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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 <t>adenylate</t> 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.
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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 <t>adenylate</t> 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.
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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: Pituitary adenylate cyclase-activating peptide (PACAP) amino acids 1–27 was purchased from Bachem AG (Switzerland) cat. No. 127317-03-7.

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