Migration Of hMSCs Research Articles

MiR-29a-3p orchestrates key signaling pathways for enhanced migration of human mesenchymal stem cells

BackgroundThe homing of human mesenchymal stem cells (hMSCs) is crucial for their therapeutic efficacy and is characterized by the orchestrated regulation of multiple signaling modules. However, the principal upstream regulators that synchronize these signaling pathways and their mechanisms during cellular migration remain largely unexplored.MethodsmiR-29a-3p was exogenously expressed in either wild-type or DiGeorge syndrome critical region 8 (DGCR8) knockdown hMSCs. Multiple pathway components were analyzed using Western blotting, immunohistochemistry, and real-time quantitative PCR. hMSC migration was assessed both in vitro and in vivo through wound healing, Transwell, contraction, and in vivo migration assays. Extensive bioinformatic analyses using gene set enrichment analysis and Ingenuity pathway analysis identified enriched pathways, upstream regulators, and downstream targets.ResultsThe global depletion of microRNAs (miRNAs) due to DGCR8 gene silencing, a critical component of miRNA biogenesis, significantly impaired hMSC migration. The bioinformatics analysis identified miR-29a-3p as a pivotal upstream regulator. Its overexpression in DGCR8-knockdown hMSCs markedly improved their migration capabilities. Our data demonstrate that miR-29a-3p enhances cell migration by directly inhibiting two key phosphatases: protein tyrosine phosphatase receptor type kappa (PTPRK) and phosphatase and tensin homolog (PTEN). The ectopic expression of miR-29a-3p stabilized the polarization of the Golgi apparatus and actin cytoskeleton during wound healing. It also altered actomyosin contractility and cellular traction forces by changing the distribution and phosphorylation of myosin light chain 2. Additionally, it regulated focal adhesions by modulating the levels of PTPRK and paxillin. In immunocompromised mice, the migration of hMSCs overexpressing miR-29a-3p toward a chemoattractant significantly increased.ConclusionsOur findings identify miR-29a-3p as a key upstream regulator that governs hMSC migration. Specifically, it was found to modulate principal signaling pathways, including polarization, actin cytoskeleton, contractility, and adhesion, both in vitro and in vivo, thereby reinforcing migration regulatory circuits.

Growth factor-loaded sulfated microislands in granular hydrogels promote hMSCs migration and chondrogenic differentiation.

Cell-based therapies for articular cartilage lesions are expensive and time-consuming; clearly, a one-step procedure to induce endogenous repair would have significant clinical benefits. Acellular heterogeneous granular hydrogels were explored for their injectability, cell-friendly cross-linking, and ability to promote migration, as well as to serve as a scaffold for depositing cartilage extracellular matrix. The hydrogels were prepared by mechanical sizing of bulk methacrylated hyaluronic acid (HAMA) and bulk HAMA incorporating sulfated HAMA (SHAMA). SHAMA's negative charges allowed for the retention of positively charged growth factors (GFs) (e.g., TGFB3 and PDGF-BB). Mixtures of HAMA and GF-loaded SHAMA microgels were annealed by enzymatic cross-linking, forming heterogeneous granular hydrogels with GF deposits. The addition of GF loaded sulfated microislands guided cell migration and enhanced chondrogenesis. Granular heterogeneous hydrogels showed increased matrix deposition and cartilage tissue maturation compared to bulk or homogeneous granular hydrogels. This advanced material provides an ideal 3D environment for guiding cell migration and differentiation into cartilage. STATEMENT OF SIGNIFICANCE: Acellular materials which promote regeneration are of great interest for repair of cartilage defects, and they are more cost- and time-effective compared to current cell-based therapies. Here we develop an injectable, granular hydrogel system which facilities cell migration from the surrounding tissue, facilitating endogenous repair. The hydrogel architecture and chemistry were optimized to increase cell migration and extracellular matrix deposition. The present study provides quantitative data on the effect of microgel size and chemical modification on cell migration, growth factor retention and tissue maturation.

High-mobility group box 1 accelerates distraction osteogenesis healing via the recruitment of endogenous stem/progenitor cells

Background aimsWhile distraction osteogenesis (DO) achieves substantial bone regeneration, prolonged fixation may lead to infections. Existing stem cell and physical therapies have limitations, requiring the development of novel therapeutic approaches. Here, we evaluated high-mobility group box 1 (HMGB1) as a novel therapeutic target for DO treatment. MethodsMicro-computed tomography (Micro-CT) analysis and histological staining of samples obtained from tibial DO model mice was performed. Transwell migration, wound healing, and proliferation assays were also performed on cultured human mesenchymal stem cells (hMSCs) and human umbilival vein endothelial cells (HUVECs). Tube formation assay was performed on HUVECs, whereas osteogenic differentiation assay was performed on hMSCs. ResultsMicro-CT analysis and histological staining of mouse samples revealed that HMGB1 promotes bone regeneration during DO via the recruitment of PDGFRα and Sca-1 positve (PαS+) cells and endothelial progenitor cells. Furthermore, HMGB1 accelerated angiogenesis during DO, promoted the migration and osteogenic differentiation of hMSCs as well as the proliferation, migration and angiogenesis of HUVECs in vitro. ConclusionsOur findings suggest that HMGB1 has a positive influence on endogenous stem/progenitor cells, representing a novel therapeutic target for the acceleration of DO-driven bone regeneration.

Therapeutic Potential of Human Mesenchymal Stem Cells Engineered with Different Concentrations of TNFα and IFNg

Aims: In this study, the authors aimed to examine the functional activities (adhesion, proliferation, and migration) of hMSCs under the effects of various concentrations of inflammatory mediators, such as TNFα or IFNg. Methods: HMSCs were exposed to various concentrations of the inflammatory mediators TNFα or IFNg, and the effects of TNFα or IFNg on the functional properties of hMSCs were determined using multiple functional assays (adhesion, proliferation, and migration). HMSC expression of genes (cytokine, chemokine, and adhesion molecule genes) was analyzed using real-time polymerase chain reaction (RT–PCR). Results: TNFα promoted their proliferation and migration especially at 100 ng/ml, while IFNg increased proliferation only at concentrations of 10 ng/ml and proliferation at 50 and 100 ng/ml. Conclusion: This study evaluated the suitability of hMSCs in treating inflammatory diseases, which are characterized by high levels of inflammatory mediators such as TNFα or IFNg, and proved that the treatment of hMSCs with TNFα or IFNg at higher concentrations has a protective effect.

Evaluation of Human Mesenchymal Stromal Cells as Carriers for the Delivery of Oncolytic HAdV-5 to Head and Neck Squamous Cell Carcinomas.

Human multipotent mesenchymal stromal cells (hMSCs) are of significant therapeutic interest due to their ability to deliver oncolytic adenoviruses to tumors. This approach is also investigated for targeting head and neck squamous cell carcinomas (HNSCCs). HAdV-5-HexPos3, a recently reported capsid-modified vector based on human adenovirus type 5 (HAdV-5), showed strongly improved infection of both hMSCs and the HNSCC cell line UM-SCC-11B. Given that, we generated life cycle-unmodified and -modified replication-competent HAdV-5-HexPos3 vector variants and analyzed their replication within bone marrow- and adipose tissue-derived hMSCs. Efficient replication was detected for both life cycle-unmodified and -modified vectors. Moreover, we analyzed the migration of vector-carrying hMSCs toward different HNSCCs. Although migration of hMSCs to HNSCC cell lines was confirmed in vitro, no homing of hMSCs to HNSCC xenografts was observed in vivo in mice and in ovo in a chorioallantoic membrane model. Taken together, our data suggest that HAdV-5-HexPos3 is a potent candidate for hMSC-based oncolytic therapy of HNSCCs. However, it also emphasizes the importance of generating optimized in vivo models for the evaluation of hMSC as carrier cells.

3D‐Bioassembly of VH‐Spheroids for Cartilage Regeneration: in Vitro Evaluation of Chondrogenesis, Fusion and Lateral Integration

AbstractLateral integration and fusion of cartilage tissue interfaces remain significantly challenging and limits success of cartilage tissue engineering (TE) strategies. In this study, self‐assembled vitreous humor (VH) spheroids are fabricated by seeding clinically relevant human articular chondrocytes (hACs) or human mesenchymal stromal cells (hMSCs) in four VH hydrogels with different glycosaminoglycans (GAG) and protein content. Quantitative and qualitative analysis of the VH spheroids revealed that hAC‐VH spheroids are dependent on the initial GAG content of VH hydrogels to achieve successful chondrogenesis. Remarkably, uniform GAG and collagen type II distribution is found in all hMSC‐VH spheroids, independent of the VH donor. HMSC‐VH spheroids are therefore further evaluated for downstream applications by tracking cellular migration, and evaluating neotissue formation at the tissue‐tissue interface of cartilage spheroids 3D‐bioassembled into an in vitro fusion model to asses fusion and integration. hMSC‐VH spheroids enhanced multidirectional cellular migration of both hACs and hMSCs toward the tissue‐tissue interface, and consequently supported dense GAG and collagen type II deposition at the integration region. Ultimately, hMSC‐seeded VH spheroids display successful chondrogenesis and endorse fusion and integration of cartilage tissue interfaces through upregulated cellular migration of clinically relevant cell sources – key elements for clinical translation of cell‐based TE strategies.

Silica Nanoparticle Internalization Improves Chemotactic Behaviour of Human Mesenchymal Stem Cells Acting on the SDF1α/CXCR4 Axis.

Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine. Despite the innate ability of hMSCs to migrate to sites of injury, homing of infused hMSCs to the target tissue is inefficient. It was shown that silica nanoparticles (SiO2-NPs), previously developed to track the stem cells after transplantation, accumulated in lysosomes leading to a transient blockage of the autophagic flux. Since CXCR4 turnover is mainly regulated by autophagy, we tested the effect of SiO2-NPs on chemotactic migration of hMSCs along the SDF1α/CXCR4 axis that plays a pivotal role in directing MSC homing to sites of injury. Our results showed that SiO2-NP internalization augmented CXCR4 surface levels. We demonstrated that SiO2-NP-dependent CXCR4 increase was transient, and it reversed at the same time as lysosomal compartment normalization. Furthermore, the autophagy inhibitor Bafilomycin-A1 reproduced CXCR4 overexpression in control hMSCs confirming the direct effect of the autophagic degradation blockage on CXCR4 expression. Chemotaxis assays showed that SiO2-NPs increased hMSC migration toward SDF1α. In contrast, migration improvement was not observed in TNFα/TNFR axis, due to the proteasome-dependent TNFR regulation. Overall, our findings demonstrated that SiO2-NP internalization increases the chemotactic behaviour of hMSCs acting on the SDF1α/CXCR4 axis, unmasking a high potential to improve hMSC migration to sites of injury and therapeutic efficacy upon cell injection in vivo.

HMSCs Migrate under the Chemotaxis of CXCL-13 and Enhance Islet B Cell Activity through p-AKT Signaling Pathway in High-Glucose Environment.

As a common clinical chronic disease, the incidence of diabetes is increasing year by year. According to the latest statistics from the International Diabetes Federation, as of 2019, the global prevalence of diabetes has reached 8.3%. This study aims to investigate the effect of CXCL-13 on the migration ability of human mesenchymal stem cells (hMSCs) and to clarify the specific molecular mechanism of the protective effect of hMSCs on islet B cells. The hMSCs were cultured in high-glucose environment, and the effect of CXCL-13 on the migration ability of hMSCs was determined by Transwell experiment. After coculture of hMSCs and islet B cells, the activity of cells was detected by CCK8 assay, the expression of Ki-67 in cells was detected by RT-PCR, and the expression of P53 was detected by Western blot to investigate the effect of hMSCs on the proliferation and apoptosis of islet B cells. The effect of hMSCs on the function of islet B cells was determined by glucose stimulated insulin secretion experiment. Transwell experiment results showed that CXCL-13 could promote the migration of hMSCs to islet B cells in high-glucose environment. The results of CCK-8 showed that the cell activity in the coculture group was significantly higher than that of the other groups, and RT-PCR showed that the expression of Ki-67 was significantly increased in the coculture group of hMSCs and islet B cells. The results of Western blot showed that the expression of P53 was significantly decreased in the coculture group, and the glucose stimulated insulin secretion test showed that insulin secretion was significantly increased. It was found that after the inhibition of ATK, cell activity was significantly reduced, and apoptosis was significantly increased. Meanwhile, the expression of Ki-67 was inhibited, the expression of P-53 was significantly increased, and insulin secretion was significantly reduced. To sum up, in a high-glucose environment, CXCL-13 effectively promoted the migration of hMSCs, and hMSCs protected the activity and function of islet B cells through Akt signaling pathway.

Discovery of chemerin as the new chemoattractant of human mesenchymal stem cells

BackgroundThe homing capacity of human mesenchymal stem cells (hMSCs) to the injured sites enables systemic administration of hMSCs in clinical practice. In reality, only a small proportion of MSCs are detected in the target tissue, which is a major bottleneck for MSC-based therapies. We still don’t know the mechanism how MSCs are chemo-attracted to certain target organ and engrafted through trans-endothelial migration. In this study, we aimed to determine the mechanism how the circulating hMSCs home to the injured liver.Methods and resultsWhen we compare the cytokine array between normal and injured mouse liver at 1-day thioacetamide (TAA)-treatment, we found that chemerin, CXCL2, and CXCL10 were higher in the injured liver than normal one. Among three, only chemerin was the chemoattractant of hMSCs in 2D- and 3D-migration assay. Analysis of the signal transduction pathways in hMSCs showed that chemerin activated the phosphorylation of JNK1/2, ERK1/2 and p38, and finally upregulated CD44, ITGA4, and MMP-2 that are involved in the transendothelial migration and extravasation of MSCs. Upstream transcription regulators of CD44, ITGA4, and MMP-2 after chemerin treatment were MZF1, GATA3, STAT3, and STAT5A. To develop chemerin as a chemoattractant tool, we cloned gene encoding the active chemerin under the CMV promoter (CMV-aChemerin). We analyzed the migration of hMSCs in the 3D model for space of the Disse, which mimics transmigration of hMSCs in the liver. CMV-aChemerin-transfected hepatocytes were more effective to attract hMSC than control hepatocytes, leading to the enhanced transendothelial migration and homing of hMSCs to liver. The homing efficiency of the intravascularly-delivered hMSCs to liver was evaluated after systemic introduction of the CMV-aChemerin plasmid packed in liposome-vitamin A conjugates which target liver. CMV-aChemerin plasmid targeting liver significantly enhanced homing efficiency of hMSCs to liver compared with control plasmid vector.ConclusionsChemerin is the newly found chemoattractant of hMSCs and may be a useful tool to manipulate the homing of the intravascularly-administered hMSC to the specific target organ.

Basement membrane proteins modulate cell migration on bovine pericardium extracellular matrix scaffold

Native bovine pericardium (BP) exhibits anisotropy of its surface ECM niches, with the serous surface (i.e., parietal pericardium) containing basement membrane components (e.g., Laminin, Col IV) and the fibrous surface (i.e., mediastinal side) being composed primarily of type I collagen (Col I). Native BP surface ECM niche anisotropy is preserved in antigen removed BP (AR-BP) extracellular matrix (ECM) scaffolds. By exploiting sideness (serous or fibrous surface) of AR-BP scaffolds, this study aims to determine the mechanism by which ECM niche influences human mesenchymal stem cells (hMSCs) migration. Human mesenchymal stem cells (hMSC) seeding on serous surface promoted more rapid cell migration than fibrous surface seeding. Gene analysis revealed that expression of integrin α3 and α11 were increased in cells cultured on serous surface compared to those on the fibrous side. Monoclonal antibody blockade of α3β1 (i.e., laminin binding) inhibited early (i.e. ≤ 6 h) hMSC migration following serous seeding, while having no effect on migration of cells on the fibrous side. Blockade of α3β1 resulted in decreased expression of integrin α3 by cells on serous surface. Monoclonal antibody blockade of α11β1 (i.e., Col IV binding) inhibited serous side migration at later time points (i.e., 6–24 h). These results confirmed the role of integrin α3β1 binding to laminin in mediating early rapid hMSCs migration and α11β1 binding to Col IV in mediating later hMSCs migration on the serous side of AR-BP, which has critical implications for rate of cellular monolayer formation and use of AR-BP as blood contacting material for clinical applications.

CRISPR-Cas9-based gene editing of human mesenchymal stromal cells to improve the outcome of cell therapy

Abstract Background The environment of the failing and infarcted myocardium drives resident and transplanted mesenchymal stromal cells (MSCs) toward a pro-inflammatory phenotype, thus restricting their survival and their reparative effects in a mechanism mediated by toll-like receptor 4 (TLR4). Hence, new approaches are needed to improve the efficacy of cell therapy for heart failure. CRISPR is a promising tool for genome editing, which raises the hope for therapeutic genome editing in the clinic. Purpose We aimed to provide a new strategy to enhance MSC-based cell therapy to improve cardiac remodeling and function. We hypothesized that ex vivo knockout (KO) of the human TLR4 gene by CRISPR would switch human-cardiac MSCs (hMSCs) to an anti-inflammatory, reparative phenotype that could prevent remodeling of the left ventricle after myocardial infarction (Fig. 1A). Methods and results We achieved up to 68% (out of 4x105 cells, R2=0.93) success rate in editing the genome of primary cardiac hMSCs taken from patients with ischemic heart disease. The deletion of TLR4 in hMSCs significantly reduced the secretion of inflammatory and extracellular-matrix (ECM) proteins, compared with unedited hMSCs, by protein mass spectrometry (Fig. 1B) and by multiplex ELISA (Fig. 1C). Additionally, edited cells secreted significantly more extracellular vesicles (EVs) than unedited hMScs (Fig. 1D, p<0.001). These EVs from edited hMSCs stimulated faster migration of hMSCs in a “wound healing” assay (p<0.001). Conclusions We show, for the first time, that CRISPR-based deletion of the TLR4 gene in hMSCs inhibits inflammatory and ECM protein secretion and facilitates a reparative response by hMSCs in vitro. This precise and efficient ex vivo gene editing could provide a newly engineered cell line to improve the outcome of hMSC-based cell therapy. Figure 1 Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): The Foundation in Memory of Seymour Fefer, Sheba Medical Center

Human mesenchymal stem cells respond differentially to platelet preparations and synthesize hyaluronic acid in nucleus pulposus extracellular matrix

BACKGROUND CONTEXTIn recent years, autologous platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMAC) have been used as treatments for disc-related pain. A better understanding of the effects of leukocyte-rich (LR) versus leukocyte poor (LP-) PRP on bone marrow derived human mesenchymal stem/progenitor cells (hMSCs) is likely to improve future research studies, clinical practice and care for patients with chronic discogenic back pain. PURPOSEThe primary aim of this study is to determine the effects of LR-PRP and LP-PRP on the proliferation and migration of hMSCs in pig nucleus pulposus (NP) extracellular matrix (ECM). The secondary aim is to characterize hMSC-dependent expression of the matrix remodeling enzymes metalloproteinases MMP-2, MMP-3, MMP-9 and tissue inhibitor of metalloproteinases TIMP-2, and to determine whether transplanted hMSCs can synthesize hyaluronic acid (HA). STUDY DESIGNControlled laboratory study. METHODSBone marrow-derived culture expanded hMSCs were seeded onto pig NP and cultured with LR-PRP, LP-PRP or serum/platelet releasate (PR). The same conditions without hMSCs were used as controls. hMSC proliferation, migration and dispersion was assessed via fluorescent microscopy, while HA synthesis, MMP-2, MMP-3, MMP-9, and TIMP-2 protein levels were assessed via enzyme linked immunosorbent assay. All funding was provided by a 501c(3) research foundation and does not have any commercial or sponsorship interests. RESULTSLP-PRP and PR cultures resulted in higher hMSC proliferation, migration, dispersion, and MMP-2 expression. LP-PRP cultures resulted in the highest HA production. LR-PRP cultures resulted in lower hMSC proliferation, negligible migration and dispersion, increased MMP-9 expression and lower HA production. CONCLUSIONSHuman bone marrow-derived hMSCs seeded onto pig NP ECM are capable of synthesizing HA, indicating a transition towards a NP cell phenotype. This process was most enhanced by LP-PRP and marked by increased hMSC proliferation, MMP-2 production, HA synthesis and reduced MMP-9 levels. CLINICAL SIGNIFICANCELP-PRP and PR, with or without hMSCs, may provide better outcomes than LR-PRP in lab investigations and clinical trials for discogenic pain. Bone marrow-derived hMSCs may hold promise as a treatment for disc degeneration.

Application of ECIS to Assess FCCP-Induced Changes of MSC Micromotion and Wound Healing Migration.

Electric cell-substrate impedance sensing (ECIS) is an emerging technique for sensitively monitoring morphological changes of adherent cells in tissue culture. In this study, human mesenchymal stem cells (hMSCs) were exposed to different concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) for 20 h and their subsequent concentration-dependent responses in micromotion and wound healing migration were measured by ECIS. FCCP disrupts ATP synthesis and results in a decrease in cell migration rates. To detect the change of cell micromotion in response to FCCP challenge, time-series resistances of cell-covered electrodes were monitored and the values of variance were calculated to verify the difference. While Seahorse XF-24 extracellular flux analyzer can detect the effect of FCCP at 3 μM concentration, the variance calculation of the time-series resistances measured at 4 kHz can detect the effect of FCCP at concentrations as low as 1 μM. For wound healing migration, the recovery resistance curves were fitted by sigmoid curve and the hill slope showed a concentration-dependent decline from 0.3 μM to 3 μM, indicating a decrease in cell migration rate. Moreover, dose dependent incline of the inflection points from 0.3 μM to 3 μM FCCP implied the increase of the half time for wound recovery migration. Together, our results demonstrate that partial uncoupling of mitochondrial oxidative phosphorylation reduces micromotion and wound healing migration of hMSCs. The ECIS method used in this study offers a simple and sensitive approach to investigate stem cell migration and its regulation by mitochondrial dynamics.

Safe and Effective Delivery of Antitumor Drug Using Mesenchymal Stem Cells Impregnated with Submicron Carriers.

An important area in modern malignant tumor therapy is the optimization of antitumor drugs pharmacokinetics. The use of some antitumor drugs is limited in clinical practice due to their high toxicity. Therefore, the strategy for optimizing the drug pharmacokinetics focuses on the generation of high local concentrations of these drugs in the tumor area with minimal systemic and tissue-specific toxicity. This can be achieved by encapsulation of highly toxic antitumor drug (vincristine (VCR) that is 20-50 times more toxic than widely used the antitumor drug doxorubicin) into nano- and microcarriers with their further association into therapeutically relevant cells that possess the ability to migrate to sites of tumor. Here, we fundamentally examine the effect of drug carrier size on the behavior of human mesenchymal stem cells (hMSCs), including internalization efficiency, cytotoxicity, cell movement, to optimize the conditions for the development of carrier-hMSCs drug delivery platform. Using the malignant tumors derived from patients, we evaluated the capability of hMSCs associated with VCR-loaded carriers to target tumors using a three-dimensional spheroid model in collagen gel. Compared to free VCR, the developed hMSC-based drug delivery platform showed enhanced antitumor activity regarding those tumors that express CXCL12 (stromal cell-derived factor-1 (SDF-1)) gene, inducing directed migration of hMSCs via CXCL12 (SDF-1)/CXCR4 pathway. These results show that the combination of encapsulated antitumor drugs and hMSCs, which possess the properties of active migration into tumors, is therapeutically beneficial and demonstrated high efficiency and low systematic toxicity, revealing novel strategies for chemotherapy in the future.

Biodegradable water-based polyurethane scaffolds with a sequential release function for cell-free cartilage tissue engineering

Three-dimensional (3D) printing technology has rapidly developed as a promising technology for manufacturing tissue engineering scaffolds. Cells used in tissue engineering are subjected to the quality management and risk of contamination, while cell-free scaffolds may not have sufficient therapeutic efficacy. In this study, water-based 3D printing ink containing biodegradable polyurethane (PU), chemokine SDF-1, and Y27632 drug-embedding PU microspheres was printed at low temperature (−40 °C) to fabricate tissue engineering scaffolds with sequential drug release function. The scaffolds containing 200 ng/ml SDF-1 and 22 wt% Y27632-encapsulated microspheres (55 µg/ml Y27632 in microspheres) (abbreviated PU/SDF-1/MS_Y scaffolds) had the optimal performance. The structural design of the scaffolds allowed each of SDF-1 and Y27632 to be released sequentially in vitro and reach the effective concentration (∼100 ng/ml and 3.38 µg/ml, respectively) after the appropriate time (24 h and 62 h, respectively). Human mesenchymal stem cells (hMSCs) seeded in the scaffolds showed significant GAG deposition in 7 days. Besides, the gradual release of SDF-1 from the PU/SDF-1/MS_Y scaffolds could induce the migration of hMSCs. Implantation of the cell-free PU/SDF-1/MS_Y scaffolds in rabbit articular cartilage defects supported the potential of the scaffolds to promote cartilage regeneration. The 3D printed scaffolds with sequential releases of SDF-1 and Y27632 may have potential in cartilage tissue engineering. Statement of SignificanceThe clinical success of tissue engineering depends highly on the quality of externally supplied cells, while cell-free scaffolds may not have sufficient therapeutic efficacy. In this manuscript, water-based 3D printing ink containing biodegradable polyurethane (PU), chemokine SDF-1, and Y27632 drug-embedding PU microspheres was printed at low temperature to fabricate tissue engineering scaffolds with sequential drug release function. The structural design of the scaffolds allowed each of SDF-1 and Y27632 to be released sequentially in vitro. SDF-1 was released earlier from the scaffolds to promote cell migration. The drug Y27632 was released later from the microspheres into the matrix of the scaffolds to induce the chondrogenic differentiation of the attracted cells. Implantation of the cell-free PU/SDF-1/MS_Y scaffolds in rabbit articular cartilage defects supported the potential of the scaffolds to promote cartilage regeneration. We hypothesized that the cell-free scaffolds may improve the clinical applicability and convenience without the use of exogenous cells or growth factor.

Magnetic ternary nanohybrids for nonviral gene delivery of stem cells and applications on cancer therapy.

Cancer toxic agent-expressing mesenchymal stem cells (MSCs), which possess inherent tumor migration and penetration capabilities, have received increasing attention in cancer therapy. To ensure that this approach is successful, safe and efficient gene delivery methods for stem cell engineering must be developed.Methods: In this study, a magnetic ternary nanohybrid (MTN) system comprising biodegradable cationic materials, nucleic acids, and hyaluronic acid-decorated superparamagnetic iron oxide nanoparticles was proposed to construct stem cells expressing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via magnetic force and receptor dual targeting.Results: The CD44/magnetic force-mediated enhanced cellular uptake of MTNs by human mesenchymal cells (hMSCs) was confirmed in vitro. Highly efficient transfection was attained using MTNs without having any detrimental effect on the tumor migration and penetration capabilities of hMSCs. TRAIL expressed by the MTN-transfected hMSCs displayed strong anticancer effects through the activation of caspase-3 apoptotic signaling. The MTN-transfected hMSCs can be clearly imaged using magnetic resonance imaging techniques in vivo. In an orthotopic xenograft cancer model, MTN-transfected TRAIL-expressing hMSCs significantly suppressed the progression of human glioma (U87MG) and prolonged the survival of the animal.Conclusions: These findings suggest the considerable potential of utilizing MTNs for effectively constructing tumor toxic agent-expressing stem cells for treating malignant cancers.

Impact of Vancomycin Treatment on Human Mesenchymal Stromal Cells During Osteogenic Differentiation

Category: Basic Sciences/Biologics Introduction/Purpose: Vancomycin is often delivered locally for surgical site infection prophylaxis. Recent reports of possible osteotoxicity have led to uncertainty concerning vancomycin’s safety in the setting of arthrodesis and bone healing. Bone formation during arthrodesis takes place as recruited human mesenchymal stromal cells (hMSCs) proliferate and differentiate into mature osteoblasts. The purpose of this research was to determine the impact of vancomycin treatment on hMSCs during osteogenic differentiation. Methods: Human MSCs were cultured in MSC growth media to an appropriate confluence. Cells were cultured for 24 hours to facilitate adherence, after which the media was aspirated and replaced with osteogenic differentiation media (Lonza, Switzerland). Osteogenic differentiation media was supplemented with vancomycin powder to yield solutions with concentrations of 0, 50, 500 & 5000 µg/mL. Fresh vancomycin powder was added with every media change. MSCs viability and proliferation were assessed via live/dead staining with 1 µM calcein-AM and 0.5 µM ethidium homodimer-1 (EthD-1) after 1, 3, and 7 days of differentiation and vancomycin treatment. Mineralization of differentiated cells was assessed via staining with 40 mM alizarin red (ARS; pH 4.1) after 21 days. Semi-quantification of the degree of mineralization was performed by measuring absorbance values at 405 nm using a microplate reader. Microscopy was used for qualitative evaluation. Results: Cell viability decreased with increasing vancomycin concentrations. Impairment of hMSC proliferation was also observed with increasing concentrations of vancomycin. MSCs treated with 5000 µg/mL vancomycin demonstrated significantly less cell growth compared to all other treatment groups (P=0.0001). Absorbance measurements from each well stained with alizarin red was used for semi-quantification of the degree of mineralization. As vancomycin concentrations were increased, absorbance levels decreased (Figure). This reduction in mineralization was also demonstrated qualitatively; with alizarin red less apparent in the wells with increasing vancomycin concentrations (Figure). Conclusion: Local vancomycin is utilized for prevention of infection, often in procedures that necessitate the formation of new bone. Bone healing requires migration, proliferation and differentiation of hMSCs. This work demonstrates impaired viability and function of hMSCs following vancomycin as well as decreased osteoblastic mineralization. Future work will require in vivo studies aimed at determining relative nonunion rates in the setting of vancomycin prophylaxis. Still, the results of this study suggest that vancomycin may be toxic to hMSCs and caution should be exercised by providers when considering vancomycin in foot and ankle patients requiring bony healing following fracture or arthrodesis.

Development of controlled release systems of biological factors for the regeneration of intervertebral disc

Purpose: The degeneration of intervertebral disc (IVD), and notably of its central part the nucleus pulposus (NP), is responsible for 40% of chronic low back pain (LBP). Recent studies have reported the existence of endogenous regenerative cells in the IVD vicinity and within the IVD. These endogenous cells have been characterized as mesenchymal stem cell-like progenitors residing in specific niches. In response to chemokines (stromal derived factor-1 (SDF-1)) these cells can be recruited and migrate to the site of injury and thus contribute to the endogenous repair process. In this context, the development of microbeads-based local delivery systems of factors involved in progenitors recruitment has recently been contemplated for IVD regenerative medicine. Because of their physicochemical and biological properties, microbeads of pullulans (PMBs) have long been investigated as drug carriers. In addition, we recently demonstrated that transforming growth factor-β1 (TGF-β1) and growth differentiation factor5 (GDF5) are potent stimulators of the differentiation of mesenchymal stem cells (MSC) into NP-like cells. In this context, the aim of this work was to develop an intradiscal pullulan microbeads-based delivery system for the controlled release of SDF-1, TGF-β1 and GDF5. This drug delivery system would be able to sequentially contribute to 1) the recruitment and mobilization of resident progenitors, 2) the differentiation of the mobilized progenitors and 3) the subsequent regeneration of NP. Methods: Chemotaxis assays were performed to determine the in vitro cell migration. Human MSCs (1250 cells/μl) were incubated with or without SDF-1 (250 ng/ml) in Transwells for 4h, migratory cells were stained by crystal violet then quantified by spectrophotometry. In parallel, PMBs were prepared by a simultaneous crosslinking protocol coupled to a water-in-oil emulsification process. Freeze-dried PMBs were incubated with GDF-5 and TGF-β1 separately (25 mg of PMBs at final concentration of 1, 2 and 4 μg/mL and in a final volume of 500 μL of PBS) for 24 h at 4°C under rotary stirring at 24 rpm. GDF-5 and TGF-β1 release assays were performed in PBS at 37°C for 21 days and concentrations were measured by ELISA. Results: SDF-1 has improved the in vitro migration of hMSCs, increasing by more than twice the number of migratory cells. GDF-5 and TGF-β1 were successfully adsorbed on PMBs with a 100% efficiency. Release experiments showed a burst release within the 1st h, at 604 ng/h and 50 ng/h for GDF-5 and TGF-β1 respectively, then the release rate decreased during 21 days with 0.6 ng/h and 0.15 ng/h during the last 7 days for GDF-5 and TGF-β1, respectively. At day 21, GDF-5 was entirely released, whereas only 40% of TGF-β1 was released. This different release profiles could be explained by the difference of molecular weight (13 kDa for GDF-5 and 25 kDa for TGF-β1). Conclusions: We have confirmed that SDF-1 improved hMSCs in vitro migration, and that PMBs are suitable microcarriers for the loading and release of GDF-5 and TGFβ-1. The loading and release capability of SDF-1 by PMBs, as well as SDF-1 bioactivity after release will be analyzed, to obtain a fast and massive recruitment of resident progenitors in vivo. Then, we will study the action of GDF-5 and TGFβ1 released from PMBs on in vitro NP differentiation, by using a 3D matrix model to mimic the NP microenvironment. Nucleopulpogenic differentiation will be evaluated by analysis of specific extracellular matrix production and gene expression markers.

Effect of CCR1 gene overexpression on the migration of bone marrow - derived mesenchymal stem cells towards hepatocellular carcinoma

Objective: To evaluate the effect of human CCR1 (hCCR1) gene overexpression on the migration of human bone marrow-derived mesenchymal stem cells (hMSCs) towards hepatocellular carcinoma (HCC), and to examine the application prospects of MSCs as gene delivery vectors in the treatment of HCC. Methods: The hCCR1 gene was subcloned into a lentiviral vector to generate the recombinant plasmid pLV-hCCR1. The pLV-hCCR1 plasmid and two other packaging plasmids were co-transfected into 293T cells using calcium phosphate, and the virus-containing supernatant was collected. hMSCs were then infected with the recombinant lentivirus, and the expression of hCCR1 mRNA and protein was analyzed by RT-PCR and Western blot, respectively. The effect of CCR1 gene overexpression on the in vitro migration of hMSCs was examined using the Transwell migration assay. Orthotopic nude mice models of HCC were established using the MHCC-97H-GFP cell line, and the mice were divided into two groups (n = 8 per group). hMSCs were then intravenously injected via the tail vein into the tumor-bearing nude mice to examine the effect of hCCR1 overexpression on the in vivo migration of hMSCs towards HCC. Unpaired Student's t-test was used for two-group comparisons, and one-way ANOVA was used for multi-group comparisons. Results: Restriction enzyme digestion and DNA sequencing demonstrated that the recombinant plasmid pLV-hCCR1 was constructed successfully. The LV-hCCR1 lentivirus packaged by 293T cells has high infection efficiency in hMSCs, and hCCR1 was overexpressed in hMSCs after LV-hCCR1 infection. Transwell migration assay showed that hCCR1-transfected hMSCs had significantly enhanced migration towards HCC cell line-derived condition medium (CM) compared with the control RFP-hMSCs [(134.8±15.7)/LPF vs (83.5±10.9)/LPF, t = 10.40, P < 0.01]. In vivo migration experiment also demonstrated that there was significantly higher number of hCCR1-hMSCs localized within the MHCC-97H-GFP xenografts than hMSCs-RFP on day 14 following intravenous injection of hMSCs in mice [(86.7±14.1)/HPF vs (54.5±9.6)/HPF, t = -7.32, P < 0.01]. Conclusion: Overexpression of CCR1 gene can significantly enhance the migration capacity of hMSCs towards HCC cells in vitro and in vivo. This study provides evidence for potential clinical application of MSCs as more effective delivery vehicles in cancer gene therapy.

Chemokine CCL15 Mediates Migration of Human Bone Marrow-Derived Mesenchymal Stem Cells Toward Hepatocellular Carcinoma.

Mesenchymal stem cells (MSCs) possess the ability to migrate toward tumor sites and are regarded as promising gene delivery vehicles for cancer therapeutics. However, the factors that mediate this tropism have yet to be completely elucidated. In this study, through cytokine array analysis, chemokine CCL15 was found to be the most abundant protein differentially expressed in hepatocellular carcinoma (HCC) cell lines compared with a normal liver cell line. Serum CCL15 levels in HCC patients determined by enzyme linked immunosorbent assay were shown to be profoundly elevated compared with healthy controls. Immunohistochemical analysis indicated that CCL15 expression was much stronger in HCC tumor tissues than in adjacent nontumor tissues. Transwell migration assay suggested that CCL15 may be involved in chemotaxis of human MSCs (hMSCs) toward HCC in vitro and that this chemotactic effect of CCL15 is mediated via CCR1 receptors on hMSCs. Orthotopic animal models of HCC were established to investigate the role of CCL15 in hMSCs migration toward HCC in vivo. Both histological and flow cytometric analysis showed that significantly fewer hMSCs localized within 97H-CCL15-shRNA xenografts compared with 97H-green fluorescent protein xenografts after intravenous delivery. Finally, the possible effects of hMSCs on HCC tumor growth were also evaluated. Coculture experiments showed that hMSCs had no apparent effect on the proliferation of HCC cells in vitro In addition, systemic administration of hMSCs did not affect HCC tumor progression in vivo. Our data in this study help to elucidate the mechanism underlying the homing capacity of hMSCs toward HCC.