosen and analyzed using ImageJ.

2.6 Immunofluorescence staining

After 14 days, NSC cultures were processed to identify the formation of neural or glial lineages. Briefly, cells were fixed in 4% paraformaldehyde at 4°C for 12 h. Subsequently, cells were washed twice with PBS and then blocked in 0.3% Triton-X solution (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 30 min. After that, cells were incubated with 3% BSA (Sigma-Aldrich) to block nonspecific binding sites. The cells were incubated overnight at 4°C with the respective primary (anti-TUBB3 and anti-GAP-43 (1:250; cat. no. PB0716; Wuhan Boster Biological Technology, Ltd.), anti-GFAP or anti-vimentin (1:250; cat. no. BM0135; Wuhan Boster Biological Technology, Ltd.), followed by incubation for 30 min at room temperature with secondary antibodies including Cy3-labeled goat anti-mouse/rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:800; Millipore EMD, Darmstadt, Germany). Finally, the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 30 min at room temperature. Fluorescence images were captured by an immunofluorescence microscope (Nikon, Tokyo, Japan).

2.7 Statistical analysis

SPSS 24.0 (SPSS company, USA) and GraphPad Prism were employed for statistical analysis. All numerical values presented in this study were mean ± standard deviation (SD). Statistical analyses were performed by one-factor analysis of variance (ANOVA) and Fisher’s protected least squares differences (FLSD) post hoc tests. All statistically processed results were determined to be significant at *P < 0.05 and **P < 0.01.

3.1 Characterization of EMSCs and SHH-EMSCs

The primary EMSCs were passaged three times within seven days after isolation until a fibroblast-like cell shape was observed. Immunofluorescence was carried out to identify some biomarkers of SHH-EMSCs, such as CD133, vimentin, and nestin (Figure 1A). The results of flow cytometry, osteogenesis, and adipogenesis revealed that EMSCs satisfy the basic principles of MSCs (Figure 1B, C, and D). Additionally, the overexpression of SHH by lentiviral vectors would not affect the properties of EMSCs (Figure 2).

3.2 Characterization of NSCs

Upon isolation and seeding, cells appeared small and round. After 24 h, the majority of cells tended to aggregate. After 3 days, a large number of spheres comprising a few dozen cells emerged, and after 5 days, typical neurospheres formed. The immunofluorescences revealed that these neurospheres are all positively expressed nestin (Figure 3A) and sox2 (Figure 3B).

3.3 SHH‑EMSCs influence NSC differentiation

To determine whether SHH-EMSCs induced NSC differentiation, we examined differentiated cells for surface marker expression. In differentiated NSCs, immunofluorescence was used to analyze the expression of GFAP and GAP43. Compared with mono-culture NSCs and EMSCs-NSCs, SHH-EMSCs-NSCs possessed a significant neuronal differentiation and extension of axons. The expression level of GFAP is higher in mono-culture NSCs, which reveals the astrocytic differentiation of NSCs. Both EMSCs and SHH-EMSCs can inhibit the astrocytic differentiation of NSCs (Figure 4). Western blot results also proved the neuronal differentiation of NSCs after co-cultured with EMSCs and SHH-EMSCs by elevating the expression of MBP, NF200, GAP43, Tuj1, and GFAP. Additionally, after co-culturing with SHH-EMSCs, the expression of con43, con47, Integrin, and smoothen is also upregulated. This revealed that the enhanced neuronal differentiation of NSCs induced by SHH-EMSCs might be related to con43-con47 gap junctions (Figure 5). These results demonstrate that SHH-EMSCs are able to not only facilitate neuronal differentiation but also suppress the astroglial commitment of NSCs.

Notably, spinal cord injury repair is a holistic process that involves multiple glial cells, including astrocytes, oligodendrocytes, and ependymocytes. The process of spinal cord injury is often accompanied by the overproliferation of astrocytes, which forms glial scarring and impedes neuronal regeneration and connectivity. However, astrocytes also play an important role in promoting myelination and myelin maintenance by clearing extracellular ions and neurotransmitters and secretion of nutrients, including pro-myelinating factors in the CNS[17]. The gap junctions (GJs), which permit the passage of ions and small molecules between cells, derived from oligodendrocyte-astrocyte (O-A) contact, are key factors during remyelination. Oligodendrocytes highly express connexin 47 (con47), con 32, and con29 (or its ortholog con31.3 in humans), while astrocytes express con43, con30, and con26[18]. Among them, the con47-con43 coupling is an essential prerequisite for the formation of O-A GJs. It has been proposed that depletion of either con43 or con47 affects the maintenance of white matter function via alterations in O-A homeostasis and plays a vital role in the chronic expansion of demyelinated plaques[19]. In this study, we showed that EMSCs significantly increased the expression of con43 and con47 and inhibited the expression of GFAP after co-culture with NSCs, indicating that EMSCs do not impede the formation of O-A coupling while inhibiting the differentiation of NSCs to astrocytes.

Although NSCs have long been considered the best seed cells for the restoration of spinal cord injury. However, more and more studies have shown that NSCs also have many limitations, such as their limited source and difficulty controlling the differentiation process in vivo. Recently, MSCs have been used in tissue engineering and regenerative medicine. Their abundant source, multi-directional differentiation ability, and immunomodulatory function make them important alternative seed cells. The immune regulatory molecules and growth factors produced by MSCs showed excellent CNS protection, such as maintaining dopaminergic neuronal activity[20], improving repair of cerebral ischemic damage[21], inhibiting axonal degeneration, and demyelination of spinal neurons[22]. Thus, MSCs are a series of promising seed cells for SCI restoration.

In addition to the MSCs-derived growth factors that facilitate SCI restoration, the immune microenvironment at the site of spinal cord injury is also critical for the survival of seeded cells. Microglia and T lymphocytes are the primary immune cells mediating the immune microenvironment. It has been shown that M2-type microglia can effectively promote nerve injury repair and improve motor neuron function after injury[3, 23]. Therefore, inhibiting the M1 polarization of microglia and prolonging the presence of M2-type microglia is a major strategy for treating spinal cord injury. MSCs can promote the conversion of M1-type microglia to M2-type through paracrine effects. Additionally, MSCs can induce the regulatable T cell (T_reg) differentiation through direct[24–26] or indirect regulation[27]. Recently, Inflammatory-licensed MSCs (IL-MSCs) have been widely used in various tissue regeneration studies owing to their more substantial paracrine effects resulting in immunosuppression properties[28]. However, IL-MSCs tend to overexpress pro-apoptotic genes such as Bax and Bid, significantly reducing the survival rate of transplanted MSCs[29]. Yu et al. primed the nasal mucosal MSCs with Lycium barbarum oligosaccharide and enhanced their paracrine effects by activating the TNFR2-PGE2 pathway without causing upregulation of pro-apoptotic genes, thus promoting spinal cord injury repair[15].

In conclusion, SHH-engineered nasal mucosal MSCs are excellent seed cells that might be applied for repairing spinal cord injury by enhancing neuronal differentiation of NSCs in the co-culture system in the future.

EMSCs Nasal mucosa-derived ectoderm mesenchymal stem cells

GFAP Glial fibrillary acidic protein

MBP Myelin basic protein

NSCs Neural stem cells

SHH Sonic hedgehog

Acknowledgments

Authors’ contributions

Funding

This study was supported by the natural science foundation of Jiangsu province (BK20220529).

Availability of data and materials

All data generated or analyzed during the current study are included in this manuscript.

Declarations

Ethical approval and consent to participate

All experimental animal protocols were performed in accordance with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and the regulations of the Jiangsu university.

Consent for publication

Not Applicable.

Competing interests

We declare that none of the authors have any conflicting interests.

Author Details

¹ Affiliated Hospital of Jiangsu University

² School of Pharmacy, Jiangsu University, Zhenjiang 212013, P.R. China

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No competing interests reported.

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Enhanced neuronic differentiation of neural stem cells by SHH gene-engineered EMSCs co-culture in vitro

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Abstract

Figures

1. Introduction

2. Materials & Methods

2.1 NSCs isolation

2.2 EMSCs preparation

2.3 SHH-GFP recombinant adenovirus transfection

2.4 Co‑culturing NSCs with EMSCs

2.5 Western blot analysis

2.6 Immunofluorescence staining

2.7 Statistical analysis

3. Results

4. Discussion

Abbreviations

Declarations

References

Additional Declarations

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