Transcriptome profiling analysis of uterus during chicken laying periods.

The avian eggshell is formed in the uterus. Changes in uterine function may have a significant effect on eggshell quality. To identify the vital genes impacting uterine functional maintenance in the chicken, uteri in three different periods (22W, 31W, 51W) were selected for RNA sequencing and bioinformatics analysis. In our study, 520, 706 and 736 differentially expressed genes (DEGs) were respectively detected in the W31 vs W22 group, W51 vs W31 group and W51 vs W22 group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated DEGs were enriched in the extracellular matrix, extracellular region part, extracellular region, extracellular matrix structural constituent, ECM receptor interaction, collagen-containing extracellular matrix and collagen trimer in the uterus (P < 0.05). Protein-protein interaction analysis revealed that FN1, LOX, THBS2, COL1A1, COL1A2, COL5A1, COL5A2, POSTN, MMP13, VANGL2, RAD54B, SPP1, SDC1, BTC, ANGPTL3 might be key candidate genes for uterine functional maintenance in chicken. This study discovered dominant genes and pathways which enhanced our knowledge of chicken uterine functional maintenance.

Whole-transcriptome RNA sequencing reveals the global molecular responses and circRNA/lncRNA-miRNA-mRNA ceRNA regulatory network in chicken fat deposition.

Fat deposition is a vital factor affecting the economics of poultry production. Numerous studies on fat deposition have been done. However, the molecular regulatory mechanism is still unclear. In the present study, the whole-transcriptome RNA sequencing in abdominal fat, back skin, and liver both high- and low-abdominal fat groups was used to uncover the competitive endogenous RNA (ceRNA) regulation network related to chicken fat deposition. The results showed that differentially expressed (DE) genes in abdominal fat, back skin, liver were 1207(784 mRNAs, 330 lncRNAs, 41 circRNAs, 52 miRNAs), 860 (607 mRNAs, 166 lncRNAs, 26 circRNAs, 61 miRNAs), and 923 (501 mRNAs, 262 lncRNAs, 15 circRNAs, 145 miRNAs), respectively. The ceRNA regulatory network analysis indicated that the fatty acid metabolic process, monocarboxylic acid metabolic process, carboxylic acid metabolic process, glycerolipid metabolism, fatty acid metabolism, and peroxisome proliferator-activated receptor (PPAR) signaling pathway took part in chicken fat deposition. Meanwhile, we scan the important genes, FADS2, HSD17B12, ELOVL5, AKR1E2, DGKQ, GPAM, PLIN2, which were regulated by gga-miR-460b-5p, gga-miR-199-5p, gga-miR-7470-3p, gga-miR-6595-5p, gga-miR-101-2-5p. While these miRNAs were competitive combined by lncRNAs including MSTRG.18043, MSTRG.7738, MSTRG.21310, MSTRG.19577, and circRNAs including novel_circ_PTPN2, novel_circ_CTNNA1, novel_circ_PTPRD. This finding provides new insights into the regulatory mechanism of mRNA, miRNA, lncRNA, and circRNA in chicken fat deposition.

Grade follicles transcriptional profiling analysis in different laying stages in chicken.

During follicular development, a series of key events such as follicular recruitment and selection are crucially governed by strict complex regulation. However, its molecular mechanisms remain obscure. To identify the dominant genes controlling chicken follicular development, the small white follicle (SWF), the small yellow follicle (SYF), and the large yellow follicle (LYF) in different laying stages (W22, W31, W51) were collected for RNA sequencing and bioinformatics analysis. There were 1866, 1211, and 1515 differentially expressed genes (DEGs) between SWF and SYF in W22, W31, and W51, respectively. 4021, 2295, and 2902 DEGs were respectively identified between SYF and LYF in W22, W31, and W51. 5618, 4016, and 4809 DEGs were respectively identified between SWF and LYF in W22, W31, and W51. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that extracellular matrix, extracellular region, extracellular region part, ECM-receptor interaction, collagen extracellular matrix, and collagen trimer were significantly enriched (P < 0.05). Protein-protein interaction analysis revealed that COL4A2, COL1A2, COL4A1, COL5A2, COL12A1, ELN, ALB, and MMP10 might be key candidate genes for follicular development in chicken. The current study identified dominant genes and pathways contributing to our understanding of chicken follicular development.

Transcriptome analysis reveals key genes and pathways associated with egg production in Nandan-Yao domestic chicken.

BACKGROUND: Egg production is a very important economic trait in chicken breeding, but its molecular mechanism is unclear until now. Nandan-Yao chicken (Gallus gallus domesticus) is a native breed in Guangxi province, China, which is famous for good meet quality, but with low egg production. METHODS: To explore the molecular regulation related to egg production, high egg production (HEP) and low egg production (LEP) were divided according to the total egg number at 55 weeks, and the concentration of serum sex hormones was tested to evaluate the physiological function of ovary and uterus. RNA sequencing (RNA-Seq) was used to explore the transcriptome from the ovary and uterus of Nandan-Yao chicken. RESULTS: The levels of serum sex hormone showed that concentrations of estradiol (E2), follicle-stimulating hormone (FSH), and luteotropic hormone (LH) were significantly higher in HEP than those in LEP (P < 0.01), while the concentration of testosterone (T) was significantly lower in HEP (P < 0.01). RNA-Seq analysis identified 901 and 2763 differentially expressed genes (DEGs) in ovary and uterus, respectively. Enrichment analysis showed that DEGs were significantly involved in the regulation of tight junction in the ovary (P < 0.05), while in uterus, DEGs were mainly enriched in the phagosome, ECM-receptor interaction, cell adhesion molecules (CAMs), focal adhesion, cardiac muscle contraction, cytokine-cytokine receptor interaction, and the regulation of MAPK signaling pathway (P < 0.05). Protein network interaction and function analyses revealed that FN1, FGF7, SOX2 identified from the ovary, and UQCRH, COX5A, FN1 from the uterus might be key candidate genes for egg production in Nandan-Yao chicken. CONCLUSIONS: Our study provided key candidate genes and pathways involved in the egg-laying process of Nandan-Yao chicken and could help to further understand the molecular mechanisms of chicken reproduction.

Genome-Wide Association Study Using Whole-Genome Sequencing Identifies a Genomic Region on Chromosome 6 Associated With Comb Traits in Nandan-Yao Chicken.

Comb traits have potential economic value in the breeding of indigenous chickens in China. Identifying and understanding relevant molecular markers for comb traits can be beneficial for genetic improvement. The purpose of this study was to utilize genome-wide association studies (GWAS) to detect promising loci and candidate genes related to comb traits, namely, comb thickness (CT), comb weight (CW), comb height, comb length (CL), and comb area. Genome-wide single-nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) in 300 Nandan-Yao chickens were detected using whole-genome sequencing. In total, we identified 134 SNPs and 25 INDELs that were strongly associated with the five comb traits. A remarkable region spanning from 29.6 to 31.4 Mb on chromosome 6 was found to be significantly associated with comb traits in both SNP- and INDEL-based GWAS. In this region, two lead SNPs (6:30,354,876 for CW and CT and 6:30,264,318 for CL) and one lead INDEL (a deletion from 30,376,404 to 30,376,405 bp for CL and CT) were identified. Additionally, two genes were identified as potential candidates for comb development. The nearby gene fibroblast growth factor receptor 2 (FGFR2)-associated with epithelial cell migration and proliferation-and the gene cytochrome b5 reductase 2 (CYB5R2)-identified on chromosome 5 from INDEL-based GWAS-are significantly correlated with collagen maturation. The findings of this study could provide promising genes and biomarkers to accelerate genetic improvement of comb development based on molecular marker-assisted breeding in Nandan-Yao chickens.

Transcriptome Analysis Identifies Candidate Genes and Signaling Pathways Associated With Feed Efficiency in Xiayan Chicken.

Feed efficiency is an important economic factor in poultry production, and the rate of feed efficiency is generally evaluated using residual feed intake (RFI). The molecular regulatory mechanisms of RFI remain unknown. Therefore, the objective of this study was to identify candidate genes and signaling pathways related to RFI using RNA-sequencing for low RFI (LRFI) and high RFI (HRFI) in the Xiayan chicken, a native chicken of the Guangxi province. Chickens were divided into four groups based on FE and sex: LRFI and HRFI for males and females, respectively. We identified a total of 1,015 and 742 differentially expressed genes associated with RFI in males and females, respectively. The 32 and 7 Gene Ontology (GO) enrichment terms, respectively, identified in males and females chiefly involved carbohydrate, amino acid, and energy metabolism. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 11 and 5 significantly enriched signaling pathways, including those for nutrient metabolism, insulin signaling, and MAPK signaling, respectively. Protein-protein interaction (PPI) network analysis showed that the pathways involving CAT, ACSL1, ECI2, ABCD2, ACOX1, PCK1, HSPA2, and HSP90AA1 may have an effect on feed efficiency, and these genes are mainly involved in the biological processes of fat metabolism and heat stress. Gene set enrichment analysis indicated that the increased expression of genes in LRFI chickens was related to intestinal microvilli structure and function, and to the fat metabolism process in males. In females, the highly expressed set of genes in the LRFI group was primarily associated with nervous system and cell development. Our findings provide further insight into RFI regulation mechanisms in chickens.

Transcriptome landscapes of differentially expressed genes related to fat deposits in Nandan-Yao chicken.

Nandan-Yao chicken is a Chinese native chicken with lower fat deposition and better meat quality. Fat deposition is a quite complex and important economic trait. However, its molecular mechanism is still unknown in chickens. In the current study, Nandan-Yao chicken was divided into two groups based on the rate of abdominal fat at 120 days old, namely the high-fat group and low-fat group. The total RNAs were isolated and sequenced by RNA sequencing (RNA-seq). After quality control, we gained 1222, 902, 784, 624, and 736 differentially expressed genes (DEGs) in abdominal fat, back skin, liver, pectoral muscle, and leg muscle, respectively. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that significantly enriched GO term and KEGG signaling pathway mainly involved cytosolic ribosome, growth development, PPAR signaling pathway, Wnt signaling pathway, and linoleic acid metabolism in abdominal fat, back skin, and liver. While in pectoral muscle and leg muscle, it is mainly enriched in phosphatidylinositol signaling system, adrenergic signaling in cardiomyocytes, cytosolic ribosome, and cytosolic part. Sixteen genes were differentially expressed in all five tissues. Among them, PLA2G4A and RPS4Y1 might be the key regulators for fat deposition in Nandan-Yao chicken. The protein-protein interaction (PPI) network analysis of DEGs showed that PCK1 was the most notable genes. The findings in the current study will help to understand the regulation mechanism of abdominal fat and intramuscular fat in Nandan-Yao chicken and provide a theoretical basis for Chinese local chicken breeding.

Metagenomic analysis reveals linkages between cecal microbiota and feed efficiency in Xiayan chickens.

The cecal microbiota plays a critical role in energy harvest and nutrient digestion, influencing intestinal health and the performance of chickens. Feed efficiency (FE) is essential for improving economic efficiency and saving social resources in chicken production and may be affected by the cecal microbiota. Therefore, to investigate the composition and functional capacity of cecum microbes related to FE in Xiayan chicken, an indigenous breed in Guangxi province, metagenome sequencing was performed on chicken cecal contents. 173 male and 167 female chickens were divided into high and low FE groups according to the residual feed intake. The cecal microbial genome was extracted and sequenced. The results showed that the genera Bacteroides, Prevotella, and Alistipes were the 3 most abundant in each cecal microbiome. The linear discriminant analysis effect size revealed 6 potential biomarkers in male and 14 in female chickens. Notably, the relative abundance of Lactobacillus in the high FE group was higher than that of the low FE group both in the male and female chickens, and the species Limosilactobacillus oris has a higher score in the high FE group of male chickens. In contrast, some potentially pathogenic microorganisms such as Campylobacter avium in females and Helicobacter pullorum in males were enriched in the low FE group. Predictive functional analysis showed that the high FE group in male chickens had a greater ability of xenobiotics biodegradation and metabolism and signaling molecules and interaction. In addition, the host sex was found to exert effects on the cecal microbial composition and function associated with FE. These results increased our understanding of the cecal microbial composition and identified many potential biomarkers related to FE, which may be used to improve the FE of the chickens.

Analysis of Population Structure and Differentially Selected Regions in Guangxi Native Breeds by Restriction Site Associated with DNA Sequencing.

Guangxi indigenous chicken breeds play a very important role in promoting the high-quality development of the broiler industry in China. However, studies on genomic information of Guangxi indigenous chicken to date remain poorly explored. To decipher the population genetic structure and differentially selected regions (DSRs) in Guangxi indigenous chickens, we dug into numerous SNPs from seven Guangxi native chickens (GX) by employing the restriction site associated with DNA sequencing (RAD-seq) technology. Another three breeds, Cobb, White Leghorn, and Chahua (CH) chicken, were used as a control. After quality control, a total of 185,117 autosomal SNPs were kept for further analysis. The results showed a significant difference in population structure, and the control breeds were distinctly separate from the Guangxi native breeds, which was also strongly supported by the phylogenetic tree. Distribution of FST indicated that there were three SNPs with big genetic differentiation (FST value all reach to 0. 9427) in GX vs. CH group, which were located on chr1-96,859,720,chr4-86,139,601, and chr12-8,128,322, respectively. Besides, we identified 717 DSRs associated with 882 genes in GX vs. Cobb group, 769 DSRs with 476 genes in GX vs. Leghorn group, and 556 DSRs with 779 genes in GX vs. CH group. GO enrichment showed that there were two significant terms, namely GPI-linked ephrin receptor activity and BMP receptor binding, which were enriched in GX vs. Leghorn group. In conclusion, this study suggests that Guangxi native chickens have a great differentiation with Cobb and Leghorn. Our findings would be beneficial to fully evaluate the genomic information on Guangxi native chicken and facilitate the application of these resources in chicken breeding.

Isolation, characterization, and SREBP1 functional analysis of mammary epithelial cell in buffalo.

Compared to cow milk, buffalo milk contains more protein, fat, and vitamin. Buffalo milk is an ideal food in human life. Sterol regulatory element-binding protein 1 (SREBP1), an important transcription factor, regulates the expression and activity of enzyme and protein involved in milk fat synthesis to influence on the synthesis and secretion of triglyceride in mammary epithelial cells. In the present study, we successfully isolated buffalo mammary epithelial cell by using enzymatic digestion, and then described the growth characteristics and expression characteristics of mammary epithelial cells. Moreover, we cloned the SREBP1 gene from total RNA isolated from milk fat globule and analyzed the function of the SREBP1 gene. After infected with shRNA-SREBP1 lentiviral particle and treated with fatty acid, the expression trend of ACACA, FABP3, FAS, SCD, ERK1, ERK2, PPARy, and Insigl genes was consistent with the expression trend of SREBP1 gene. These results suggested that SREBP1 gene is a central transcription factor in regulating milk fat synthesis and SREBP1 gene may act on ERK1/ERK2 signaling pathway to regulate the expression of PPARy gene. The current study will provide a theoretical basis for further reveal the molecular mechanism of milk fat synthesis in buffalo mammary epithelial cells. PRACTICAL APPLICATIONS: This study aim to separate and analysis characterization of mammary epithelial cell in buffalo. Compared to cow milk, buffalo milk contains more protein, fat, and vitamin. Buffalo milk is an ideal food in human life. This study will provide a theoretical basis for further research on the molecular mechanism of milk fat synthesis in buffalo mammary epithelial cells.

Hypoxia enhances the therapeutic potential of superparamagnetic iron oxide-labeled adipose-derived stem cells for myocardial infarction.

Adipose-derived stem cells (ASCs) induce therapeutic angiogenesis due to pro-angiogenic cytokines secretion. Superparamagnetic iron oxide (SPIO) nanoparticles are critical for magnetic resonance (MR) tracking of implanted cells. Hypoxia is a powerful stimulus for angiogenic activity of ASCs. In this study, we investigated whether therapeutic potency could be enhanced by implantation of hypoxia-preconditioned SPIO-labeled ASCs (SPIOASCs) into the infarcted myocardium. ASCs and SPIOASCs were cultured under 2% O2 (hypoxia) or 95% air (normoxia). Cells were intramyocardially injected into the infarcted myocardium after 48-h culture. We found that hypoxia culture increased the mRNA expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in ASCs and SPIOASCs. The VEGF protein in the conditioned medium was significantly higher in hypoxic ASCs and SPIOASCs than in normoxic ASCs and SPIOASCs. The capillary density and left ventricular contractile function in the infarcted myocardium were significantly higher 4 weeks after implantation with hypoxic ASCs and SPIOASCs than with normoxic ASCs and SPIOASCs. Improvement in the capillary density and left ventricle function didn't differ between hypoxic ASCs-transplanted rats and hypoxic SPIOASCs-transplanted rats. Hypoxic culture enhanced the angiogenic efficiency of ASCs. It was concluded that implantation of hypoxic ASCs or SPIOASCs promotes therapeutic angiogenesis and cardiac function recovery in the infarcted myocardium. SPIO labeling does not impact the beneficial effect of hypoxic ASCs.

The effects of activin A on the migration of human breast cancer cells and neutrophils and their migratory interaction.

Activin A belongs to the superfamily of transforming growth factor beta (TGFß) and is a critical regulatory cytokine in breast cancer and inflammation. However, the role of activin A in migration of breast cancer cells and immune cells was not well characterized. Here, a microfluidic device was used to examine the effect of activin A on the migration of human breast cancer cell line MDA-MB-231 cells and human blood neutrophils as well as their migratory interaction. We found that activin A promoted the basal migration but impaired epidermal growth factor (EGF)-induced migration of breast cancer cells. By contrast, activin A reduced neutrophil chemotaxis and transendothelial migration to N-Formyl-Met-Leu-Phe (fMLP). Finally, activin A promoted neutrophil chemotaxis to the supernatant from breast cancer cell culture. Collectively, our study revealed the different roles of activin A in regulating the migration of breast cancer cells and neutrophils and their migratory interaction. These findings suggested the potential of activin A as a therapeutic target for inflammation and breast cancers.

Preservation of Myocardial Perfusion and Function by Keeping Hypertrophied Heart Empty and Beating for Valve Surgery: An In Vivo MR Study of Pig Hearts.

Objectives. Normothermic hyperkalemic cardioplegia arrest (NHCA) may not effectively preserve hypertrophied myocardium during open-heart surgery. Normothermic normokalemic beating perfusion (NNBP), keeping hearts empty-beating, was utilized as an alternative to evaluate its cardioprotective role. Materials and Methods. Twelve hypertrophied pig hearts at 58.6 ± 7.2 days after ascending aorta banding underwent NNBP and NHCA, respectively. Near infrared myocardial perfusion imaging with indocyanine green (ICG) was conducted to assess myocardial perfusion. Left ventricular (LV) contractile function was assessed by cine MRI. TUNEL staining and western blotting for caspase-3 cleavage and cardiac troponin I (cTnI) degradation were conducted in LV tissue samples. Results. Ascending aortic diameter was reduced by 52.7% ± 0.4% at approximately fifty-eight days after banding. LV wall thickness was significantly higher in aorta banding than in sham operation. Myocardial blood flow reflected by maximum ICG absorbance value was markedly higher in NNBP than in NHCA. The amount of apoptotic cardiomyocyte was significantly lower in NNBP than in NHCA. NNBP alleviated caspase-3 cleavage and cTnI degradation associated with NHCA. NNBP displayed a substantially increased postoperative ejection fraction relative to NHCA. Conclusions. NNBP was better than NHCA in enhancing myocardial perfusion, inhibiting cardiomyocyte apoptosis, and preserving LV contractile function for hypertrophied hearts.

Corrigendum to "Preservation of Myocardial Perfusion and Function by Keeping Hypertrophied Heart Empty and Beating for Valve Surgery: An In Vivo MR Study of Pig Hearts".

[This corrects the article DOI: 10.1155/2017/4107587.].

Hypoxia Enhances the Therapeutic Potential of Superparamagnetic Iron Oxide-labeled Adipose-derived Stem Cells for Myocardial Infarction / 华中科技大学学报（医学）（英德文版）

Adipose-derived stem cells (ASCs) induce therapeutic angiogenesis due to pro-angiogenic cytokines secretion.Superparamagnetic iron oxide (SPIO) nanoparticles are critical for magnetic resonance (MR) tracking of implanted cells.Hypoxia is a powerful stimulus for angiogenic activity of ASCs.In this study,we investigated whether therapeutic potency could be enhanced by implantation of hypoxia-preconditioned SPIO-labeled ASCs (SPIOASCs) into the infarcted myocardium.ASCs and SPIOASCs were cultured under 2％ O2 (hypoxia) or 95％ air (normoxia).Cells were intramyocardially injected into the infarcted myocardium after 48-h culture.We found that hypoxia culture increased the mRNA expression of hypoxia-inducible factor-1 alpha (HIF-lαt) and vascular endothelial growth factor (VEGF) in ASCs and SPIOASCs.The VEGF protein in the conditioned medium was significantly higher in hypoxic ASCs and SPIOASCs than in normoxic ASCs and SPIOASCs.The capillary density and left ventricular contractile function in the infarcted myocardium were significantly higher 4 weeks after implantation with hypoxic ASCs and SPIOASCs than with normoxic ASCs and SPIOASCs.Improvement in the capillary density and left ventricle function didn't differ between hypoxic ASCs-transplanted rats and hypoxic SPIOASCs-transplanted rats.Hypoxic culture enhanced the angiogenic efficiency of ASCs.It was concluded that implantation of hypoxic ASCs or SPIOASCs promotes therapeutic angiogenesis and cardiac function recovery in the infarcted myocardium.SPIO labeling does not impact the beneficial effect of hypoxic ASCs.

A Window of MHM Demethylation Correlates with Key Events in Gonadal Differentiation in the Chicken.

The MHM (male hypermethylated) region on the chicken Z chromosome is of interest in terms of sex determination both because hypermethylation of the promoter in males results in female-specific expression, and in females the non-coding transcripts accumulate in the region of a locus that encodes a key candidate gene for testis differentiation in birds - DMRT1. However, it has yet to be established whether MHM plays a role in either sex determination or gonadal development in chicken. Here, we sought to establish the MHM methylation pattern during chicken gonadal development and determine how this pattern correlates with the expression profiles of genes involved in sex determination and gonadal differentiation. As expected, we found that MHM methylation levels were higher in male than in female gonads at most stages of development. We also showed that there was no significant difference in the methylation patterns between the right and left gonad and that the pattern of methylation in female gonads was not affected by sex reversal. However, we did find an unexpected window where MHM promoter methylation levels were very low in both males and females. This developmental window coincided with a stage of gonad development where there is a dramatic increase in expression of key testis genes in males and of key ovary genes in females. The current study raises the possibility that the non-coding RNA transcripts of MHM may affect testis and ovary differentiation by influencing sex-specific gene expression.

Externally Applied Static Magnetic Field Enhances Cardiac Retention and Functional Benefit of Magnetically Iron-Labeled Adipose-Derived Stem Cells in Infarcted Hearts.

: Although adipose-derived stem cells (ASCs) hold the promise of effective therapy for myocardial infarction, low cardiac retention of implanted ASCs has hindered their therapeutic efficiency. We investigated whether an externally applied static magnetic field (SMF) enhances cardiac localization of "magnetic" cells and promotes heart function recovery when ASCs are preloaded with superparamagnetic iron oxide (SPIO) nanoparticles. The influence of SMF (0.1 Tesla) on the biological activities of SPIO-labeled ASCs (SPIOASCs) was investigated first. Fifty-six female rats with myocardial infarction underwent intramyocardial injection of cell culture medium (CCM) or male SPIOASCs with or without the subcutaneous implantable magnet (CCM-magnet or SPIOASC-magnet). Four weeks later, endothelial differentiation, angiogenic cytokine secretion, angiogenesis, cardiomyocyte apoptosis, cell retention, and cardiac performance were examined. The 0.1-Tsela SMF did not adversely affect the viability, proliferation, angiogenic cytokine secretion, and DNA integrity of SPIOASCs. The implanted SPIOASCs could differentiate into endothelial cell, incorporate into newly formed vessels, and secrete multiple angiogenic cytokines. Four weeks after cell transplantation, the number of cardiac SPIOASCs was significantly increased, vascular density was markedly enlarged, fewer apoptotic cardiomyocytes were present, and heart contractile function was substantially improved in the SPIOASC-magnet treated rats in comparison with the SPIOASC-treated rats. The SPIOASCs could differentiate into endothelial cells, incorporate into vessels, promote angiogenesis, and inhibit ischemic cardiomyocyte apoptosis. An externally applied SMF offered a secure environment for biological properties of SPIOASCs, increased the cardiac retention of implanted magnetic SPIOASCs, and further enhanced heart function recovery after myocardial infarction. SIGNIFICANCE: This pilot proof-of-concept study suggests that a 0.1-Tesla static magnetic field does not adversely affect the viability, proliferation, angiogenic cytokine secretion, or DNA integrity of the superparamagnetic iron oxide-labeled adipose-derived stem cells (SPIOASCs). Implantation of adipose-derived stem cells promotes myocardial neovascularization and inhibits ischemic cardiomyocyte apoptosis through endothelial differentiation, incorporation into vessels, and paracrine factor secretion. An externally applied static magnetic field enhanced myocardial retention of intramyocardially injected "magnetic" SPIOASCs and promoted cardiac function recovery after myocardial infarction. With further preclinical optimization, this approach may improve the outcome of current stem cell therapy for ischemic myocardial infarction.

Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields.

After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF), Adipose-derived Stem Cells (ASCs) and those labeled by superparamagnetic iron oxide (SPIO) nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT) assay, proliferation by cell counting and bromodeoxyuridine (BrdU) incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factor Beta 1 (TGF-ß1), genetic markers comprising Stem Cell Antigen-1 (Sca1), Octamer-4 (Oct-4), ATP-binding Cassette Subfamily B Member 1 (ABCB1), adipogenic marker genes containing Lipoprotein Lipase (LPL), Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ), and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1) and Osterix (OSX). Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling.

Adipose-derived stem cells from both visceral and subcutaneous fat deposits significantly improve contractile function of infarcted rat hearts.

Adipose-derived stem cells (ASCs) from subcutaneous and visceral adipose tissues have been studied individually. No studies have compared their abilities in treatment of heart failure. This study was designed to evaluate whether ASCs from the two sources could provide a long-term improvement of cardiac function in infarcted hearts. Rat subcutaneous and visceral adipose tissues were excised for isolation of ASCs. Morphology, yield, proliferation, surface markers, differentiation, and cytokine secretion of the subcutaneous ASCs (S-ASCs) and visceral ASCs (V-ASCs) were analyzed. Then a rat model of myocardial infarction (MI) was established by a coronary occlusion. Seven days after occlusion, S-ASCs (n = 22), V-ASCs (n = 22), and Dulbecco's modified Eagle medium (DMEM, n = 20) were injected into the infarct rim, respectively. Cardiac function was then monitored with MRI for up to 6 months. The hearts were then removed for histological assessments. The yield of V-ASCs per gram of the visceral adipose depot was significantly greater than that of S-ASCs in 1 g of the subcutaneous adipose depot. On the other hand, the S-ASCs showed a greater proliferation rate and colony-forming unit relative to the V-ASCs. In addition, the infarcted hearts treated with either S-ASCs or V-ASCs showed a significantly greater left ventricular ejection fraction (LVEF) than those treated with DMEM at 4 weeks and 6 months following the cell/DMEM transplantation. Moreover, the infarct sizes of both S-ASC- and V-ASC-treated hearts were significantly smaller than that in the DMEM-treated hearts. MRI showed the implanted ASCs at the end of 6 months of recovery. Despite the differences in cell yield, proliferation, and colony formation capacity, both S-ASCs and V-ASCs provide a long-lasting improvement of cardiac contractile function in infarcted hearts. We conclude that the subcutaneous and visceral adipose tissues are equally effective cell sources for cell therapy of heart failure.

Assessment of three techniques for delivering stem cells to the heart using PET and MR imaging.

BACKGROUND: Stem cell therapy has a promising potential for the curing of various degenerative diseases, including congestive heart failure (CHF). In this study, we determined the efficacy of different delivery methods for stem cell administration to the heart for the treatment of CHF. Both positron emission tomography (PET) and magnetic resonance imaging (MRI) were utilized to assess the distribution of delivered stem cells. METHODS: Adipose-derived stem cells of male rats were labeled with super-paramagnetic iron oxide (SPIO) and 18 F-fluorodeoxyglucose (FDG). The left anterior descending coronary artery (LAD) of the female rats was occluded to induce acute ischemic myocardial injury. Immediately after the LAD occlusion, the double-labeled stem cells were injected into the ischemic myocardium (n = 5), left ventricle (n = 5), or tail vein (n = 4). In another group of animals (n = 3), the stem cells were injected directly into the infarct rim 1 week after the LAD occlusion. Whole-body PET images and MR images were acquired to determine biodistribution of the stem cells. After the imaging, the animals were euthanized and retention of the stem cells in the vital organs was determined by measuring the cDNA specific to the Y chromosome. RESULTS: PET images showed that retention of the stem cells in the ischemic myocardium was dependent on the cell delivery method. The tail vein injection resulted in the least cell retention in the heart (1.2% ± 0.6% of total injected cells). Left ventricle injection led to 3.5% ± 0.9% cell retention and direct myocardial injection resulted in the highest rate of cell retention (14% ± 4%) in the heart. In the animals treated 1 week after the LAD occlusion, rate of cell retention in the heart was only 4.5% ±1.1%, suggesting that tissue injury has a negative impact on cell homing. In addition, there was a good agreement between the results obtained through PET-MR imaging and histochemical measurements. CONCLUSION: PET-MR imaging is a reliable technique for noninvasive tracking of implanted stem cells in vivo. Direct injection of stem cells into the myocardium is the most effective way for cell transplantation to the heart in heart failure models.