Applications of extraembryonic tissue-derived cells in vascular tissue regeneration.

Vascular tissue engineering is a promising approach for regenerating damaged blood vessels and developing new therapeutic approaches for heart disease treatment. To date, different sources of cells have been recognized that offer assistance within the recovery of heart supply routes and veins with distinctive capacities and are compelling for heart regeneration. However, some challenges still remain that need to be overcome to establish the full potential application of these cells. In this paper, we review the different cell sources used for vascular tissue engineering, focusing on extraembryonic tissue-derived cells (ESCs), and elucidate their roles in cardiovascular disease. In addition, we highlight the intricate interplay between mechanical and biochemical factors in regulating mesenchymal stem cell (MSC) differentiation, offering insights into optimizing their application in vascular tissues.

An in situ hydrogel-forming scaffold loaded by PLGA microspheres containing carbon nanotube as a suitable niche for neural differentiation.

The cell-extracellular matrix (ECM) interactions are known to have a strong impact on cell behaviors in neural tissues. Due to complex physiology system and limited regenerative capacity of nervous system, neural tissue engineering has attracted attention as a promising strategy. In this study, we designed a hydrogel loaded by poly (lactic-co-glycolic acid) (PLGA) microspheres containing carbon nanotubes (CNT) and the biochemical differentiation factors, as a scaffold, in order to replicate the neural niche for stem cell growth (and/or differentiation). Different formulations from Hyaluronic acid (H), Poloxamer (P), Ethoxy-silane-capped poloxamer (PE), and cross-linked Alginate (Alg) were utilized as an in situ gel structure matrix to mirror the mechanical properties of the ECM of CNS. Subsequently, conductivity, surface morphology, size of microspheres, and CNT dispersion in microsphere were measured using two probes electrical conductometer, scanning electron microscopy (SEM), dynamic light scattering (DLS), and Raman spectroscopy, respectively. According to SEM and fluorescent microscopy images, CNTs increased the porosity of polymeric structure, which, in turn, facilitated the adhesion of stem cells on the surface of microspheres compared with control. Microstructure and rheological behaviors of different gel compositions were investigated using SEM and parallel-plate oscillatory rheometer, respectively. The MTT assay showed the toxicity profile of hydrogels was appropriate for cell transplantation. The confocal images illustrated the 3D platform of P15%H10% and P20%H5% gel formulations containing the PLGA-CNT microspheres, which allows the proliferation of neural stem cells (NSCs) derived from MSC. The results of real-time PCR and immunocytochemistry showed neuronal differentiation capacity of cultured NSCs derived from MSC in the alginate gel that contained PLGA-CNT microspheres as well as other control groups. The dispersion of the CNT-PLGA microspheres, covered by NSCs, into alginate gel in the presence of induction factors was found to notably enhance the expression of Sox2-SYP and ß-Tubulin III neuronal markers.

In vivo bone regeneration using a bioactive nanocomposite scaffold and human mesenchymal stem cells.

Due to the osteoconductive role of bioceramics, use of these bioactive nanocomposite scaffolds that can maintain their structural integrity during bone tissue repair is one of the major goals of tissue engineering. Herein, a nanofibrous poly-L-lactic acid (PLLA) scaffold was fabricated by electrospinning and then gelatin and hydroxyapatite nanoparticles (nHA) were coated over the surface of the scaffold. Osteoconductivity of the fabricated nano-composite scaffolds was then studied while grafted on the rat calvarial defects. Our results indicated that the coating of PLLA scaffold with nHA and gelatin increased the adhesion and growth of the human bone marrow derived mesenchymal stem cells (BM-MSCs) and also significantly increased the level of mineralization over a week culture period. The results of radiographic and histological studies showed that the newly created bone tissue at the defect site was significantly higher in animals treated with nanocomposite scaffolds than the empty scaffolds and control groups. This increase in the defect reconstruction was significantly increased after culturing BM-MSCs on the scaffolds, especially nanocomposite scaffolds. It can be concluded that the combination of nanocomposite scaffolds and BM-MSCs could be a very good candidate for treatment of bone lesions and could be considered as a bony bioimplant.

Electrical stimulation induces differentiation of human cardiosphere-derived cells (hCDCs) to committed cardiomyocyte.

Logistic complexities of heart transplantation embossed the necessity of utilizing novel methods, which enable heart regeneration. Human cardiosphere-derived cells (hCDCs) are taken into consideration as a promising cell resource in cell therapy in recent years. In this study, we designed an electrochemical stimulation system, which sends square pulses to the hCDCs and records their electrical response. Morphology, viability and differentiation of hCDCs are monitored at certain time courses of the treatment. Differentiating hCDCs aligned perpendicularly with respect to the direction of applied electric current, and obtained a spindle-like morphology, while they remained viable. At the same time, specific cardiac marker genes including GATA4, cTnT and α-MHC showed a considerable up-regulation. Our findings confirm that hCDCs differentiate to committed cardiomyocytes when hCDCs receive an electrical energy of 0.06 - 0.12 Wh. This amount of electrical energy could be applied to the stem cells using versatile electrical stimulation patterns via commercially available devices.

DKK1 expression is suppressed by miR-9 during induced dopaminergic differentiation of human trabecular meshwork mesenchymal stem cells.

Wnt/ß-catenin pathway has been recently identified as one of the key players in dopaminergic (DA) neuron differentiation. DKK1, the potent inhibitor of the Wnt/ß-catenin pathway, is expressed in a precisely controlled manner in ventral midbrain during brain development, however the molecular mechanism underlying this regulation is still unknown. Here we show that human trabecular meshwork mesenchymal stem cells (TM-MSCs) can be used as an efficient tool for in vitro differentiation of DA neurons. After differentiating TM-MSCs to DA neuron-like cells, ß-catenin protein accumulation was increased in the nucleus, indicating the increased activity of Wnt/ß-catenin pathway in the time-window of DA differentiation. Interestingly, DKK1 transcript level was reduced dramatically after DA induction in TM-MSCs which was accompanied by an increase in the in silico-predicted MIR9 and MIR101 levels. Measuring DKK1 expression level after overexpressing either MIR9 or MIR101 and performing luciferase assay alongside, revealed that both miR-9 and miR-101 suppress DKK1 expression and that miR-9 exerts a direct inhibitory effect on 3'UTR regulatory region. Therefore miR-9 and miR-101 might explain, at least in part, the underlying regulatory mechanism of DKK1 reduction and resulting Wnt/ß-catenin pathway activation during DA neuron differentiation process.

Fucosylated umbilical cord blood hematopoietic stem cell expansion on selectin-coated scaffolds.

Despite the advantages of transplantation of umbilical cord blood's (UCB's) hematopoietic stem cells (uHSCs) for hematologic malignancy treatment, there are two major challenges in using them: (a) Insufficient amount of uHSCs in a UCB unit; (b) a defect in uHSCs homing to bone marrow (BM) due to loose binding of their surface glycan ligands to BM's endothelium selectin receptors. To overcome these limitations, after poly l-lactic acid (PLLA) scaffold establishment and incubation of uHSCs with fucosyltransferase-VI and GDP-fucose, ex vivo expansion of these cells on selectin-coated scaffold was done. The characteristics of the cultured fucosylated and nonfucosylated cells on a two-dimensional culture system, PLLA, and a selectin-coated scaffold were evaluated by flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony-forming unit (CFU) assay, and CXCR4 expression at the messenger RNA and protein levels. According to the findings of this study, optimized attachment to the scaffold in scanning electron microscopy micrograph, maximum count of CFU, and the highest 570 nm absorption were observed in fucosylated cells expanded on selectin-coated scaffolds. Furthermore, real-time polymerase chain reaction showed the highest expression of the CXCR4 gene, and immunocytochemistry data confirmed that the CXCR4 protein was functional in this group compared with the other groups. Considered together, the results showed that selectin-coated scaffold could be a supportive structure for fucosylated uHSC expansion and homing by nanotopography. Fucosylated cells placed on the selectin-coated scaffold serve as a basal surface for cell-cell interaction and more homing potential of uHSCs. Accordingly, this procedure can also be considered as a promising technique for the hematological disorder treatment and tissue engineering applications.

Zinc silicate mineral-coated scaffold improved in vitro osteogenic differentiation of equine adipose-derived mesenchymal stem cells.

In current study we aimed to coat the PLLA scaffold with zinc (Zn) silicate mineral nanoparticles. Then, using equine adipose-derived stem cells (ASCs) we intended to compare the osteogenic induction potency of Zn silicate mineral-coated PLLA scaffold with uncoated PLLA scaffold and tissue culture plastic (TCPS). Adipose tissues were collected from 3 horses, and isolation of ASCs was achieved by enzymatic digestion. PLLA scaffold was successfully prepared using a phase separation method and coated with Zn silicate mineral nanoparticles. The coating efficiency was then characterized by scanning electron microscopy and further evaluated with the application of fourier transform infrared microscopic imaging. Viability and growth characteristics of ASCs on TCPS, uncoated and coated PLAA scaffolds were investigated by MTT assay. Alizarin Red staining was performed for determination of calcium deposition following the osteogenic induction. Furthermore, other common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium content, as well as osteogenic (Runx2, ALP, osteonectin, and collagen I) marker genes were also evaluated. Our data showed that Zn silicate mineral nanoparticles was coated successfully on PLLA scaffold and such scaffold had no detrimental effect on cell growth rate as indicated by MTT assay. Moreover, ASCs that differentiated on Zn silicate mineral-coated PLLA scaffold indicated higher ALP activity, more calcium content, and higher expression of bone-related genes than that on uncoated PLLA scaffold and TCPS. Adequate proliferation rate and higher expression of osteogenic markers of stem cells, provides this scaffold as a suitable substrate to support proliferation and differentiation of ASCs in equine.

Osteogenic differentiation of Wharton's jelly-derived mesenchymal stem cells cultured on WJ-scaffold through conventional signalling mechanism.

Wharton's jelly-derived extracellular matrix (WJ-ECM) has attracted researcher's attention for its biomedical applications. Previously, we fabricated a biomimetic spongy scaffold from decellularized WJ-ECM and, in this study, we sought to examine the osteogenic inductive potential of this scaffold and its underlying mechanism. To address this question, mesenchymal stem cells (MSCs) were isolated from WJ using a mechanical method and cultured on the scaffold, under dynamic condition, for over 21 days in the presence or absence of osteogenic medium. The status of signalling pathways involved in the osteogenic differentiation and the expression profile of integrins in the WJ-derived MSCs (WJ-MSCs) were examined. WJ-MSCs displayed differentiation capacities and expressed surface antigens, characteristics of MSCs. Histologically, WJ-MSCs seeded on the scaffold showed a proper cellular attachment, penetration and migration. They also exhibited a higher degree of alkaline phosphatase activity, calcium deposition and osteogenic gene expression, than those cultured in 2D condition. The expression of Wnt, BMP and TGF-ß signalling target genes together with that of α2, αv and ß1 integrins was increased in WJ-MSCs in both presence and absence of osteogenic induction medium. Taken together, our results demonstrate that WJ-derived scaffold induces osteogenic differentiation of WJ-MSCs, possibly through activating integrins and subsequently conventional intracellular signalling pathways.

Nanotopographical cues of electrospun PLLA efficiently modulate non-coding RNA network to osteogenic differentiation of mesenchymal stem cells during BMP signaling pathway.

Application of stem cells in combination with nanofibrous substrates is an interesting biomimetic approach for enhanced regeneration of damaged tissues such as bone and cartilage. The investigation of the complex interplay between nanotopographical cues of niche and noncoding RNAs in stem cells fate is an effective tool to find a new strategy for enhancing the induction of osteogenesis. In this study, we investigated the effects of aligned and random orientations of nanofibers as a natural ECM-mimicking environment on the network of noncoding RNA in mesenchymal stem cells. Aligned and randomly oriented Ploy (L-lactide) PLLA scaffolds were fabricated via electrospinning. Human Adipose Tissue-Derived Mesenchymal Stem Cells (hASCs) were isolated from adipose tissue and were cultured on surfaces of these scaffolds. Their capacity to support hMSCs proliferation was also investigated by MTT assay and the expression of c-Myc gene. Then, after 7, 14 and 21 days, the osteogenic commitment of hMSCs and the miRNA regulatory network in BMP signaling pathway were evaluated by measuring alkaline phosphatase (ALP) activity, extracellular calcium deposition, and bone-related gene activation by Real-Time PCR. Furthermore, osteogenic differentiation was evaluated with regard to their noncoding RNA network. Our results for the first time showed an interaction between nanotopographical cues and miRNA activity in hMSCs. We found that the nanotopographical cues could be used to influence the osteogenic differentiation process of hMSCs through the modulation of lncRNAs and miR-125b as negative regulators of osteogenesis as well as the H19 modulator BMP signaling pathway that acts as a miRNA sponge. Moreover, we also demonstrated for the first time that MEG3 as a long noncoding RNA is controlled by miR-125b and microRNA-triggered lncRNA decay mechanism. This strategy seems to be an important tool for controlling stem cell fate in engineered tissues and provide new insights into most biocompatible scaffolds for bone-graft substitutes.

The osmolyte type affects cartilage associated pathologic marker expression during in vitro mesenchymal stem cell chondrogenesis under hypertonic conditions.

Stem cells' fate during in vitro differentiation is influenced by biophysicochemical cues. Osmotic stress has proved to enhance chondrocyte marker expression, however its potent negative impacts had never been surveyed. We questioned whether specific osmotic conditions, regarding the osmolyte agent, could benefit chondrogenesis while dampening undesired concomitant hypertrophy and inflammatory responses. To examine the potential side effects of hypertonicity, we assessed cell proliferation as well as chondrogenic and hypertrophic marker expression of human Adipose Derived-MSC after a two week induction in chondrogenic media with either NaCl or Sorbitol, as the osmolyte agent to reach a +100 mOsm hypertonic condition. Calcium deposition and TNF-α secretion as markers associated with hypertrophy and inflammation were then assayed. While both hyperosmotic conditions upregulated chondrogenic markers, sorbitol had a nearly three times higher chondro-promotive effect and a lesser hypertrophic effect compared to NaCl. Also, a significantly lesser calcium deposition was observed in sorbitol hypertonic group. NaCl showed an anti-proinflammatory effect while sorbitol had no effect on inflammatory markers. The ossification potential and cartilage associated pathologic markers were affected differentially by the type of the osmolyte. Thus, a vigilant application of the osmotic agent is inevitable in order to avoid or reduce undesired hypertrophic and inflammatory phenotype acquisition by MSC during chondrogenic differentiation. Our findings are a step towards developing a more reliable chondrogenic regimen using external hypertonic cues for MSC chondrogenesis with potential applications in chondral lesions cell therapy.

Generation of high-yield insulin producing cells from human-induced pluripotent stem cells on polyethersulfone nanofibrous scaffold.

Transplantation of islet is a promising method in treatment of patients with type 1 diabetes mellitus (T1DM), however, is limited by islet shortage. The aim of this study was to prepare a polyethersulfone (PES) nanofibrous scaffolds to evaluate the pancreatic differentiation of human induced pluripotent stem cells (hiPSCs). The differentiation process in tissue culture dishes and PES scaffolds was evaluated at mRNA and protein level by RT-qPCR and immunofluorescence assay, respectively. The functionality of differentiated cells was determined by insulin and C-peptide release in response to glucose challenges. The results of this study showed that cells cultured on PES nanofibrous scaffolds exhibit more pancreatic ß-cell characteristics as they express more pancreatic tissue-specific genes and proteins. Furthermore, the immunoassay showed that differentiated cells in both culture plates and PES scaffolds groups are functional and secrete C-peptide and insulin in response to glucose challenges. Altogether, the results of this study demonstrated that PES nanofibrous scaffold could provide the microenvironment that promotes the differentiation of induced pluripotent stem cells (iPSCs) into insulin producing cells.

Enhanced chondrogenesis of human bone marrow mesenchymal Stem Cell (BMSC) on nanofiber-based polyethersulfone (PES) scaffold.

Mesenchymal stem cells (MSC) from bone marrow hold great potential as a cell source for cartilage repair. The objective of our study was differentiation of MSC toward chondrocyte by using Nanofiber-based polyethersulfone (PES) scaffold and also enhanced chondrogenic differentiation of BMSC in vitro. MSCs were harvested from bone marrow of human and PES scaffold was fabricated via Electrospinning. The isolated cells were cultured on the PES scaffold and scaffold free method. After 21days, Real-time PCR was performed to evaluate the cartilage-specific genes in the mRNA levels. Also, in order to confirm our results, we have done immunocytochemistry and SEM imaging. Flowcytometry confirmed the nature of the isolated adherent cells. Immunocytochemistry and SEM imaging confirmed the differentiation of MSC toward chondrocyte. Also, real time PCR showed a significant increased gene expression of collagen type II and aggrecan on the PES scaffold method when compared to the mRNA levels measured in scaffold free method. Down regulation of Collagen type I was observed in PES scaffold compared to scaffold free at day 21. Also, both methods showed a similar pattern of expression of SOX9. Our results showed that PES scaffold maintains BMSC proliferation and differentiation, and can significantly enhance chondrogenic differentiation of BMSC. PES scaffold seeded BMSC showed the highest capacity for differentiation into chondrocyte-like cells.

Improved stem cell therapy of spinal cord injury using GDNF-overexpressed bone marrow stem cells in a rat model.

The use of stem cell base therapy as an effective strategy for the treatment of spinal cord injury (SCI) is very promising. Although some strategy has been made to generate neural-like cells using bone marrow mesenchymal stem cells (BMSCs), the differentiation strategies are still inefficiently. For this purpose, we improved the therapeutic outcome with utilize both of N-neurotrophic factor derived Gelial cells (GDNF) gene and differentiation medium that induce the BMSCs into the neural-like cells. The differentiated GDNF overexpressed BMSCs (BMSCs-GDNF) were injected on the third day of post-SCI. BBB score test was performed for four weeks. Two weeks before the end of BBB, biotin dextranamin was injected intracrebrally and at the end of the fourth week, the tissue was stained. BBB scores were significantly different in BMSCs-GDNF injected and control animals. Significant difference in axon counting was observed in BMSCs-GDNF treated animals compared to the control group. According to the results, differentiated BMSCs-GDNF showed better results in comparison to the BMSCs without genetic modification. This study provides a new strategy to investigate the role of simultaneous in stem cell and gene therapy for future neural-like cells transplantation base therapies for SCI.