Magnetic-activated Cell Sorting Research Articles

Bovine muscle satellite cells in calves and cattle: A comparative study of cellular and genetic characteristics for cultivated meat production

This study compared the cellular and genetic characteristics of bovine skeletal muscle satellite cells (SMSCs) from Hanwoo (a Korean native cattle breed), including calves and mature cattle. SMSCs were isolated using magnetic-activated cell sorting (MACS) from tissue samples of six Hanwoo (three calves and three mature cattle) using the CD29 antibody. Calves’ SMSCs exhibited significantly faster growth rates than did those from cattle (P < 0.01), with a doubling time of 2.43 days. Genetic analysis revealed higher MyoD and Pax7 expression in SMSCs from calves during proliferation than in those from mature cattle (P < 0.001). However, FASN and PLAG1 expression levels were higher in mature cattle than in calves during both proliferation and differentiation (P < 0.001). These findings highlight the need for strategies to improve bovine muscle cell growth to produce competitive cultivated meat at a competitive price.

Single-cell RNA-seq and bulk-seq identify RAB17 as a potential regulator of angiogenesis by human dermal microvascular endothelial cells in diabetic foot ulcers.

Angiogenesis is crucial in diabetic wound healing and is often impaired in diabetic foot ulcers (DFUs). Human dermal microvascular endothelial cells (HDMECs) are vital components in dermal angiogenesis; however, their functional and transcriptomic characteristics in DFU patients are not well understood. This study aimed to comprehensively analyse HDMECs from DFU patients and healthy controls and find the potential regulator of angiogenesis in DFUs. HDMECs were isolated from skin specimens of DFU patients and healthy controls via magnetic-activated cell sorting. The proliferation, migration and tube-formation abilities of the cells were then compared between the experimental groups. Both bulk RNA sequencing (bulk-seq) and single-cell RNA-seq (scRNA-seq) were used to identify RAB17 as a potential marker of angiogenesis, which was further confirmed via weighted gene co-expression network analysis (WGCNA) and least absolute shrink and selection operator (LASSO) regression. The role of RAB17 in angiogenesis was examined through in vitro and in vivo experiments. The isolated HDMECs displayed typical markers of endothelial cells. HDMECs isolated from DFU patients showed considerably impaired tube formation, rather than proliferation or migration, compared to those from healthy controls. Gene set enrichment analysis (GSEA), fGSEA, and gene set variation analysis (GSVA) of bulk-seq and scRNA-seq indicated that angiogenesis was downregulated in DFU-HDMECs. LASSO regression identified two genes, RAB17 and CD200, as characteristic of DFU-HDMECs; additionally, the expression of RAB17 was found to be significantly reduced in DFU-HDMECs compared to that in the HDMECs of healthy controls. Overexpression of RAB17 was found to enhance angiogenesis, the expression of hypoxia inducible factor-1α and vascular endothelial growth factor A, and diabetic wound healing, partially through the mitogen-activated protein kinase/extracellular signal-regulated kinase signalling pathway. Our findings suggest that the impaired angiogenic capacity in DFUs may be related to the dysregulated expression of RAB17 in HDMECs. The identification of RAB17 as a potential molecular target provides a potential avenue for the treatment of impaired angiogenesis in DFUs.

Simultaneous and Multimodal Antigen-Binding Profiling and Isolation of Rare Cells via Quantitative Ferrohydrodynamic Cell Separation.

Simultaneous cell profiling and isolation based on cellular antigen-binding capacity plays an important role in understanding and treating diseases. However, fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are not able to meet this need, due to their requirement for a large quantity of target cells and the limitation stemming from bimodal separation. Here we report a microfluidic method, termed quantitative ferrohydrodynamic cell separation (qFCS), that achieved multimodal rare cell sorting and simultaneous antigen profiling at a ∼30,000 cell min-1 throughput with a 96.49% recovery rate and a 98.72% purity of recovered cells. qFCS profiles and sorts cells via cellular magnetic content of the magnetically labeled cells, which correlates to cellular antigen-binding capacity. By integrating cellular magnetophoresis and diamagnetophoresis in biocompatible ferrofluids, we demonstrate that the resulting qFCS device can accurately profile and isolate rare cells even when present at ∼1:50,000 target to background cells frequency. We show that the qFCS device could accurately profile and isolate T lymphocytes based on a low-expression CD154 antigen and allow on-device analysis of cells after processing. This method could address the need for simultaneous and multimodal rare cell isolation and profiling in disease diagnostics, prognostics, and treatment.

Cell sorting after tracer injection to elucidate allocation of cellular FDG uptake in different cell types and different microglial activation states

AbstractBackground2‐deoxy‐2‐[18F]fluoro‐d‐glucose positron‐emission‐tomography (FDG‐PET) is widely used to study cerebral glucose metabolism. Radiosorting revealed that the FDG‐PET signal is directly influenced by microglial glucose uptake in mouse models and we found similar associations in patients with neurodegenerative diseases. Now, we questioned the allocation of cellular FDG uptake in the rodent brain.MethodSingle cell [18F]FDG uptake as assessed by magnetic activated cell sorting (MACS) was multiplied with published total cell numbers in the mouse brain, i.e. 71e+6 neurons or 7% (≈ 7.6e+6) microglia cells to estimate the overall uptake per cell fraction. Estimated cellular microglial [18F]FDG uptake as assessed by MACS was cross‐validated with observed %‐FDG‐PET reductions after microglia depletion. Interstitial and vessel [18F]FDG was estimated by interstitial compartment volume and assumed equilibrium of non‐phosphorylated extra‐ and intracellular [14C]Deoxyglucose. Synaptic [18F]FDG uptake was estimated via an enriched synaptosome fraction relative to [18F]FDG uptake of whole‐brain homogenate. A microglia proliferation index of 1.3 was assumed for the whole brain in the Aβ mouse model.ResultMicroglia cells accounted for 8.6% of the overall brain [18F]FDG uptake in wild‐type mice and for 29.9% of the overall brain [18F]FDG uptake in PS2APP mice, confirmed by similar alterations after microglia depletion in PET. Synapses contributed to 13.2‐17.1% of the overall brain [18F]FDG uptake whereas interstitial (5.9‐7.7%) and vessel (2.6‐3.4%) [18F]FDG uptake was of minor magnitude. Neuronal and astrocyte cell bodies accounted for &lt;10% of the overall brain [18F]FDG uptake, but a proportion of 42‐55% was not recovered and likely allocated in lost cell processes, oligodendrocytes and other cell types.ConclusionMicroglial FDG uptake alterations are the source of FDG‐PET changes in models of neurodegenerative diseases. Our data should increase the awareness to consider glia cells as key contributors of FDG uptake and the detailed proportions of these cellular contributions in healthy brains and under disease conditions deserve deeper investigation.

Activated NK cells reprogram MDSCs via NKG2D-NKG2DL and IFN-γ to modulate antitumor T-cell response after cryo-thermal therapy

BackgroundMyeloid‐derived suppressor cells (MDSCs) can potently inhibit T-cell activity, promote growth and metastasis of tumor and contribute to resistance to immunotherapy. Targeting MDSCs to alleviate their protumor functions and immunosuppressive...

Tumor-associated neutrophils and macrophages exacerbate antidrug IgG-mediated anaphylactic reaction against an immune checkpoint inhibitor

BackgroundWith the increased use of immune checkpoint inhibitors (ICIs), side effects and toxicity are a great concern. Anaphylaxis has been identified as a potential adverse event induced by ICIs. Anaphylaxis...

Natural Killer Cells Improve Cognitive Function and Support to Induce Amyloid Beta Clearance by Functional Recovery of Impaired Microglia in Natural Killer Cell‐treated Alzheimer’s Diseases Mouse Model

AbstractBackgroundDysfunction of microglia that plays a pivotal role in amyloid beta clearance is recognized to be involved in pathological progression of the Alzheimer’s diseases. Natural killer (NK) cells participate in homeostatic immune‐modulation and immune‐surveillance by secreting immune‐boosting signaling molecules. These molecules can serve as cell‐based therapeutics for AD. In this study, we found that the activated NK cells eliminated Aβ plaques, resulting in a marked improvement in behavior in mice with AD. This improvement may be associated with the recovery in microglial function due to the upregulation of phagocytic activity and autophagy genes.MethodNK cells were isolated, using a negative selection process, from the spleens of BALB/c mice and subsequently expanded to 1×108 cells.ResultThe cultured NK cells were intravenously administered into APP/PS1 mutant mice. There was dramatic reduction of Aβ deposition in the cortex and dentate gyrus areas of the brain in these mice following the NK cell treatment. While phosphate‐buffered saline APP/PS1 control mice exhibited learning deficits in the escape platform, the NK cell‐treated mice showed reduced escape latency in the Morris water maze test. Mouse microglia, in the AD model, were isolated using CD11b positive magnetic activated cell sorting method. RNA sequencing was performed using total RNA isolated from mouse brain hippocampus tissue, to explore differential gene expression levels. Hierarchical cluster heat maps revealed the recovered gene populations in the NK‐treated microglial group. The percentage of genes upregulated in the total gene ontology groups were significantly altered in NK treated/sham ratio group (i.e., 171 upregulated immune response‐related genes). The homeostatic and disease‐associated microglia (DAM)‐related genes that were increased in microglia isolated from APP/PS1 mouse brain were downregulated after NK treatment. KEGG pathway analysis projected the autophagy signaling pathways as critical pathways associated with amyloid beta clearance. Our analysis showed that treatment of mice with NK cells recovered gene signatures involved in phagocytic activity.ConclusionThese findings show that NK cells, which are known to have low in vivo toxicity, could be a potential therapeutic option for AD that could be rapidly translated to the clinic.

Immunomodulatory effects of long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) on porcine monocytes (CD14 +) immune response in vitro

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are omega-3 long-chain polyunsaturated fatty acids (n-3 PUFA) found mostly in fish oil. They have been commonly used as dietary integrators in human and animal nutrition, modulating the immune system, mostly by exerting anti-inflammatory activities as demonstrated by in vivo and in vitro studies. The precise mechanisms of action at the background of EPA and DHA immunomodulatory activity are still not fully elucidated. Moreover, no information on their effects on porcine monocytes immune response is available yet. To cover this gap, the study aimed to evaluate DHA and EPA's in vitro impact on porcine monocytes (CD14 +) defensive functions. Briefly, monocytes were isolated from the blood of twenty-six healthy pigs, using a magnetic-activated cell sorting technique (MACS). Monocytes were first treated with increasing concentrations of DHA and EPA (25, 50, 100 and 200 µM) and apoptosis and viability were measured to assess potential cytotoxic effects. Once determined EPA and DHA subtoxic working concentrations (25, 50 and 100 µM), their effects on chemotaxis, phagocytosis and total, intracellular and extracellular reactive oxygen species (ROS) production were evaluated. DHA and EPA only decreased porcine monocytes viability at the highest concentration (200 µM), but their apoptosis was unaffected. DHA (100 µM) decreased the cells’ chemotaxis, while EPA (25 µM) increased their intracellular ROS production after 60 min under non-inflammatory or resting conditions and at 90 min under pro-inflammatory conditions (PMA challenge). EPA (50 µM) decreased monocytes’ intracellular ROS levels only under resting conditions at 30 min. No effects were observed on porcine monocytes phagocytic capacity. In conclusion, this study demonstrates that DHA and EPA can exert differential in vitro immunomodulatory effects in pigs, by dampening monocytes chemotaxis and potentiating their oxidative burst, respectively. Thus, our results suggest these n-3 PUFA might exert both anti-inflammatory and/or immune-enhancing effects in pigs.

Ex Vivo Culture with Albumin Greatly Increases the Immunosuppressive Subset of T Lymphocytes

Emerging studies suggest that the expression of co-inhibitory receptors such as PD1, TIM3, TIGIT, and LAG3 on the T cell surface is important in regulating T cell response. However, the low number of cells expressing such co-inhibitory receptors in peripheral blood severely limits their clinical potential in treating GVHD. In this study, we introduce a novel method to culture and obtain a high number of CD3+PD1+TIM3+ lymphocytes that also exert strong immunosuppression. We demonstrate that culturing CD3+ cells isolated from G-CSF mobilized peripheral blood stem cells (G-PBSCs) with human serum albumin (HSA) greatly increases the number of immunosuppressive CD3+ cells. Also, we confirm that the CD3+PD1+TIM3+ lymphocytes are the most potent subset of the CD3+ cells that induce immunosuppression and inhibit T cell proliferation. Methods. The donors were subcutaneously injected with G-CSF (10μg/kg) for five days. G-PBSCs were collected from the donors using a COBE spectra cell separator, and CD3+ cells were isolated by positive selection by magnetic-activated cell sorting (MACS) using CD3 Dynabeads™. Isolated CD3+ cells were cultured with 5% HSA for 4 days (1x105 cells/mL). PD1, TIM3, LAG3, and TIGIT expressions were analyzed using flow cytometry (FACS). CD3+, CD3+PD1+, and CD3+PD1+TIM3+ lymphocytes were sorted and cultured with irradiated allo-MNCs for three days (Mixed Lymphocyte Reaction; MLR). 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was used as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure T cell proliferation. Results. We discovered that treating G-PBSC cells with 5% HSA resulted in a dramatic increase in the number of immunosuppressive lymphocytes after the HSA treatment. In untreated G-PBSCs, there were very low numbers of CD3+PD1+TIM3+, CD3+PD1+LAG3+, and CD3+PD1+TIGIT+ lymphocytes (0.9±0.3x103, 0.1±0.1x103, and 0.3±0.2x103, respectively). In G-PBSCs, the HSA treatment markedly increased the cell numbers (47.6±1.9x103, 7.8±3.0x103, and 0.7±0.3x103, respectively) while resulting in a mild increase in the total number of CD3+ cells (1.2-fold increase, 1x105 to 1.2±0.4x105). Our data also demonstrated significant percent changes in subpopulations of CD3+ lymphocytes. In untreated G-PBSCs, very low percentages of cells were CD3+PD1+TIM3+, CD3+PD1+LAG3+, and CD3+PD1+TIGIT+ (9.0±3.1%, 1.2±0.1% and 3.2±2.8% in G-PBSC, respectively). After the HSA treatment, the percentages of the immunosuppressive lymphocytes in G-PBSCs markedly increased to 39.7±9.1%, 6.5±1.3%, and 0.6±0.4%, respectively. In particular, CD3+PD1+TIM3+ lymphocytes demonstrated the greatest increase in the cell number and the percentage after the HSA treatment, increasing by ~52 fold. Next, we tested whether the HSA-treatment induced proliferation of immunosuppressive T cells translated into increased immunosuppression. To do this, we used CSFE-MLR, co-cultured the CD3+ G-PBSCs with allo-MNCs, and analyzed the number of unstimulated T cells. The HSA-treated CD3+ G-PBSCs significantly increased the levels of unstimulated T cells compared to the untreated G-PBSCs (% of unstimulated T cells in untreated cells vs. in HSA-treated cells; 0.5±0.1 vs. 15.4±11.7), indicating that the HSA treatment increased CD3+ cell-mediated immunosuppression. Lastly, we tested the immunosuppressive effects of CD3+PD1+ and CD3+PD1-lymphocytes in HSA-treated CD3+ G-PBSCs. Both CD3+PD1+ and CD3+PD1+TIM3+ lymphocytes demonstrated significantly enhanced levels of immunosuppression compared to CD3+PD1-lymphocytes and CD3+PD1+TIM3- lymphocytes, respectively (% of unstimulated T cells in CD3+PD1+ vs. in CD3+PD1-lymphocytes, 27.9±5.5 vs. 4.3±7.5, and % of unstimulated T cells in CD3+PD1+TIM3+ vs. in CD3+PD1+TIM3- lymphocytes, 46.1±5.5 vs. 17.4±7.4). Collectively, these data suggest that PD1 and TIM3 are key markers in the HSA-dependent increase in immunosuppression. Conclusion. The ex vivo culture of CD3+ G-PBSCs with HSA markedly increases both the number and the effect of immunosuppressive CD3+ lymphocytes. Our analysis also indicates that the increased immunosuppression is likely due to the proliferation of CD3+PD1+TIM3+ lymphocytes. Taken together, our data suggest that the ex vivo HSA treatment of CD3+ G-PBSCs shows promising therapeutic potential for treating patients with GVHD.

CD44v6+ Hepatocellular Carcinoma Cells Maintain Stemness Properties through Met/cJun/Nanog Signaling.

Cancer stem cells (CSCs) are characterized by their self-renewal and differentiation abilities. CD44v6 is a novel CSC marker that can activate various signaling pathways. Here, we hypothesized that the HGF/Met signaling pathway promotes stemness properties in CD44v6+ hepatocellular carcinoma (HCC) cells via overexpression of the transcription factor, cJun, thus representing a valuable target for HCC therapy. Magnetic activated cell sorting was used to separate the CD44v6+ from CD44v6- cells, and Met levels were regulated using lentiviral particles and the selective Met inhibitor, PHA665752. An orthotopic liver xenograft tumor model was used to assess the self-renewal ability of CD44v6+ cells in immunodeficient NOD/SCID mice. Luciferase reporter and chromatin immunoprecipitation assays were also conducted using cJun-overexpressing 293 T cells to identify the exact binding site of cJun in the Nanog promoter. Our data demonstrate that CD44v6 is an ideal surface marker of liver CSCs. CD44v6+ HCC cells express higher levels of Met and possess self-renewal and tumor growth abilities. Xenograft liver tumors were smaller in nude mice injected with shMet HCC cells. Immunohistochemical analysis of liver tissue specimens revealed that high Met levels in HCC cells were associated with poor patient prognosis. Further, a cJun binding site was identified 1700 bp upstream of the Nanog transcription start site and mutation of the cJun binding site reduced Nanog expression. In conclusion, the HGF/Met signaling pathway is important for maintenance of stemness in CD44v6+ HCC cells by enhancing expression of cJun, which binds 1700 bp upstream of the Nanog transcription start site.

Utility of Magnetic Activated Cell Sorting (MACS) in Assisted Reproduction

Utility of Magnetic Activated Cell Sorting (MACS) in Assisted Reproduction

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Characterization of a Stemness-Optimized Purification Method for Human Dental-Pulp Stem Cells: An Approach to Standardization.

Human dental pulp stem cells (hDPSCs) are promising for oral/craniofacial regeneration, but their purification and characterization is not yet standardized. hDPSCs from three donors were purified by magnetic activated cell sorting (MACS)-assisted STRO-1-positive cell enrichment (+), colony derivation (c), or a combination of both (c/+). Immunophenotype, clonogenicity, stemness marker expression, senescence, and proliferation were analyzed. Multilineage differentiation was assessed by qPCR, immunohistochemistry, and extracellular matrix mineralization. To confirm the credibility of the results, repeated measures analysis and post hoc p-value adjustment were applied. All hDPSC fractions expressed STRO-1 and were similar for several surface markers, while their clonogenicity and expression of CD10/44/105/146, and 166 varied with the purification method. (+) cells proliferated significantly faster than (c/+), while (c) showed the highest increase in metabolic activity. Colony formation was most efficient in (+) cells, which also exhibited the lowest cellular senescence. All hDPSCs produced mineralized extracellular matrix. Regarding osteogenic induction, (c/+) revealed a significant increase in mRNA expression of COL5A1 and COL6A1, while osteogenic marker genes were detected at varying levels. (c/+) were the only population missing BDNF gene transcription increase during neurogenic induction. All hDPSCs were able to differentiate into chondrocytes. In summary, the three hDPSCs populations showed differences in phenotype, stemness, proliferation, and differentiation capacity. The data suggest that STRO-1-positive cell enrichment is the optimal choice for hDPSCs purification to maintain hDPSCs stemness. Furthermore, an (immuno) phenotypic characterization is the minimum requirement for quality control in hDPSCs studies.

OPTIMAL INTERVENTION TO OBTAIN SPERMS WITH GOOD DNA QUALITY – ROLE OF MACS VS MICROFLUIDICS IN SPERM SORTING

In Individuals with raised Sperm DNA Fragmentation Index (DFI), which sperm selection technique, Magnetic Activated Cell Sorting (MACS) or Microfluidics Sperm Sorting (MF) helps obtain sperms with good DNA quality.

SPERM PROCESSING BY MACS DOES NOT IMPROVE REPRODUCTIVE OUTCOMES IN CYCLES USING VITRIFIED DONOR OOCYTES WHEN COMPARED TO STANDARD SEMEN PROCESSING

The aim of this study is to retrospectively assess pregnancy, live birth rates (LBR) and cumulative live birth rates (CLBR) in intracytoplasmic sperm injection (ICSI) cycles using vitrified donor oocytes, comparing those using semen processed according to standard procedure versus those processing by magnetic-activated cell sorting (MACS).

The potential benefits of dinitrophenol combination with chemotherapy in the treatment of ovarian cancer.

2,4-dinitrophenol (DNP), an uncoupling mitochondrial agent, has been identified as a source of oxidative stress and linked to the pathogenesis of ovarian cancer. In this study, we determine the cytotoxic effect of DNP alone or in combination with chemotherapies in ovarian cancer cells. We utilized human ovarian cancer cell lines SKOV-3 and MDAH-2774 with their chemoresistant counterparts. Cancer stem cells (CSCs) were isolated from SKOV-3 utilizing magnetic-activated cell sorting technique for CD44+/CD117+ cells. Human normal primary ovarian epithelial (NOEC) and HOSEpiC cell lines were used as a control. Cells were treated with and without chemotherapy (Taxotere 0.3μM or cisplatin 50 μM), with or without increasing doses of DNP (0.125, 0.25, or 0.5 mM) for 24 hours followed by evaluation of cell viability and IC50 utilizing MTT assay. For determination of synergism, Facombination index plots were created using the CompuSyn software. All data were run in triplicates and analyzed by t-test. DNP treatment of ovarian cancer and chemoresistant ovarian cancer cell lines as well as CSCs resulted in decreased cell viability in a dose dependent manner with no effect on normal cells. Combination of DNP with chemotherapy synergistically enhances cytotoxicity of chemotherapeutics in all ovarian cancer cells as compared to chemotherapy alone. Our data indicates the potential of the addition of DNP to the arsenal of drugs available to treat ovarian cancer, whether alone or in combination with chemotherapies. The synergistic effects of DNP in reducing the required amount of chemotherapy, is critical for the alleviation of harmful side effects.

MicroRNA-1 Induces Transdifferentiation of Peripheral Blood CD34+ Cells into Cardiomyocytes-like Cells

BACKGROUND: Transdifferentiation is a method to provide cells sources for cellular cardiomyoplasty. CD34+ cells are potential cells sources because these cells can differentiate into cardiomyocytes through several mechanisms. MicroRNA (miR-1) is known to have the ability to inhibit the expression of histone deacetylase 4 (HDAC4). HDAC4 is a gene that essentially contributes in cardiomyocytes differentiation. However, the study reporting an evidence that miR-1 can induce transdifferentiation of CD34+ peripheral blood cells into mature cardiomyocytes is limited.METHODS: CD34+ cells were taken from peripheral blood and isolated using a magnetic-activated cell sorting (MACS) method in vitro. Mature mimics of miR-1 were transfected into isolated CD34+ cells and then incubated for 48 hours for quantification of HDAC4 mRNA using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). On the fifth day after miR-1 transfection, cardiomyocyte-like cells were identified based on their morphology and cardiac troponin expression using immunocytochemistry.RESULTS: Transfection of miR-1 in CD34+ isolated cells decreased HDAC4 gene expression by -0.54 fold at second day and caused a significant increase in percentage of cardiac troponin positive cells (median: 31.34; p&lt;0.05) at fifth-day post-transfection. The efficiency of transdifferentiation was 32%. The miR-1 transfection had a significant negative relationship with HDAC4 gene expression (B=-1.000; p=0.001). HDAC4 gene expression had a negative and significant relationship with the percentage of cardiac troponin-positive cells (B=-0.701; p=0.001).CONCLUSION: This study suggests that miR-1 can induce transdifferentiation of peripheral blood CD34+ cells into cardiomyocytes-like cells by decreasing HDAC4 gene expression.KEYWORDS: transdifferentiation, microRNA-1, CD34, cardiomyocyte, HDAC4

OS10.7.A Activation of the CCR8-ACP5 axis by human microglia/macrophage derived CCL18 promotes glioma growth

Abstract Background Glioblastoma multiforme is a highly malignant primary brain tumor with an average survival of 14 months and very limited therapeutic options. Glioma associated microglia and macrophages (GAMs) foster tumor growth by releasing several cytokines, which have only partly been identified. Here, we studied the chemokine (C-C motif) ligand 18 (CCL18), a chemokine which is only expressed in human, but not rodent GAMs, in a novel ex-vivo brain slice model including transplantation of human induced pluripotent stem cells (iPSC) derived human microglia (iMGL) and human glioma cells in to murine brain slices, which had been depleted of intrinsic murine microglia before. Material and Methods After establishing the humanized ex-vivo brain slice model, we performed immunohistochemical analysis (IHC) of growth and invasiveness, qrtPCR on glioma cells isolated by magnetic-activated cell sorting (MACS), functional assays measuring invasiveness, proliferation, migration and colony formation of glioma cells in vitro and in slice experiments. Corresponding studies on tumor growth and invasiveness were performed after treatment with a CCL18 neutralizing antibody, a CCR8 neutralizing antibodies and knockdown of CCR8, ACP5 (Acid Phosphatase 5) and PITPNM3 with small interfering RNA (siRNA) and short hairpin RNA (shRNA). QrtPCR, IHC and Westernblot analysis were performed on primary glioma specimens. We also conducted bioinformatic analyses, based on the TCGA GBM, GLIOVIS and GEPIA databases. Results We observed that CCL18 was highly expressed in GAMs, whereas CCR8 was only expressed in glioma cells. We identified the chemokine (C-C motif) receptor 8 (CCR8) as a functional receptor for CCL18 and ACP5 as an important down-stream signaling component in glioma cells. Activation of the CCL18/CCR8/ACP5 signaling pathway in human glioblastoma was associated with enhanced tumor growth and invasiveness. Conclusion GAMs derived CCL18 promoted glioma growth by activation of the CCR8/ACP5 axis in human glioma cells and therefore is a potential therapeutic target.

Shared and Non-Shared sIgA-Coated and -Uncoated Bacteria in Intestine of Mother-Infant Pairs.

The infant gut microbiota is critical for promoting and maintaining early-life health. The study aimed to analyze the composition of sIgA-coated and sIgA-uncoated bacterial communities at genus level and lactobacilli and bifidobacterial communities at species level in human breast milk (HBM) and infant and maternal feces. Eleven pregnant women were recruited successfully. HBM; infant feces during colostrum, transition, and mature stages; and maternal feces within the mature stage were collected. sIgA-coated and sIgA-uncoated bacteria were separated with magnetic-activated cell sorting. Then, 16S rRNA sequencing, bifidobacterial groEL gene sequencing, and lactobacilli groEL gene sequencing were performed to analyze the bacterial community. PCoA revealed that the compositions of sIgA-coated and sIgA-uncoated bacteria were different among HBM and infant and maternal feces. Higher relative abundance of sIgA-uncoated Bifidobacterium was found in the three lactation stages in infant feces compared to the corresponding HBM, and a higher relative abundance of sIgA-uncoated Faecalibacterium was found in maternal feces compared to HBM and infant feces. For bifidobacterial community, sIgA-coated and sIgA-uncoated B. longum subsp. infantis and B. pseudocatenulatum was dominant in infant feces and maternal feces, respectively. The relative abundance of sIgA-uncoated B. longum subsp. infantis was significantly higher in infant feces compared to that in maternal feces. For the Lactobacillus community, L. paragasseri and L. mucosae were dominant in infant and maternal feces, respectively. HBM and infant and maternal feces showed distinct diversity and composition of both sIgA-coated and sIgA-uncoated bacteria at genus level. Infant and maternal feces showed similar composition of Bifidobacterium at species level. The same Bifidobacterium species could be detected both in sIgA-coated and -uncoated form. This article provided deeper understanding on the microbiota profile in HBM and infant and maternal feces.

Survivin; a novel therapeutic target that correlates with survival of autoreactive T lymphocytes obtained from patients with ankylosing spondylitis

Ankylosing spondylitis (AS) is progressive immune-mediated arthritis. Persistent autoreactivity of T cells with an up-regulated Survivin expression is strongly implicated in AS immunopathogenesis. Besides, Survivin can inhibit proapoptotic caspase 9 activations. Moreover, microRNAs are small non-coding RNAs that are dysregulated in various diseases, in which their altered expression could modulate Survivin expression. The primary goal of this study was to assess the role of Survivin and its-targeting microRNAs in the immunopathogenesis of AS disease. For this aim, peripheral blood mononuclear cells (PBMCs) were isolated from 15 patients with AS and healthy matched controls using Ficoll-Hypaque. T cells were obtained using the magnetic-activated cell sorting (MACS) method. After that, the expression levels of Survivin, Caspase 9, and specific miRNAs were determined using qT-qPCR. Also, the expression of Survivin and Caspase 9 at protein levels was determined by western blotting. Then, the isolated T cells were co-cultured with interleukin (IL)-2 and muromonab-CD3 (OKT-3) for active-induced cell death (AICD) induction, Survivin siRNA for inhibition of Survivin expression, and their combination to assess the implication of Survivin expression in autoreactive T lymphocytes' resistance to apoptosis by determining the rate of apoptosis by Flowcytometry assay. The results showed that Survivin was up-regulated while Caspase 9 was downregulated in patients with AS. It was also revealed that microRNAs that directly or indirectly target the Survivin mRNA were dysregulated in patients with AS. It was also revealed that T cells obtained from AS patients were more resistant to apoptosis induction than those obtained from healthy people. In summary, the results obtained from this study showed that dysregulation of Survivin and Survivin-targeting miRNAs in T lymphocytes obtained from AS patients contribute to their resistance to apoptosis, suggesting the future development of targeted therapies for AS.