Swine Mesenchymal Stem Cells Research Articles

Metabolic Syndrome Induces Epigenetic Alterations in Mitochondria-Related Genes in Swine Mesenchymal Stem Cells.

Autologous mesenchymal stem/stromal cells (MSCs) have demonstrated important therapeutic effects in several diseases. Cardiovascular risk factors may impair MSC mitochondrial structure and function, but the underlying mechanisms remain unknown. We hypothesized that metabolic syndrome (MetS) induces epigenetic alterations in mitochondria-related genes in swine MSCs. Pigs were fed a Lean or MetS diet (n = 6 each) for 16 weeks. MSCs were collected from subcutaneous abdominal fat, and DNA hydroxymethylation (5 hmC) profiles of mitochondria-related genes (MitoCarta-2.0) were analyzed by hydroxymethylated DNA immunoprecipitation and next-generation sequencing (hMeDIP-seq) in Lean- and MetS-MSCs untreated or treated with the epigenetic modulator vitamin (Vit)-C (n = 3 each). Functional analysis of genes with differential 5 hmC regions was performed using DAVID6.8. Mitochondrial structure (electron microscopy), oxidative stress, and membrane potential were assessed. hMeDIP-seq identified 172 peaks (associated with 103 mitochondrial genes) with higher and 416 peaks (associated with 165 mitochondrial genes) with lower 5 hmC levels in MetS-MSCs versus Lean-MSCs (≥2-fold, p < 0.05). Genes with higher 5 hmC levels in MetS + MSCs were primarily implicated in fatty acid metabolism, whereas those with lower 5 hmC levels were associated with electron transport chain activity. Vit-C increased 5 hmC levels in mitochondrial antioxidant genes, improved mitochondrial structure and membrane potential, and decreased oxidative stress. MetS alters 5 hmC levels of mitochondria-related genes in swine MSCs. Vit-C modulated 5 hmC levels in these genes and preserved mitochondrial structure and function in MetS-MSCs. These observations may contribute to development of strategies to overcome the deleterious effects of MetS on MSCs.

Metabolic Syndrome Impairs 3D Mitochondrial Structure, Dynamics, and Function in Swine Mesenchymal Stem Cells.

Transplantation of autologous mesenchymal stem cells (MSCs) is an effective therapy for several diseases. Mitochondria modulate several important aspects of MSC function, but might be damaged by comorbidities and cardiovascular risk factors. We hypothesized that metabolic syndrome (MetS) compromises 3D mitochondrial structure, dynamics, and function in swine adipose tissue-derived MSCs. Domestic pigs were fed a Lean or MetS diet (n= 6 each) for 16weeks. MSCs were collected from subcutaneous abdominal fat and their mitochondria analyzed using state-of-the-art Serial Block Face Electron Microscopy and 3D reconstruction. Mitochondrial dynamics (fusion/fission) were assessed by mRNA sequencing and Western blotting, and bioenergetics by membrane potential (TMRE), cytochrome-c oxidase (COX)-IV activity, and Seahorse Analyzer. Expression of mitochondria-associated microRNAs (mitomiRs) was measured by quantitative polymerase chain reaction (qPCR). MetS pigs developed obesity, hypertension, insulin resistance, and hyperlipidemia. Mitochondrial density was similar between the groups, but 3D mitochondrial and matrix volumes were lower in MetS-MSCs versus Lean-MSCs. Mitochondrial fission was higher, but fusion lower in MetS-MSCs versus Lean-MSCs, as were membrane potential, COX-IV activity, and ATP production. Contrarily, expression of the mitomiRs miR15a, miR-137, and miR-181c, which target mitochondrial genes that support mitochondrial structure, energy pathways, and dynamics, was higher in MetS-MSCs compared to Lean-MSCs, suggesting a potential to modulate their expression. MetS damages MSC 3D mitochondrial structure, dynamics, and function, and may modulate genes encoding for mitochondrial proteins. These observations support development of mitoprotective strategies to preserve the regenerative potency of MSCs and their suitability for autologous transplantation in patients with MetS.

Metabolic syndrome alters expression of insulin signaling-related genes in swine mesenchymal stem cells

AimsMetabolic syndrome (MetS) is associated with insulin resistance (IR) and impaired glucose metabolism in muscle, fat, and other cells, and may induce inflammation and vascular remodeling. Endogenous reparative systems, including adipose tissue-derived mesenchymal stem/stromal cells (MSC), are responsible for repair of damaged tissue. MSC have also been proposed as an exogenous therapeutic intervention in patients with cardiovascular and chronic kidney disease (CKD). The feasibility of using autologous cells depends on their integrity, but whether in MetS IR involves adipose tissue-derived MSC remains unknown. The aim of this study was to examine the expression of mRNA involved in insulin signaling in MSC from subjects with MetS. MethodsDomestic pigs consumed a lean or obese diet (n=6 each) for 16weeks. MSC were collected from subcutaneous abdominal fat and analyzed using high-throughput RNA-sequencing for expression of genes involved in insulin signaling. Expression profiles for enriched (fold change>1.4, p<0.05) and suppressed (fold change<0.7, p<0.05) mRNAs in MetS pigs were functionally interpreted by gene ontology analysis. The most prominently upregulated and downregulated mRNAs were further probed. ResultsWe identified in MetS-MSC 168 up-regulated and 51 down-regulated mRNAs related to insulin signaling. Enriched mRNAs were implicated in biological pathways including hepatic glucose metabolism, adipocyte differentiation, and transcription regulation, and down-regulated mRNAs in intracellular calcium signaling and cleaving peptides. Functional analysis suggested that overall these alterations could increase IR. ConclusionsMetS alters mRNA expression related to insulin signaling in adipose tissue-derived MSC. These observations mandate caution during administration of autologous MSC in subjects with MetS.

Magnetic resonance hypointensive signal primarily originates from extracellular iron particles in the long-term tracking of mesenchymal stem cells transplanted in the infarcted myocardium

PurposeThe long-lasting hypointensities in cardiac magnetic resonance (CMR) were believed to originate from superparamagnetic iron oxide (SPIO)-engulfed macrophages during long-term stem cell tracking. However, the iron clearance capacity of the ischemic heart was limited. Therefore, we speculated that the extracellular SPIO particles may also be involved in the generation of false-positive signals.Methods and resultsMale swine mesenchymal stem cells (MSCs) were incubated with SPIO for 24 hours, and SPIO labeling had no significant effects on either cell viability or differentiation. In vitro studies showed that magnetic resonance failed to distinguish SPIO from living SPIO-MSCs or dead SPIO-MSCs. Two hours after the establishment of the female swine acute myocardial infarction model, 2×107 male SPIO-labeled MSCs (n=5) or unlabeled MSCs (n=5) were transextracardially injected into the infarcted myocardium at ten distinct sites. In vivo CMR with T2 star weighted imaging-flash-2D sequence revealed a signal void corresponding to the initial SPIO-MSC injection sites. At 6 months after transplantation, CMR identified 32 (64%) of the 50 injection sites, where massive Prussian blue-positive iron deposits were detected by pathological examination. However, iron particles were predominantly distributed in the extracellular space, and a minority was distributed within CD68-positive macrophages and other CD68-negative cells. No sex-determining region Y DNA of donor MSCs was detected.ConclusionCMR hypointensive signal is primarily caused by extracellular iron particles in the long-term tracking of transplanted MSCs after myocardial infarction. Consideration should be given to both the false-positive signal and the potential cardiac toxicity of long-standing iron deposits in the heart.

Survival of porcine mesenchymal stem cells over the alginate recovered cellular method

Although previous studies have established alginate as a three-dimensional scaffold for chondrogenic differentiation of mesenchymal stem cells (MSCs), little is known about the cytotoxic effects of the polymerizing and chelating reagents used in the alginate recovered cellular method. Swine MSCs (sMSC) were suspended in the polymerizing agent, CaCl(2), and viability was assessed using a trypan blue exclusion assay at intervals over 2 h. MSCs were also suspended in two different chelating agents, 55 mM sodium citrate and 50 mM EDTA, and viability was calculated. Finally, sMSCs and human MSCs were encapsulated and cultured in vitro. The sMSC were collected at day 4 and the cells were recovered by chelation. Encapsulated hMSCs were cultured with TGFβ3 and IGF-1 and assayed by qRT-PCR for collagen types I and II and sox9 with encapsulated human dermal fibroblasts and chondrocytes as controls recovered on days 0, 7, and 14. Only 53% of sMSCs were viable after 10 min in CaCl(2), the standard polymerization period, whereas 94% of encapsulated sMSCs in polymerized alginate were alive at 15 days. After 20 min in chelating agents, sodium citrate and EDTA, only 46% and 17% of the cells, were viable, respectively. The alginate recovered sMSCs yielded 12% survival. Human MSCs upregulated cartilage-specific collagen type II over the 14-day culture. Collagen I remained unchanged in the hMSCs. Alginate is a suitable agent for chondrodifferentiation of MSCs and can be dissolved by chelation, but the agents involved in hydrogel polymerization and cell recovery should be altered to improve MSC survival.

Study on MR tracking of magnetically labeled swine mesenchymal stem cells after autologous transplantation into acute injured liver in vivo

Objective To evaluate in vivo tracking of swine mesenchymal stem cells (MSCs) la-beled with super paramagnetic iron oxide (SPIO) in intraportal transplantation by a clinical 1.5T MR.Methods MSCs were isolated from swine and cultured as well as expanded, which were then incuba-ted with SPIO (Feridex I. V.). Prussian blue staining was performed for showing intracelluar irons.To establish a swine model of acute liver necrosis, 0.5 g/kg of D-galactosamine was administrated to 10 pigs. MSCs(labeled cells in six, unlabeled cells in four)were injected into liver via portal veins. MR imaging was performed with a clinical 1.5T MR immediately before and at 6 h, 3 d, 7 d, 14 d after transplantation, respectively. Results Prussian blue staining of SPIO labeled MSCs could be effec-tively labeled and the labeling efficiency was almost 100%. Signal intensity loss in liver by SPIO labe-ling on FFE sequence persisted until 14 days after transplantation. Histological analysis by Prussian blue staining showed homing of labeled MSCs in liver after 14 days, primarily distributing in hepatic sinusoids and liver parenchyma. Conclusion MSCs can be labeled with SPIO in vitro successfully.MRI can monitor magnetically labeled MSCs transplanted into liver. Key words: Stem cell transplantation; Mesenchymal stem cell; Super paramagnetic iron ox-ide; Feridex; Magnetic resonance imaging

Immunogenicity and Immune Modulation of Osteogenic Differentiated Mesenchymal Stem Cells From Banna Minipig Inbred Line

Mesenchymal stem cells (MSCs) represent a good choice for cell transplantation due to their multilineage differentiation ability and low immunogenicity. Our previous in vitro studies indicated that undifferentiated swine MSCs show low immunogenicity suppressing the proliferative responses of human peripheral blood lymphocyte to several antigens. In this study, we investigated the immunogenicity and immune modulation ability of osteogenic differentiated MSCs. SLA class I (P1, P14) was detectable by reverse-transcriptase polymerase chain reaction on both differentiated and undifferentiated MSCs. SLA class II (SLA-DRA, SLA-DQA) was only detectable on differentiated MSCs mixed lymphocyte reaction assays demonstrated that both differentiated and undifferentiated MSCs failed to stimulate proliferative responses by human peripheral blood lymphocytes (hPBLs). Furthermore, as undifferentiated MSCs, osteogenic differentiated MSCs also suppressed hPBL proliferation to phytohemaglutinin.