Monolayer Mesenchymal Stem Cells Research Articles

Self-assembled adipose-derived mesenchymal stem cells as an extracellular matrix component- and growth factor-enriched filler.

The clinical application of mesenchymal stem cells (MSCs) is attracting attention due to their excellent safety, convenient acquisition, multipotency, and trophic activity. The clinical effectiveness of transplanted MSCs is well-known in regenerative and immunomodulatory medicine, but there is a demand for their improved viability and regenerative function after transplantation. In this study, we isolated MSCs from adipose tissue from three human donors and generated uniformly sized MSC spheroids (∼100µm in diameter) called microblocks (MiBs) for dermal reconstitution. The viability and MSC marker expression of MSCs in MiBs were similar to those of monolayer MSCs. Compared with monolayer MSCs, MiBs produced more extracellular matrix (ECM) components, including type I collagen, fibronectin, and hyaluronic acid, and growth factors such as vascular endothelial growth factor and hepatocyte growth factor. Subcutaneously injected MiBs showed skin volume retaining capacity in mice. These results indicate that MiBs could be applied as regenerative medicine for skin conditions such as atrophic scar by having high ECM and bioactive factor expression.

Equine Platelet Lysate Gel: A Matrix for Mesenchymal Stem Cell Delivery.

A variety of bioscaffolds have been developed as carriers for the delivery of mesenchymal stem cells (MSCs), however, many of them are unable to provide direct cell nourishment, a critical factor for survival and retention of MSCs at the site of delivery. Platelet lysate is a plasma-derived product rich in growth factors that can be turned into a gel matrix following the addition of calcium chloride. Our objective was to characterize growth factor and cytokine release of equine platelet lysate gel (ePL gel) encapsulated with MSCs over time and to measure the viability and proliferation of ePL gel-encapsulated MSCs for up to 14 days. The release of interleukin-1β (IL-1β), interleukin-10 (IL-10), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), and platelet-derived growth factor BB (PDGF-BB), as well as fibrinogen degradation, were measured from ePL gel with and without equine bone marrow-derived MSCs and compared with MSCs in monolayer. MSC proliferation and viability within the gel were assessed up to 14 days. Compared with monolayer MSC cultures, significantly higher concentrations of IL-1β, IL-10, and TGF-β were measured from supernatants collected from ePL gel containing MSCs at various time points. Significantly lower concentrations of PDGF-BB were measured in the supernatant when MSCs were incorporated in ePL gel while VEGF tended to be increased compared with MSCs in monolayer. Incorporation in ePL gel for up to 14 days did not appear to affect viability and proliferation rates of MSCs as these were found to be similar to those measured in monolayer cell culture. ePL gel may have the potential to serve as bioscaffold for MSC delivery since it appears to support the proliferation and viability of MSCs for up to 14 days.

18F- based Quantification of the Osteogenic Potential of hMSCs.

In bone tissue engineering, there is a constant need to design new methods for promoting in vitro osteogenic differentiation. Consequently, there is a strong demand for fast, effective and reliable methods to track and quantify osteogenesis in vitro. In this study, we used the radiopharmacon fluorine-18 (18F) to evaluate the amount of hydroxylapatite produced by mesenchymal stem cells (MSCs) in a monolayer cell culture in vitro. The hydroxylapatite bound tracer was evaluated using µ-positron emission tomography (µ-PET) scanning and activimeter analysis. It was therefore possible to determine the amount of synthesized mineral and thus to conclude the osteogenic potential of the cells. A Student’s t-test revealed a highly significant difference regarding tracer uptake between the osteogenic group and the corresponding control group (µ-PET p = 0.043; activimeter analysis p = 0.012). This tracer uptake showed a highly significant correlation with the gold standard of quantitative Alizarin Red staining (ARS) (r2 = 0.86) as well as with the absolute calcium content detected by inductively coupled plasma mass spectrometry (r2 = 0.81). The results showed that 18F labeling is a novel method to prove and quantify hydroxyapatite content in MSC monolayer cultures. The mineral layer remains intact for further analysis. This non-destructive in vitro method can be used to rapidly investigate bone tissue engineering strategies in terms of hydroxylapatite production, and could therefore accelerate the process of implementing new strategies in clinical practice.

Evidence that TNF-β suppresses osteoblast differentiation of mesenchymal stem cells and resveratrol reverses it through modulation of NF-κB, Sirt1 and Runx2.

It has been established that inflammation plays an important role in bone formation and bone loss. Although a lot is known about the role of TNF-α in bone health, very little is understood about TNF-β, also called lymphotoxin. In this report, we examine the effect of TNF-β on osteogenic differentiation of mesenchymal stem cells (MSCs) and its modulation by resveratrol. Monolayer and high-density cultures of MSCs were treated with osteogenic induction medium with/without TNF-β, Sirt1 inhibitor nicotinamide (NAM), antisense oligonucleotides against Sirt1 (ASO) and/or Sirt1 stimulator resveratrol. We found that TNF-β inhibits, in a similar way to NAM or Sirt1-ASO, the early stage of osteogenic differentiation of MSCs and this was accompanied with downregulation of bone-specific matrix, β1-integrin, Runx2 and with upregulation of NF-κB phosphorylation and NF-κB-regulated gene products involved in the inflammatory, degradative processes and apoptosis. However, resveratrol reversed TNF-β- and NAM-suppressed MSCs osteogenesis by activation of Sirt1 and Runx2 that led to osteoblast differentiation. Furthermore, downregulation of Sirt1by mRNA inhibited the effect of resveratrol, highlighting the important impact of this enzyme in the TNF-β signaling pathway. Finally, resveratrol was able to manifest its effect both by suppression of TNF-β-induced NF-κB and through direct activation of the Sirt1 and Runx2 pathway. Thus, through these studies, we present a mechanism by which a T cell-derived cytokine, TNF-β can affect bone formation through modulation of MSCs differentiation that involves NF-κB, Sirt1, Runx2 and resveratrol reversed TNF-β-promoted impairments in MSCs osteogenesis.

Varied approach of using MSCs for bovine embryo in vitro culture

The objective of the present study was to evaluate the effect of porcine Mesenchymal Stem Cells (MSCs) secreted factors on bovine in vitro embryo development by using MSCs in different culture systems: SOF medium, SOF medium conditioned by MSCs in monolayer and in reverse drop and by embryo culture in co-culture with MSCs. Statistically highly significant differences were noted between the number of blastocysts derived cultures in all tested culture systems. The in vitro culture in SOF turned out to be the most optimal. Statistically highly significant differences were observed in the number of blastocyst obtained between SOF and SOF in co-culture with MSCs (p < 0.0001), and between SOF and SOF conditioned (monolayer and drop) (p < 0.00001). The trials to produce blastocysts in SOF conditioned by MSCs in reverse drops and monolayer failed. The blastocysts were obtained and analysed by TUNEL only in two out of four experimental groups: SOF and SOF in co-culture with MSCs. There were no significant differences between any of analysed blastocysts’ groups neither in the total number of nuclei nor in the apoptotic features. Neither medium conditioning by MSCs in monolayer and in reverse drop nor embryo culture in co-culture with MSC turned out to be effective.

Varied Approach of Using MSCs for Bovine Embryo In Vitro Culture

ABSTRACTThe objective of the present study was to evaluate the effect of porcine mesenchymal stem cells (MSCs) secreted factors on bovine in vitro embryo development using MSCs in different culture systems: SOF medium, SOF medium conditioned by MSCs in monolayer, and in reverse drop and by embryo culture in coculture with MSCs. Statistically highly significant differences were noted between the number of blastocysts derived cultures in all tested culture systems. The in vitro culture in SOF turned out to be the most optimal. Statistically highly significant differences were observed in the number of blastocyst obtained between SOF and SOF in coculture with MSCs (P < 0.0001), and between SOF and SOF conditioned (monolayer and drop) (P < 0.00001). The trials to produce blastocysts in SOF conditioned by MSCs in reverse drops and monolayer failed. The blastocysts were obtained and analyzed by TUNEL only in two out of four experimental groups: SOF and SOF in coculture with MSCs. There were no significant differences between any of analyzed blastocysts’ groups neither in the total number of nuclei nor in the apoptotic features. Neither medium conditioning by MSCs in monolayer and in reverse drop nor embryo culture in coculture with MSC turned out to be effective.

The Effects of Hyperacute Serum on the Elements of the Human Subchondral Bone Marrow Niche.

Mesenchymal stem cells (MSCs) are widely used in laboratory experiments as well as in human cell therapy. Their culture requires animal sera like fetal calf serum (FCS) as essential supplementation; however, animal sera pose a risk for clinical applications. Human blood derivatives, for example, platelet-rich plasma (PRP) releasates, are potential replacements of FCS; however, it is unclear which serum variant has the best effect on the given cell or tissue type. Additionally, blood derivatives are commonly used in musculoskeletal diseases like osteoarthritis (OA) or osteonecrosis as “proliferative agents” for the topical MSC pool. Hyperacute serum (HAS), a new serum derivative, has been designed to approximate the natural coagulation cascade with a single-step, additive-free preparation method. We investigated the effects of HAS on monolayer MSC cultures and in their natural niche, in 3D subchondral bone and marrow explants. Viability measurements, RT-qPCR evaluation for gene expression and flow cytometry for cell surface marker analysis were performed to compare the effects of FCS-, PRP-, or HAS-supplemented culture media. Monolayer MSCs showed significantly higher metabolic activity following 5 days' incubation in HAS, and osteoblast-specific mRNA expression was markedly increased, while cells also retained their MSC-specific cell surface markers. A similar effect was observed on bone and marrow explants, which was further confirmed with confocal microscopy analysis. Moreover, markedly higher bone marrow preservation was observed with histology in case of HAS supplementation compared to FCS. These findings indicate possible application of HAS in regenerative solutions of skeletal diseases like OA or osteonecrosis.

An efficient, non-viral dendritic vector for gene delivery in tissue engineering.

Recent developments within the field of tissue engineering (TE) have shown that biomaterial scaffold systems can be augmented via the incorporation of gene therapeutics. The objective of this study was to assess the potential of the activated polyamidoamine dendrimer (dPAMAM) transfection reagent (SuperfectTM) as a gene delivery system to mesenchymal stem cells (MSCs) in both monolayer and 3D culture on collagen based scaffolds. dPAMAM-pDNA polyplexes at a mass ratio (M:R) 10:1 (dPAMAM : pDNA) (1 ug pDNA) were capable of facilitating prolonged reporter gene expression in monolayer MSCs which was superior to that facilitated using polyethylenimine (PEI)-pDNA polyplexes (2 ug pDNA). When dPAMAM-pDNA polyplexes (1 ug pDNA) were soak loaded onto a collagen-chondroitin sulphate (CS) scaffold prolonged transgene expression was facilitated which was higher than that obtained for a PEI-pDNA polyplex (2 ug pDNA) loaded scaffold. Transgene expression was dependent on the composite nature of the collagen scaffold with varying expression profiles obtained from a suite of collagen constructs including a collagen alone, collagen-CS, collagen-hydroxyapatite, collagen-nanohydroxyapatite and collagen-hyaluronic acid scaffold. Therefore, the dPAMAM vector described herein represents a biocompatible, effective gene delivery vector for TE applications which, via matching with a particular composite scaffold type, can be tailored for regeneration of various tissue defects.

Spheroidal formation preserves human stem cells for prolonged time under ambient conditions for facile storage and transportation.

Human stem cells are vulnerable to unfavorable conditions, and their transportation relies on costly and inconvenient cryopreservation. We report here that human mesenchymal stem cells (MSC) in spheroids survived ambient conditions (AC) many days longer than in monolayer. Under AC, the viability of MSC in spheroids remained >90% even after seven days, whereas MSC in monolayer mostly died fast. AC-exposed MSC spheroids, after recovery under normal monolayer culture conditions with controlled carbon dioxide and humidity contents, resumed typical morphology and proliferation, and retained differentiating and immunosuppressive capabilities. RNA-sequencing and other assays demonstrate that reduced cell metabolism and proliferation correlates to the enhanced survival of AC-exposed MSC in spheroids versus monolayer. Moreover, AC-exposed MSC, when injected as either single cells or spheroids, retained therapeutic effects invivo in mouse colitis models. Spheroidal formation also prolonged survival and sustained pluripotency of human embryonic stem cells kept under AC. Therefore, this work offers an alternative and relatively simple method termed spheropreservation versus the conventional method cryopreservation. It shall remarkably simplify long-distance transportation of stem cells of these and probably also other types within temperature-mild areas, and facilitate therapeutic application of MSC as spheroids without further processing.

Exposure of a tendon extracellular matrix to synovial fluid triggers endogenous and engrafted cell death: A mechanism for failed healing of intrathecal tendon injuries

ABSTRACTAim: The purpose of this study was to investigate the effect of normal synovial fluid (SF) on exposed endogenous tendon-derived cells (TDCs) and engrafted mesenchymal stem cells (MSCs) within the tendon extracellular matrix. Methods: Explants from equine superficial digital flexor (extra-synovial) and deep digital flexor tendons (DDFTs) from the compressed, intra-synovial and the tensile, extra-synovial regions were cultured in allogeneic or autologous SF-media. Human hamstring explants were cultured in allogeneic SF. Explant viability was assessed by staining. Proliferation of equine monolayer MSCs and TDCs in SF-media and co-culture with DDFT explants was determined by alamarblue®. Non-viable Native Tendon matrices (NNTs) were re-populated with MSCs or TDCs and cultured in SF-media. Immunohistochemical staining of tendon sections for the apoptotic proteins caspase-3, −8, and −9 was performed. Results: Contact with autologous or allogeneic SF resulted in rapid death of resident tenocytes in equine and human tendon. SF did not affect the viability of equine epitenon cells, or of MSCs and TDCs in the monolayer or indirect explant co-culture. MSCs and TDCs, engrafted into NNTs, died when cultured in SF. Caspase-3, −8, and −9 expression was the greatest in SDFT explants exposed to allogeneic SF. Conclusions: The efficacy of cells administered intra-synovially for tendon lesion repair is likely to be limited, since once incorporated into the matrix, cells become vlnerable to the adverse effects of SF. These observations could account for the poor success rate of intra-synovial tendon healing following damage to the epitenon and contact with SF, common with most soft tissue intra-synovial pathologies.

Long-Duration Three-Dimensional Spheroid Culture Promotes Angiogenic Activities of Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) offer significant therapeutic promise for various regenerative therapies. However, MSC-based therapy for injury exhibits low efficacy due to the pathological environment in target tissues and the differences between in vitro and in vivo conditions. To address this issue, we developed adipose-derived MSC spheroids as a novel delivery method to preserve the stem cell microenvironment. MSC spheroids were generated by suspension culture for 3 days, and their sizes increased in a time-dependent manner. After re-attachment of MSC spheroids to the plastic dish, their adhesion capacity and morphology were not altered. MSC spheroids showed enhanced production of hypoxia-induced angiogenic cytokines such as vascular endothelial growth factor (VEGF), stromal cell derived factor (SDF), and hepatocyte growth factor (HGF). In addition, spheroid culture promoted the preservation of extracellular matrix (ECM) components, such as laminin and fibronectin, in a culture time- and spheroid size-dependent manner. Furthermore, phosphorylation of AKT, a cell survival signal, was significantly higher and the expression of pro-apoptotic molecules, poly (ADP ribose) polymerase-1 (PARP-1) and cleaved caspase-3, was markedly lower in the spheroids than in MSCs in monolayers. In the murine hindlimb ischemia model, transplanted MSC spheroids showed better proliferation than MSCs in monolayer. These findings suggest that MSC spheroids promote MSC bioactivities via secretion of angiogenic cytokines, preservation of ECM components, and regulation of apoptotic signals. Therefore, MSC spheroid-based cell therapy may serve as a simple and effective strategy for regenerative medicine.

Suspension-Expansion of Bone Marrow Results in Small Mesenchymal Stem Cells Exhibiting Increased Transpulmonary Passage Following Intravenous Administration.

Mesenchymal stem cells (MSCs) have been extensively explored in a variety of regenerative medicine applications. The relatively large size of MSCs expanded in tissue culture flasks leads to retention in the microcirculation of the lungs following intravenous delivery, reducing their capacity to reach target sites. We explored whether the expansion of whole marrow in suspension cultures would yield smaller MSCs with increased capacity to traverse the pulmonary microcirculation compared with traditional monolayer cultures. We tested this hypothesis using rat marrow in a suspension bioreactor culture with fibronectin-coated microcarriers, leading to sustained expansion of both the microbead-adherent cells, as well as of a nonadherent cell fraction. Magnetic depletion of CD45(+) cells from the bioreactor cultures after 5 weeks led to a highly enriched CD73(+)/CD90(+)/CD105(+) MSC population. The bioreactor-grown MSCs were significantly smaller than parallel monolayer MSCs (15.1 ± 0.9 μm vs. 18.5 ± 2.3 μm diameter, p<0.05). When fluorescently labeled bioreactor-grown MSCs were intravenously injected into rats, the peak cell concentration in the arterial circulation was an order of magnitude higher than similarly delivered monolayer-grown MSCs (94.8 ± 29.6 vs. 8.2 ± 5.6/10(6) nucleated blood cells, respectively, p<0.05). At 24 h after intravenous injection of the LacZ-labeled bioreactor-grown MSCs, there was a significant threefold decrease in the LacZ-labeled MSCs trapped in the lungs, with a significant increase in the cells reaching the spleen and liver in comparison to their monolayer MSC counterparts. Bioreactor-grown whole marrow cell cultures yielded smaller MSCs with increased capacity to traverse the pulmonary microcirculation compared with traditionally expanded monolayer MSCs. This may significantly improve the capacity and efficiency of these cells to home to injury sites downstream of the lungs.

Mesenchymal stem cell spheroids exhibit enhanced in-vitro and in-vivo osteoregenerative potential.

BackgroundMesenchymal stem cells (MSCs) are a favored cell source for regenerative medicine because of their multilinage potential. However, the conventional monolayer technique used to culture MSCs, inadequately overcomes their low differentiation capacity. Culture of MSCs in multicellular spheroids, more accurately mimics the in-vivo microenvironment; thus, resolving this problem. In this study, we assessed whether the osteoregenerative potential of MSC spheroids is greater than that of monolayer MSCs.ResultsMSC spheroids were generated from rat MSCs (rMSCs) using low-binding plates. Real-time reverse transcription-polymerase chain reaction and immunocytochemical analysis indicated that osteogenic properties were accelerated in MSC spheroids compared with monolayer rMSCs when treated with an osteoblast-inducer reagent for 7 days. Moreover, increased calcium deposition was visualized in MSC spheroids using Alizarin red staining. In a rat calvarial defect model, micro-computed tomography and histological assays showed that MSC spheroid-engrafted defects experienced enhanced bone regeneration.ConclusionsOur in-vitro and in-vivo results reveal that MSCs in the spheroid culture exhibit enhanced osteoregenerative efficiency compared with monolayer MSCs.

The Potential of Umbilical Cord Derived Mesenchymal Stem Cells in Intervertebral Disc Repair

Introduction Nucleus pulposus (NP) is the center and major compartment of intervertebral disc (IVD). Mesenchymal stem cells (MSCs) are a type of stem cell source under intensive investigation for their potential to regenerate NP. MSCs have been identified from various sources with different characteristics. There are indications that fetal or close to fetal tissue sources contain cells with relatively undifferentiated phenotype with respect to MSCs from adult sources. Moreover, evidences have shown that umbilical cord-derived MSCs (CMSCs) may have better chondrogenic differentiation potential than bone marrow-derived MSCs (BMSCs).1 We hypothesize CMSCs might be a suitable stem cell source for NP regeneration. The aim of this research is to analyze the paracrine effect of MSCs on NP cells, and compare the effect of BMSCs and CMSCs in an attempt to identify a better MSC source for future clinical application. Materials and Methods Human BMSCs, CMSCs, and degenerated NP cells (three batches each) were isolated and characterized from patients undergoing spinal fusion and patients at caesarean delivery, respectively, after IRB approval was acquired. Conditioned media (CM) was collected after 48 hours exposure to MSC monolayer. Cell proliferation and cytotoxicity were assessed by MTT assay after 1, 3, and 7 days in MSC-CM. Proteoglycan content of NP cells in both types of MSC-CM were measured by DMMB assay after 14 days in culture. Gene expression of degeneration-related molecules of NP cells in MSC-CM, including CDH2, CD55, FBLN1, Sox9, KRT19, KRT18, and MGP, were determined by real-time RT-PCR. Protein expression of KRT19 in degenerated NP cells before and after MSC-CM treatment was examined by immunocytochemistry and confocal microscopy. All results were normalized to the control group in which the NP cells were cultured in basal medium. Results Human BMSCs and CMSCs that we isolated satisfied the minimum criteria of MSCs; that is, they were CD73(+), CD105(+), CD146(+), CD14(−), CD45(−), C34(−), and had tri-lineage differentiation potency. The overall metabolic activities of NP cells measured by MTT reading were significantly enhanced in MSC-CM than that in control basal medium, especially in CMSC-CM. This is accompanied by a slight increase in proteoglycan production. We demonstrated that MMP12, MGP, and KRT19 are the major differential expressed genes between scoliotic and degenerated human NP cells. We found that MGP and MMP12 were significantly downregulated, while KRT19 expression was significantly upregulated, in NP cells treated by MSC-CM, especially CMSC-CM. The increased KRT19 expression in NP cells was also confirmed at protein level by confocal microscopy. Conclusion This is the first comparative study that discussed how different sources of MSCs affect the biological activities of cultured NP cells through paracrine effect. MGP2 and KRT193 has recently been reported to be associated with IVD degeneration. In this study, MSC-CM effectively upregulated KRT19 while downregulated MMP12 and MGP, suggesting that MSC-CM may promote a recovery of NP cell phenotype. In line with this finding is that MSC-CM treatment also enhanced overall cell metabolic activities, reduced apoptosis, and enhanced proteoglycan production of NP cells in culture. In all aspects tested, CMSC-CM showed stronger effect than BMSC-CM, suggesting that CMSC is a superior source of MSCs for future clinical application for IVD regeneration. Disclosure of Interest None declared References Wang L, Tran I, Seshareddy K, Weiss ML, Detamore MS. A comparison of human bone marrow-derived mesenchymal stem cells and human umbilical cord-derived mesenchymal stromal cells for cartilage tissue engineering. Tissue Eng Part A 2009;15(8):2259–2266 Lee CR, Sakai D, Nakai T, et al. A phenotypic comparison of intervertebral disc and articular cartilage cells in the rat. Eur Spine J 2007;16(12):2174–2185 Rutges J, Creemers LB, Dhert W, et al. Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: potential associations with aging and degeneration. Osteoarthritis Cartilage 2010;18(3):416–423

Three-Dimensional Spheroid-Cultured Mesenchymal Stem Cells Devoid of Embolism Attenuate Brain Stroke Injury After Intra-Arterial Injection

The therapeutic effect of mesenchymal stem cells (MSCs) in tissue repair/regeneration is substantially dampened by the loss of primitive properties and poor engraftment to target organs. In this study, the multipotency and cell sizes of human MSCs, which had been expanded in monolayer culture for several passages, were dramatically restored after an episode of three-dimensional (3D) spheroid culture. Unlike MSCs derived from monolayer, which caused embolism and blindness, MSCs derived from 3D spheroids did not cause vascular obstructions, after intra-carotid artery infusion in rats. Importantly, intra-carotid infusion of 1 million 3D spheroid MSCs in rats 24 h after middle cerebral artery occlusion and reperfusion resulted in engraftment of the cells into the lesion and significant (over 70%) reduction of infarct size along with restoration of neurologic function. Moreover, the enhanced effect of spheroid MSCs was coincided with significantly increased differentiation of the MSCs into neurons and markedly increased number of endogenous glial fibrillary acidic protein-positive neural progenitors in the peri-infarct boundary zone. However, the similarly administered monolayer MSCs resulted in a modest functional improvement. Our results suggest that 3D MSCs, in combination with intra-carotid delivery, may represent a novel therapeutic approach of MSCs for stroke.

Substrate-dependent gene regulation of self-assembled human MSC spheroids on chitosan membranes

BackgroundThree-dimensional (3D) multicellular spheroids of mesenchymal stem cells (MSCs) are generally regarded to have beneficial properties over MSCs in monolayer. Recent literatures have documented that MSCs can self-assemble into 3D spheroids with a greater capacity for differentiation into various cell types when grown on chitosan (CS), a biopolymer. The genomic modulation occurring in these MSC spheroids is thus of essential importance for understanding their uniqueness and therapeutic potentials. In this study, 3D spheroids self-assembled from human umbilical cord MSCs grown on CS membranes were analyzed by mRNA as well as microRNA microarrays, which helped identify the critical signaling events that may alter the cellular functions during the spheroid forming process.ResultsGenes screened from mRNA and microRNA cross-correlation analyses were further confirmed with the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. Results revealed the regulation of a significant number of calcium-associated genes, which suggested the crucial role of calcium signaling in CS-derived MSC spheroids. In addition, many genes associated with the multilineage differentiation capacities and those associated with the antiinflammatory and antitumor properties of MSCs were upregulated. The genetic modulation was significantly more remarkable and endured longer for MSC spheroids derived on CS substrates compared to those derived on a non-adherent (polyvinyl alcohol) substrate.ConclusionsBased on the study, the culture substrates used to prepare 3D MSC spheroids may predefine their properties through cell-substrate interaction.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-10) contains supplementary material, which is available to authorized users.

Effect of various co-culture systems on embryo development in ovine

Considering the advent of mesenchymal stem cells (MSCs) as a new source of somatic cells in embryo co-culture system, the current study was aimed to compare in vitro embryo development using embryonic MSCs monolayer with embryonic fibroblast cells (EFCs), oviductal epithelial cells (OECs), and cell-free culture system. The IVM/IVF presumptive sheep zygotes were randomly cultured in different culture conditions as follows: (1) SOFaaBSA medium for the whole culture period (SOF, n = 371), (2) SOFaaBSA medium for the first 3 days followed by co-culturing with MSCs for the next 5 days (SOF-MSCs, n = 120), (3) co-culturing with MSCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (MSCs-SOF, n = 133), (4) co-culturing with MSCs for the whole culture period (MSCs, n = 212), (5) SOFaaBSA medium for the first 3 days followed by co-culturing with EFCs for the next 5 days (SOF-EFCs, n = 132), (6) co-culturing with EFCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (EFCs-SOF, n = 165), (7) co-culturing with EFCs for the whole culture period (EFCs, n = 236), and (8) co-culturing with OECs for the whole culture period (OECs, n = 255). One-Way ANOVA by multiple pairwise comparisons using Tukey&amp;rsquo;s test was performed. Co-culturing in MSCs group had no superiority over EFCs and OECs groups. Though, when co-culturing with MSCs and EFCs was limited to the first 3 days of culture, the embryo development indices were improved compared to the other co-cultured groups. Considering both the hatching rate and total cell number, the application of MSCs for the first 3 days of culture (MSCs-SOF) was superior to the other co-culture and SOF groups. &amp;nbsp;

Cytotoxicity of Local Anesthetics on Human Mesenchymal Stem Cells in Vitro

The purpose of this study was to investigate the cytotoxic potency of local anesthetics on human mesenchymal stem cells (MSCs) before and after chondrogenic differentiation. MSCs were exposed to equal and equipotent concentrations of bupivacaine, ropivacaine, and mepivacaine for 1 hour. Cell viability, apoptosis, and necrosis were determined using flow cytometry and live/dead staining. After chondrogenic differentiation, MSC viability was determined in aggregates exposed to equipotent concentrations of the named agents, applying fluorescence microscopy. All local anesthetics showed detrimental cytotoxic effects on MSC monolayer cultures in a concentration- and time-specific manner. Minimum viability rates were found 96 hours after a 1-hour exposure. Bupivacaine 0.5% caused a reduction of vital MSCs to 5% ± 1%. Sixteen percent ± 2% viable cells were detected after treatment with 0.75% ropivacaine. Exposure to 2% mepivacaine decreased vitality rates to 1% ± 0%. Ropivacaine was significantly less cytotoxic than were bupivacaine and mepivacaine. Immediate cell death was mainly caused by necrosis followed by apoptosis afterward. Viability rates of MSCs embedded in cartilaginous tissue after chondrogenic differentiation were not reduced by local anesthetic treatment. Local anesthetics are cytotoxic to MSCs in a concentration-, time-, and agent-dependent manner in monolayer cultures but not in whole-tissue probes. MSCs are applied for treatment of cartilage defects. Intra-articular application of local anesthesia is a common procedure in pain management and has shown chondrotoxic effects. Therefore, it is crucial to evaluate the impact of local anesthetics on human MSCs and regenerative cartilage tissue engineering.

Low-Level Shear Stress Induces Human Mesenchymal Stem Cell Migration Through the SDF-1/CXCR4 Axis Via MAPK Signaling Pathways

Mesenchymal stem cells (MSCs) are able to home and migrate into damaged tissues and are thus, considered an optimal therapeutic strategy for clinical use. We previously demonstrated that higher shear stress (>2 Pa) hindered human MSC (hMSC) migration, whereas lower shear stress (0.2 Pa) induced cell migration through mitogen-activated protein kinase (MAPK) pathways. Here the mechanisms underlying shear stress-induced hMSC migration have been studied further. An MSC monolayer was mechanically wounded and subsequently exposed to low-level shear stress of 0.2 Pa. Image analysis was performed to quantify cell migration speeds under both flow and static conditions. hMSCs along both upstream- and downstream edges of the wound migrated at a similar speed to cover the wounded area under static conditions, whereas shear stress induced cells along the downstream edge of the wound to migrate significantly faster than those along the upstream edge. We also found that shear stress upregulated the secretion of stromal-derived factor-1 (SDF-1), which stimulated its receptor CXCR4 expression in hMSCs until the cells covered the wounded area. A CXCR4 antagonist repressed both cell migration and activation of c-Jun N-terminal kinase (JNK) and p38 MAPK but did not affect extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. When MAPK activation in upstream- and downstream hMSCs was evaluated separately, ERK1/2 was activated earlier in downstream than in upstream cells. These results indicate that the SDF-1/CXCR4 axis mediates shear stress-induced hMSC migration through JNK and p38 MAPK pathways and that the difference in migration speeds between upstream- and downstream cells may be due to ERK1/2 activation.

Cyclic tensile strain upon human mesenchymal stem cells in 2D and 3D culture differentially influences CCNL2, WDR61 and BAHCC1 gene expression levels

It has been shown that tensile strain can alter cell behaviour. Evidence exists to confirm that human mesenchymal stem cells can be encouraged to differentiate in response to tensile loading forces. We have investigated the short-term effects of cyclic tensile strain (3%, 1 Hz) on gene expression in primary human mesenchymal stem cells in monolayer and whilst encapsulated in a self-assembled peptide hydrogel. The main aims of the project were to gain the following novel information: (1) to determine if the genes CCNL2, WDR61 and BAHCC1 are potentially important mechanosensitive genes in monolayer, (2) to determine if these genes showed the same differential expression in a 3D environment (either tethered to RGD or simply encapsulated within a hydrogel (with RGE motif)) and (3) to determine whether the mesenchymal stem cells would survive within the hydrogels over several days whilst enduring dynamic culture. In the monolayer system, real-time PCR confirmed CCNL2 was significantly downregulated after 1 h strain and 2 h latency (post strain). BAHCC1 was significantly downregulated after 1 h strain (both 2 h and 24 h latency). WDR61 followed the same trend in 2D culture. After 24 h strain and 2 h latency, BAHCC1 was significantly upregulated. We found that both types of peptide hydrogel supported viable mesenchymal stem cells over 48 h. Results of the 3D dynamic culture did not correspond with those of the 2D dynamic culture, where the BAHCC1 gene was not expressed in the 3D experiments. The disparity in the differential gene expression observed between the 2D and 3D culture systems may partly be a result of the different cellular environments in each. It is likely that cells cultured within an intricate 3D architecture respond to mechanical cues in a different and more complex manner than do cells in 2D monolayer, as is illustrated by our gene expression data.