Genetically modified MSCs into neuraland endothelial cell-like cells inducedby a branched peptide amphiphile hydrogel

doi:10.21203/rs.3.rs-1822746/v3

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Genetically modified MSCs into neuraland endothelial cell-like cells inducedby a branched peptide amphiphile hydrogel

https://doi.org/10.21203/rs.3.rs-1822746/v3

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Background： Spinal cord injury (SCI) remain one of the great clinical challenges because of their considerable long-term disability potential. In this study, A branched amphiphilic peptide (B-PA) segments were constructed and self-assembled into hydrogels to support bone marrow derived mesenchymal stromal cells (BMSCs) encoding Recombinant Rat VEGF165（VEGF165）, neurotrophins-3(NT-3)and angiopoietin-1(ANG-1) genes. In addition, B-PAS promoted the transdifferentiation of BMSCs into nerve and vascular endothelial cells.

Methods：Self-assembly of b-PA into a nanofiber hydrogel was triggered by the culture medium and analysed by transmission electron microscopy (TEM). Rat BMSCs were cultivated using the differential adhesion method and assessed by flow cytometry. Cells were inoculated into the hydrogel and investigated using calcein-AM/PI, CCK-8, and cell adhesion assays. Cells transfected with adenoviral vectors of biocarrier (Adv-Bic) and adenoviral vectors of NT-3 gene (AdvNT-3) were cultured for 7 days and evaluated by immunofluorescence and real time quantitative reverse transcription-PCR.

Results：The molecular weight and purity of b-PA were 2191.72 and >95%, respectively. Self-assembled nanofibers had a diameter of 5-8 nm and a length ranging from hundreds of nanometres to micrometres. The resulting CD29+/44+ cells could differentiate into osteoblasts and fat cells, and the hydrogel supported cell growth, proliferation and adhesion. Cells in the hydrogel overexpressed VEGF₁₆₅, NT-3 and Ang-1, and NSE and Nestin markers of CD31+/34+ cells were also abundant.

Conclusions： The hydrogel has potential as a tissue engineering scaffold with good cytocompatibility, and can induce genetically modified BMSCs to transdifferentiate into neural and endothelial cell (EC)-like cells. The article have selected preprint on the research platform, this article has been assigned a DOI, The DOI is:10.21203/rs.3.rs-1822746/v1.

differentiation

branched amphiphilic peptide

self-assembly

MSCs

genetic modification

Tissue engineering (TE) consists of three elements; scaffolds, seed cells, and various growth factors and signal peptides [1-4]. It was a good technique to fabricated the neural tissue engineering scaffolds with a number of desirable characteristics by amphiphilic peptide self-assembly[5-8],andemerged as attractive candidates for regenerative medicine [9]. The basic principle involved peptide self-assembly into a three-dimensional porous nanofiber hydrogel under the action of acid or cell culture medium (containing calcium, magnesium or hydrogen plasma) via non-covalent intramolecular bonds (hydrogen bonds, hydrophobic interactions, and van der Waals forces) [10, 11]. This approach could achieve good biocompatibility, and degradation products are amino acids that was able to be utilised by cells [12].

The self-assembling amphiphilic peptides (SAPs) synthesised in a previous study was assembled into a three-dimensional porous hybrid nanofiber hydrogel, and an active IKVAV(Isoleucine-Lysine-Valine-Alanine-Valine) motif located on the surface of nanofibers was shown to regulate neuronal survival and maturation [13] . However, this peptide had few active motifs and a restricted structure that made binding receptors on the cell surface and complex biological functions difficult, and the poor hydrogel stability and low performance of this self-assembled peptide make it difficult to use it widely [14]. The co-assembling peptides (Co-SAP) hydrogel scaffolds with complicated structures and limited functions composed of different types of synthesized amphiphile peptides[15, 16].The hydrogels supported from cross-linked self-assembling peptide (cross-SAPs) were fabricated using a one-pot in situ gelation system, and need complicated multi-step chemical reactions with toxic by-products [17-21].

Branched amphiphilic peptides (b-PAs) were a new type of peptide scaffold developed in recent years that were branched via lysine residues, harbour multiple functional groups, and could bind receptors to perform certain biological functions [22-29]. If they were to be deployed for neural tissue engineering scaffolds providing biomimetic microenvironments favoring adherence and growth and tuning the differentiation of cells, further manipulation was clearly required.

In the present study, We reported here the design of a bioactive hydrogel using self-assembly of novel b-PAs containing RGD and IKVAV motifs with the aim of creating a material with several features for tissue engineering applications. This self-formed hydrogel from b-PAs had the potential of supporting the genetically-modified MSCs with VEGF165, NT-3 and Ang-1 adhesion and growth, and could promote morphological changes of cells and differentiate into neural-like cells and endothelial cell-like cells. The structure of the hydrogel was examined by transmission electron microscopy (TEM), and calcein-AM/PI was performed to evaluate biocompatibility. Finally, the immunocytochemistry and qRT-PCR were used to investigate cell differentiation.

b-PA synthesis

Firstly, b-PAs were synthesised using the Fmoc/PyBOP method as previously described [30, 31]. Fmoc was removed with a solution of 30% piperidine in dimethyl formamide (DMF), and the peptide chain was cleaved from the resin using a peptide-cutting reagent (81.5 : 5 : 5 : 5 : 2.5 trifluoroacetic acid : crystalline phenol : water : ethanedithiol : methyl ethyl sulphide). Peptide chain elongation was carried out on a 431A automatic synthesiser (Applied Biosystems, USA), and 2 M Fmoc-amino acids were gradually added to the C-terminus according to the polypeptide sequence. The carboxyl group at the C-terminus of the second peptide was activated, and condensation with the amino group of the 2 M N-α-Fmoc-N-ε-4-Mtt--l-lysine of the first polypeptide yielded a polypeptide with a protected branched side chain that was cleaved, concentrated, cooled, and precipitated with cold diethyl ether. The precipitate was collected by centrifugation, dried under vacuum, and characterised by mass spectrometry (MS) using a Finnigan LCQ instrument (Thermo Fisher, USA) and high-performance liquid chromatograph (HPLC) on an Agilent 1100 instrument (Agilent, USA).

Gel preparation

A 10 mg sample of lyophilised b-PA powder was added to 400 μL of 0.1 M NaOH solution,400 μL of sterile double distilled water was added dropwise, and the pH of the peptide solution was 9.5. A 10 μL sample of 0.1 M HCl was gradually added dropwise to adjust the pH to 8.0, and double distilled water was continuously added dropwise until the total volume of the peptide solution was 1 mL. At the critical point of peptide self-assembly, its concentration was 1wt% (weight/volume). Using this method, 5 wt%, 1 wt%, and 0.5 wt% peptide solutions were separately prepared.

A 200 μL sample of peptide solution was dropped into an equal volume of calcium ion (Ca²⁺)-containing dulbecco's modified eagle medium (DMEM)/F₁₂ medium[32, 33] at pH 7.40, and a translucent hydrogel was formed in the vial after several seconds. Culture medium with a pH of 8.0 was added, and the hydrogel gradually dissolved. A 10 μL sample of the hydrogel was placed on a metal grid, dried at the critical point of dehydration, stained with phosphotungstic acid, and observed by TEM using a Hitachi HT7700 instrument (Hitachi, Japan).

Cell isolation and culture

All animal experiments were approved by the Animal Experimental Ethics Committee of Nanchang University and were performed as described previously[34-40]. Cells were harvested from rat bone marrow and cultured in a 75 cm² tissue culture dish in cell culture incubator at 37°C with 5% CO₂ (Thermo Fisher, China) at a cell density of 1×10⁶ cell/ml. After 48 h the culture medium was changed, and it was then changed once every 2 days. When cells in the culture dish were evenly spread and confluence reached 80-90%, they were passaged at a ratio of 1:2.

The cells adjusted to the density of 1×10⁶ cell/ml at the third passage were incubated with primary antibody for 40 min with saturating concentrations of monoclonal antibodies CD29, CD29-FITC, CD34-PE, CD44, CD44-PE, CD45 or CD45-FITC (Bio-legend, USA). After the cells were washed three times in buffer and centrifuged at 200 g for five minutes, they were resuspended in ice cold PBS and incubated with the FITC-labeled and phycoerythrin (PE)-labeled secondary antibody (HarveyBio, USA) for 30 minutes in the dark at 4 degrees centigrade. Cell fluorescence was evaluated by flow cytometry analysis using an Attune NxT instrument (Invitrogen, USA).

The cell adjusted to the density of 2×10⁵cell/ml at the third passage were placed in a six-well plate. The cells were divided into three groups when cell confluence reached 80%. The group A were added into osteogenic low-glucose DMEM containing 10% fetal bovine serum, 0.1 μM dexamethasone, 50 μM ascorbic acid, 10 mM β-glycerophosphate. Group B were cultured with adipogenic low-glucose DMEM containing 10% fetal bovine serum, 1 μM dexamethasone, 200 μM indometacin, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 μM insulin. Group C were only incubated in low-glucose DMEM. The medium was changed once every 3 days and cells were cultured for 2 weeks. Group A and C were subjected to Alizarin Red staining, and translucent lipid droplets in group B and C were identified by Oil Red O staining.

Calcein-AM/PI assay

A 200 μL sample of 1 wt% b-PA was placed in each well of a 24-well plate, an equal volume of cell suspension at a cell density of 1×10⁵cell/ml was added to each well. Next, 4 μM calcein-acetoxymethyl methacrylate (calcein-AM) and 2 μM propidium iodide (PI) (Dojindo Laboratories, Japan) were added after 1, 3 and 7 days, and live/dead cells were stained and incubated at 37°C for 30 min, and counted using inverted fluorescence microscope (OlympusCX23, Japan).

Virus construction and optimal MOI

Two adenoviral vectors [41, 42], one carrying VEGF165 and Ang-1 gene (Adv-Bic), and the other carrying NT-3 gene (AdvNT-3), were constructed using Cytomegalovirus (CMV) as a promoter. Adenoviral vectors were amplified using human embryonic kidney 293 cells and purified using cesium chloride gradient ultracentrifugation. The vector titer of mother liquor was adjusted to be 1×10¹²-1×10¹³ VG/mL. Green fluorescent protein (GFP)-labelled Adv-Bic and red fluorescent protein (RFP)-labelled AdvNT-3 were constructed in the same way.

Third passage cells were diluted to 1×10⁶ cell/ml in a 96-well plate, and 100 μL (1×10⁴ cells) of cell suspension was added to each well and incubated overnight. Adv-Bic (GFP-labelled) and AdvNT-3 (RFP-labelled) virus solutions were prepared at six different multiplicity of infection (MOI) concentrations in Eppendorf (EP) tubes, and each sample was diluted to 100 μL of complete culture with MOI values of 3, 10, 30, 100, 300 or 1000. These virus solutions were added to the 96-well plate and incubated for 4-8 h. After cell adherence, culture medium was discarded, 100 μL of complete culture medium was added to each well, and plates were further incubated. After 2 days, Select the most suitable MOI value according to the fluorescent cell ratio under the fluorescence microscope.

Gene transfection

Third passage cells were diluted to 1×10⁶ cell/ml in a 24-well plate with 200 μL (2×10⁵ cells) of cell suspension per well, and the Adv-Bic and AdvNT-3 genes were transfected into cells at the optimal MOI value (100). Then an equal volume of 1wt% b-PA peptide was added into the wells and a three-dimensional hydrogel encapsulating cells was formed within seconds (3D cultures). The hydrogel was adjusted to a pH of 7.35-7.45. After 7 days of culture, the pH of the hydrogel was adjusted to ~9.0, the gel was dissolved, and paraformaldehyde (40 g/L) was added for fixation for 30 min. The immunocytochemistry labelling technique was employed. Briefly, after blocking with serum at room temperature for 20 min, primary antibody was added and incubated at 4°C overnight, samples were washed three times with phosphate-buffered saline (PBS), and secondary antibody was added and incubated for 50 min at room temperature. After three more washes with PBS, DAPI staining solution was added and incubated in the dark for 10 min at room temperature, and cells were observed under a fluorescence microscope.

Primary antibodies (Beijing Zhongshan Golden Bridge Biotech, China) and dilutions were rat polyclonal antibody recognizing VEGF165 (1:200), CD31 (1:500), CD34 (1:500), Ang-1 (1:500) and Nestin (1:300); rabbit polyclonal antibody NT-3 (1:200) and neuron specific enolase (NSE)(1:300). Secondary antibodies (Boster Biotech, China) and dilutions were CY3-labllled goat anti-rabbit IgG (1:300) and Alexa Fluor 488-labelled goat anti-rabbit IgG (1:400).

qRT-PCR

Third passage cells were diluted to 1×10⁶ cell/ml and added to a six-well plate. 2 mL sample of cell suspension (containing 2×10⁶ cells) was added to each well. Advb-bic and AdvNT-3 cells were transfected with optimal MOI value (EG), and the rest were transfected with blank virus (CG). Then the genetically modified cell suspension were added with equal volume of 1wt% b-PA and incubated with at 37°C with 5% CO₂. After 7 days of culture, the pH of the hydrogel was adjusted to ~9.0, then the hydrogel was dissolved. Collected cells were washed with PBS twice, then TRIzol reagent (Invitrogen) was added.After extraction with chloroform, the aqueous phase was transferred to a new tube. The RNA in the aqueous phase was precipitated with isopropanol, washed with 75% ethanol and dried at room temperature. After dissolving with diethyl pyrocarbonate water, extracted RNAs were placed in a -20°C refrigerator for subsequent use. Extracted RNA was reverse transcribed into cDNA according to the instructions. qRT-PCR was performed using ABI 7500 RT-PCR (Applied Biosystems, USA). The specific reaction conditions were as follows: pre-denaturation at 95°C for 15 min, denaturation at 94°C for 15 s, 55°C for 30 s, extension at 72°C for 30 s, for a total of 40 cycles. Fluorescence was collected at 75-80°C and finally analyzed at 65-95°C for melting curve analysis.

Primers used in this study were as follows: GAPDH(F:5’-AGTGCCAGCCTCGTCTCATA-3’, R: 5’-AGAGAAGGCAGCCCTGGTAA-3’),VEGF165(F:5’-GGGAGCAGAAAGCCCATG AA-3’, R: 5’-AGATGTCCACCAGGGTCTCA-3’), NT-3(F:5’-TCCTCTCCTTACCCAGCATCT-3’, R:5’-GTTCGGTCATTCAGTCTCGC-3’), Ang-1(F:5’-TCGCTGCCATTCTGACTCAC-3’, R: 5’-CCGTCGTGTTCTGGAAGAATG-3’), CD31(F:5’-CGTTCAACAGAGCCAGCATT-3’, R: 5’-GGCAATGACCACTCCAATGA-3’), CD34(F:5’-ACACTCCACCTGGAACACCA-3’, R: 5’-AGCACATGGCTCAGTGGTTG-3’), NSE(F:5’-AAGTTGGCCATGCAGGAGTT-3’, R: 5’-AGTGGCATCCTTGCCGTACT-3’), Nestin(F:5’-TCGCTCAGATCCTGGAAGGT-3’, R: 5’-CAGGTGTCTGCAACCGAGAG-3’); The mRNA expression levels of all samples were known after correction by GAPDH expression. Gene expression was calculated using the 2-ΔΔCt method.

Statistical analysis

Statistical analysis of variance and t-test was performed using SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation. P < 0.05 was considered statistically significant.

b-PA characterisation

The structure of the synthesised b-PA molecule is shown in Figure 1. It can be seen by MS that the peak of the impure peptide is much lower than that of the target peptide; the average molecular weight was 2236 and 2304, compared with 2191.72 for the target peptide (Figure 2a). HPLC peak 3 showed that the purity of the synthetic target peptide was 95.9% (Figure 2b). Peaks 1 and 2 were impurities with concentrations <5%.

Gel detection

The hydrogels self-assembled from 5 wt% and 1wt% b-PA solution adhered to the vial wall, and did not detach from the bottom of vial when inverted (Figure 3c). By contrast, the 0.5 wt% peptide solution formed a thin flowing gel that did not adhere to the base of the inverted vial. TEM analysis showed that b-PA self-assembled into nanofibers with a high aspect ratio, the nanofiber diameter ranged from 5 to 8 nm and the length from hundreds of nanometres to micrometres. The nanofibers interweaved to form a grid-like structure (Figure 3e). These self-assembled nanofibers from 5 wt% b-PA were tightly arranged, with small inter-fiber spaces, whereas nanofibers from 0.5 wt% b-PA were more loosely arranged, with large inter-fiber spaces (Figure 3d).

Cell identification

After 48 h of cell culture, inverted microscopy showed that a small number of spindle cells began to adhere. At 3 days after inoculation, multiple cell colonies composed of massive long spindle cells were scattered at the bottom of the culture dish (Figure 4a). After 7 days of culture, cells proliferated and were showed different appearances of a large fusiform, polygonal or flattened mat (Figure 4b). The single cell morphology passed on to the 2^nd and 3^rd generations was typically long fusiform or spindle-shaped, and formed a radial or vortex arrangement (Figure 4c,d).

CD14, CD29, CD34, CD44, CD45 and CD90 were mesenchymal antigen markers [43], and CD34 and CD45 were haematopoietic stem cell marker antigens [44]. The results of flow cytometry showed that the cell surface molecules CD29 (Figure5a, b) and CD44 (Figure5c, d) were positively expressed with ratios of 97.8% and 99.8% respectively. The positive expression rates of antigens CD34 (Figure5e, f) and CD45 derived from haematopoietic stem cell were 1.61% and 1.71% (Figure5g, h).

The plain cells at the third passage were induced by osteogenic and adipogenic medium for 2 weeks, and stained with Alizarin Red and Oil Red O, revealing a large number of calcium nodules in group A (Figure 6a), and lipid droplets in group B cells (Figure 6b). However the cells only incubated in medium in group C failed to be stained by Alizarin Red or Oil Red O, which indicated the cells had no a capacity of osteogenic and adipogenic differentiation.

Cell viability

The results of fluorescence microscopy showed that 1wt% b-PA was self-assembled into the hydrogel with addition of cell suspensions at a cell density of 1×10⁵cell/ml. On the first day of culture, green fluorescent (living) cells were accompanied by some red fluorescent (dead) cells, and green fluorescence increased and red fluorescence decreased at the third day of culture, On the seventh day, green fluorescence covered the entire field of view, and no red fluorescence was observed. As the culture time was prolonged, the green fluorescence of living cell increased significantly (Figure 7a, c, e), and the red fluorescence decreased significantly (Figure 7b, d, f).

Optimal MOI value

Adv-Bic and AdvNT-3 were simultaneously transfected into BMSCs at different MOI values. After 2 days of culture, fluorescence microscopy showed that cells transfected with the NT-3 gene exhibited red fluorescence, and that with Adv-Bic displayed green fluorescence. With increasing MOI value, both green and red fluorescence increased correspondingly; when the MOI was 100, the peaks were reached for both green and red fluorescence (Figure 8a-h), and the transfection efficiency was about 65%. when the MOI was 300, some cells were clearly dead, and when the MOI was 1000, a large number of cells were dead.

Immunofluorescence

The cells were transfected with Adv-Bic and NT-3 genes and cultured within the hydrogel from 1wt% b-PA in vitro for 7 days, and were identified by immunocytochemistry. The fluorescence microscope showed the cells were positively expressed for VEGF165, NT-3, Ang-1, as evidenced by red, green, red fluorescence (Figure 9a, b and c). The cells within the hydrogel were able to be differentiated into endothelial cell-like cells, and the CD31 and CD34 markers were positively expressed with red fluorescence (Figure 9d and e). On the other hand, the self-assembled hydrogel from b-PA also promoted the cells to be differentiated into neural-like cells whose markers Nestin and NSE were positively expressed as red (Figure 9f) and green fluorescence (Figure 9g) respectively.

qRT-PCR detection

Neuron-specific enolase (NSE) was used as a marker of the neuron, and Nestin was a marker protein of neural stem cell (NSC) [45-48]. CD31 was an important endothelial cell surface membrane molecules, which was used to identify endothelial cells and angiogenesis [49-51]. Expression levels of VEGF165, NT-3, Ang-1, CD31, CD34, NSE and Nestin in EG were significantly higher than that in CG (p <0.05; Figure 10), which indicated that the hydrogel from 1wt% b-PA supported the cells to proliferate and differentiate into NSC, neural-like cells and endothelial cell-like cells.

We developed a b-PA containing RGD and IKVAV motifs that was self-assembled into a hydrogel. The RGD motif had adhesion to MSCs [52], and the IKVAV motify promoted neural precursor cell adhesion, differentiation, and axon growth, and inhibited glial cell adhesion and differentiation [53]. The carboxy terminal of the peptide included three lysine branching units to provide steric bulk, and separated RGD and IKVAV from each other, improved their binding to integrin receptors.

The solid phase synthesis method used to prepare b-PA was a third-generation peptide synthesis technology, and the side chains of the amino acids involved in the reaction were protected [54] to reduce the production of by-products. MS analysis showed that the average molecular weight of b-PA was consistent with the theorhetical molecular weight of the designed peptide, confirming generation of the correct target, and the peptide purity was >95%. A 1 wt% solution of b-PA was self-assembled to form a viscoelastic three-dimensional nanofiber hydrogel via weak hydrogen bonds, triggered by medium with a pH of 7.4 (including Ca²⁺). The hydrogel formed from 1 wt% b-PA had a high water content and adhered to the bottom of inverted vials. Meanwhile, the 0.5 wt% b-PA solution self-assembled to form a flowing hydrogel with poor viscoelasticity. TEM observation showed that when the concentration of the peptide solution increased slowly, the denser was the arrangement between nanofibers, and the smaller the gaps between them, which was not conducive to cell survival. Therefore, the concentration of b-PA in subsequent experiments was 1 wt%, and 5 wt% and 0.5 wt% solutions were discarded.

MSCs expressed surface antigens such as CD29, CD44, CD71 and CD90, and do not expressed antigens such as CD11a, CD14, CD34, CD38 and CD45 [55]. Cells prepared in the present study presented fibroblast-like appereance, and could divide and proliferate. Cells expressed CD44 and CD29 positive markers, but not express CD34 or CD45, and displayed multi-directional differentiation potential, consistent with MSCs [56, 57]. Cells had a typical long spindle shape, expressed surface antigen CD29+/44+, the positive rate was >95%, they displayed good cell uniformity and high purity, and could differentiate into osteoblasts and adipocytes, all consistent with the known characteristics of MSCs.

1wt% b-PA was added into cell suspension and self-assembled into nanofiber hydrogel encapsulating the cells. Calcein-AM/PI was performed to evaluate the biological properties of self-assmbling hydrogel. Live cells were fluorescently labelled with calcein-AM that emitted green fluorescence (excitation, 490nm, emission, 515nm), and dead cells stained with PI displayed red fluorescence (excitation, 535 nm; emission, 617 nm). Calcein-AM/PI assay results revealed many green fluorescently-labelled living cells on the surface and within the interior of the b-PA gel, and few dead cells. The self-assembled hydrogel from 1wt% b-PA supported cell proliferation, and was more favourable for cell adhesion and proliferation. These results showed that the self-supported hydrogel from b-PA had good cell compatibility, excellent material-cell interface properties, supported cell proliferation and adhesion, and the b-PA structure was easy to design and modify.

Using gene transfection technology, adv-bic and AdvNT-3 were transfected into BMSCs together by adenovirus. After 2 days of culturing, red and green fluorescence were observed under a microscope, and transfection was confirmed to be successful. When the MOI was 100, transfected cells were in the healthiest state and fluorescent expression was strongest. When the MOI was ≥300, cell death was observed, further demonstrating that an MOI of 100 was optimal. MSCs modified with non-fluorescently-labelled Adv-Bic and AdvNT-3 were inoculated into the b-PA hydrogel at the optimal MOI value and cultured in vitro for 1 week. Immunofluorescence analysis subsequently confirmed that VEGF165, NT-3 and Ang-1 were positively expressed, indicating that the b-PA hrdrogel supported gene-modified BMSCs overexpressing VEGF165, NT-3 and Ang-1.

VEGF was the strongest growth factor known to induce angiogenesis [58]. There were four subtypes (VEGF121, 165, 189 and 206), of which VEGF165 was the most active. By binding to Flk1, VEGF₁₆₅ promoted vascular regeneration by inducing MSCs to differentiate into endothelial cells, and to infiltrate endothelial cells [59, 60]. However, VEGF could cause side effects such as increased microvascular permeability and nerve tissue oedema [60]. Ang-1 counteracted VEGF-mediated increases in vascular permeability, regulated endothelial cell survival, inhibits exudation of inflammatory mediators, and reduced oedema [61-63]. NT-3 induced differentiation of neural precursor cells into neurons via tyrosine kinase receptor 3 (NTPK-3/TrkC), and induces differentiation of MSCs into functional neurons [64, 65].

In our experiments, the gene-modified MSCs within the self-assembled from b-PA expressed VEGF165, NT-3 and Ang-1, and could be differentiated into neural and endothelial cell-like cells. The results of qRT-PCR showed that expression levels of VEGF165, NT-3, Ang-1, CD31+/34+, NSE and Nestin were significantly higher than those of the control group, further demonstrating that b-PA hydrogel could be used as a scaffold to promote the differentiation of cells into nerves and blood vessels.

In summary, the b-PA was designed and synthesized in solid-phase method and was self-assembled into a three-dimensional nanofiber hydrogel. The genetical modified MSCs by VEGF₁₆₅, NT-3 and Ang-1 were inoculated into three-dimensional hydrogel, and were differentiated into neural-like cells and endothelial cell-like cells with alteration in the phenotype. This novel self-assembled hydrogel from b-PA might be used for the scaffolds of neural tissue engineering for the repair of spinal cord injury in the future.

Author(s’) disclosure

All authors declare no conflicts of interest.

Ethical conduct of research

Animal Experimental Ethics Committee of Nanchang University approved the study. All participants gave their written informed consent.

Financial & competing interests disclosure

This study was supported by the National Natural Science Foundation of China (30801158 and 81360271), the Natural Science Foundation of Jiangxi Province (20151BAB205051, 20151BAB205055 and 20181BAB205020), the Education Department of Jiangxi Province (GJJ09086 and GJJ14054); and the Health Department of Jiangxi Province (20081078).The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Contributions

SYL conceptualized, designed, and performed the experiments and data analysis, and drafted the manuscript. LYX and YW performed data analysis and revised the manuscript; YW, ZTT and JXH performed experiments. WK contributed reagents/materials and revised the manuscript. YPY conceptualized and designed the experiments and revised the manuscript. Final manuscript read and approved by all authors.

Acknowledgment

It has been previously presented as a preprint in this source: https://www.researchsquare.com/article/rs-1822746/v1.

Yixiu Li, Peiyi Yin, Wang Yu et al. Genetically modified MSCs into neural and endothelial cell-like cells induced by a branched peptide amphiphile hydrogel, 18 July 2022, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-1822746/v1]

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Genetically modified MSCs into neuraland endothelial cell-like cells inducedby a branched peptide amphiphile hydrogel

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