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PACAP polarizes MSC towards an anti-inflammatory (MSC2) phenotype. Conditioned medium from naïve and PACAP-treated MSC was analyzed for chemokines and cytokines secretion, utilizing Proteome Profiler Mouse Cytokine Array (R&D Systems). The following chemokine and anti-inflammatory cytokines IL-2, IL-3, IL-4, IL-27, IP10, IL-1ra, RANTES, SDF-1, CCL2(JE), CCL1 (i-309), G-CSF, BLC were upregulated, while pro-inflammatory cytokines were downregulated (IL-6, IL-1a, IFN-ɣ and soluble ICAM-1). Analysis was performed on pooled samples (n = 3) for each culture.

Journal: International Journal of Molecular Sciences

Article Title: Polarized Anti-Inflammatory Mesenchymal Stem Cells Increase Hippocampal Neurogenesis and Improve Cognitive Function in Aged Mice

doi: 10.3390/ijms24054490

Figure Lengend Snippet: PACAP polarizes MSC towards an anti-inflammatory (MSC2) phenotype. Conditioned medium from naïve and PACAP-treated MSC was analyzed for chemokines and cytokines secretion, utilizing Proteome Profiler Mouse Cytokine Array (R&D Systems). The following chemokine and anti-inflammatory cytokines IL-2, IL-3, IL-4, IL-27, IP10, IL-1ra, RANTES, SDF-1, CCL2(JE), CCL1 (i-309), G-CSF, BLC were upregulated, while pro-inflammatory cytokines were downregulated (IL-6, IL-1a, IFN-ɣ and soluble ICAM-1). Analysis was performed on pooled samples (n = 3) for each culture.

Article Snippet: Cytokine expression was detected in 100 μL of serum or pooled conditioned media using the Proteome Profiler Mouse Cytokine Array Kit (R&D Systems, Cat no. SC018), according to the manufacturer’s protocol.

Techniques:

Systemic administration of polarized MSC to aged mice normalizes systemic chemokine levels. Intravenous injection of polarized anti-inflammatory MSC (pMSC, N = 3–4) reduces the levels of plasma chemokines that are associated with aging and inflammation to levels of young (3 months old) mice (N = 7–8) compared with vehicle-treated aged control mice (N = 3), as detected by Proteome Profiler Mouse Cytokine Array (R&D Systems). Bars in the graph present mean ± SE. * p < 0.05, ** p < 0.01. One-way ANOVA. ns = non-significant.

Journal: International Journal of Molecular Sciences

Article Title: Polarized Anti-Inflammatory Mesenchymal Stem Cells Increase Hippocampal Neurogenesis and Improve Cognitive Function in Aged Mice

doi: 10.3390/ijms24054490

Figure Lengend Snippet: Systemic administration of polarized MSC to aged mice normalizes systemic chemokine levels. Intravenous injection of polarized anti-inflammatory MSC (pMSC, N = 3–4) reduces the levels of plasma chemokines that are associated with aging and inflammation to levels of young (3 months old) mice (N = 7–8) compared with vehicle-treated aged control mice (N = 3), as detected by Proteome Profiler Mouse Cytokine Array (R&D Systems). Bars in the graph present mean ± SE. * p < 0.05, ** p < 0.01. One-way ANOVA. ns = non-significant.

Article Snippet: Cytokine expression was detected in 100 μL of serum or pooled conditioned media using the Proteome Profiler Mouse Cytokine Array Kit (R&D Systems, Cat no. SC018), according to the manufacturer’s protocol.

Techniques: Injection, Clinical Proteomics, Control

Polarized PACAP-treated MSC maintain mesenchymal phenotype. ( A ). Immunophenotyping of PACAP-treated MSC in flow cytometry presents positive expression of the mesenchymal markers CD106 (>50%), CD29 (>80%), CD44 (>70%), CD73 (>60%) and sca-1 (>40%) but negative expression of the hematopoietic markers CD45 (<8%), and CD11b (<5%). Graphs represent flow cytometry histograms for the expression of the different markers. The negative control histogram is presented with the blue filled histogram. ( B ). Both naïve (N = 4) and PACAP-treated MSC (N = 3) expressed detectable levels of VPAC2 receptor mRNA, as detected in real-time PCR. Since the activation of Toll-like receptor 3 (TLR3) is an established marker of the MSC anti-inflammatory phenotype (MSC2) , we administered pituitary adenylate cyclase-activating peptide (PACAP), a neuropeptide with anti-inflammatory properties that is known to upregulate TLR3 and vice versa with TLR4, at 20 nM for 4 days to establish the anti-inflammatory MCS phenotype (MSC2). PACAP treatment of MSC in vitro (pMSC, N = 5) did not increase significantly the expression of TLR3 ( C ) or TLR4 ( D ) but increased the TLR3/TLR4 gene expression ratio ( E ) compared with naïve MSC (N = 4), as detected in real-time PCR. All graphs present mean ± SE. * p < 0.05, Student t -test. ns = non-significant.

Journal: International Journal of Molecular Sciences

Article Title: Polarized Anti-Inflammatory Mesenchymal Stem Cells Increase Hippocampal Neurogenesis and Improve Cognitive Function in Aged Mice

doi: 10.3390/ijms24054490

Figure Lengend Snippet: Polarized PACAP-treated MSC maintain mesenchymal phenotype. ( A ). Immunophenotyping of PACAP-treated MSC in flow cytometry presents positive expression of the mesenchymal markers CD106 (>50%), CD29 (>80%), CD44 (>70%), CD73 (>60%) and sca-1 (>40%) but negative expression of the hematopoietic markers CD45 (<8%), and CD11b (<5%). Graphs represent flow cytometry histograms for the expression of the different markers. The negative control histogram is presented with the blue filled histogram. ( B ). Both naïve (N = 4) and PACAP-treated MSC (N = 3) expressed detectable levels of VPAC2 receptor mRNA, as detected in real-time PCR. Since the activation of Toll-like receptor 3 (TLR3) is an established marker of the MSC anti-inflammatory phenotype (MSC2) , we administered pituitary adenylate cyclase-activating peptide (PACAP), a neuropeptide with anti-inflammatory properties that is known to upregulate TLR3 and vice versa with TLR4, at 20 nM for 4 days to establish the anti-inflammatory MCS phenotype (MSC2). PACAP treatment of MSC in vitro (pMSC, N = 5) did not increase significantly the expression of TLR3 ( C ) or TLR4 ( D ) but increased the TLR3/TLR4 gene expression ratio ( E ) compared with naïve MSC (N = 4), as detected in real-time PCR. All graphs present mean ± SE. * p < 0.05, Student t -test. ns = non-significant.

Article Snippet: To characterize the mesenchymal phenotype of polarized MSC, PACAP-treated MSC were immunophenotyped by FACS analysis (FACSCalibur with CellQuest software, Becton Dickinson, Franklin lakes, NJ, USA) using the mouse multipotent mesenchymal stromal cell marker antibody panel (cat No. SC018; R&D systems, Minneapolis, MN, USA), as we previously described [ ].

Techniques: Flow Cytometry, Expressing, Negative Control, Real-time Polymerase Chain Reaction, Activation Assay, Marker, In Vitro, Gene Expression