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Comparative miRNA-Based Fingerprinting Reveals Biological Differences in Human Olfactory Mucosa- and Bone-Marrow-Derived Mesenchymal Stromal Cells.

Lindsay SL, Johnstone SA, McGrath MA, Mallinson D, Barnett SC - Stem Cell Reports (2016)

Bottom Line: Previously we reported that nestin-positive human mesenchymal stromal cells (MSCs) derived from the olfactory mucosa (OM) enhanced CNS myelination in vitro to a greater extent than bone-marrow-derived MSCs (BM-MSCs). miRNA-based fingerprinting revealed the two MSCs were 64% homologous, with 26 miRNAs differentially expressed.The lower expression of miR-140-5p in OM-MSCs correlated with higher secretion of CXCL12 compared with BM-MSCs.Nestin-positive OM-MSCs could therefore offer a cell transplantation alternative for CNS repair, should these biological behaviors be translated in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection, Inflammation and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Sir Graeme Davies Building, 120 University Place, Glasgow G12 8TA, UK.

No MeSH data available.


Related in: MedlinePlus

CXCL12 Action May Be via OPC Process Extension and Microglial Polarization(A) CXCR4 expression (green) in oligodendrocytes (OPCs, red O4), microglia (MG) and astrocytes (GFAP, red). White arrows show microglia positive for CXCR4. Scale bar represents 50 μm.(B) CXCR4 and CXCR7 protein levels in microglia (MG) and OPCs (n = 2, both cell types). GAPDH used as loading control.(C) Graphical representation of western blot analysis. Microglia and OPCs show differing expression of various CXCR4 kDa bands. MG preferentially express the 50 kDa band (n = 4), while OPCs predominantly express the 45 kDa band (n = 6) (mean ± SEM, ∗∗p < 0.05, two-way ANOVA, Sidak's multiple comparison).(D) OPC differentiation was quantified using markers for OPCs (NG2), oligodendrocytes (O4), and mature oligodendrocytes (PLP) after CXCL12 treatment (n = 3), and CM from OM-MSCs (n = 3 patient samples) and BM-MSCs (n = 3 patient samples) compared with control (n = 3, mean ± SEM). No difference in marker expression was found.(E) Process extension of OPCs assessed by O4. OM-MSC-CM (OM-CM, n = 3 patient samples), BM-MSC-CM (BM-CM, n = 3 patient samples), and CXCL12 treatment (n = 3) significantly increased the number of complex processes and reduced the number of simple processes formed compared with control media. OM-MSC-CM caused a significant increase in membranous process formation (mean ± SEM, ∗∗∗p < 0.001, two-way ANOVA, Tukey's multiple comparison).(F) Quantification of the PLP-positive cell area of OPCs grown on inert nanofibers and treated with OM-MSC-CM (n = 3, patient samples), BM-MSC-CM (n = 3, patient samples), or CXCL12 (n = 3). There was a significantly greater cell area in the presence of OM-CM and CXCL12 (mean ± SEM, ∗p < 0.05 one-way ANOVA, Tukey multiple comparisons). Example images of OPCs (red PLP, blue DAPI) incubated with inert nanofibers. Scale bar represents 100 μm.(G and H) Graphical representation of western blot analysis (H) of arginase I and iNOS after treatment with CXCL12 (n = 4), CM from OM-MSCs (n = 4 patient samples) and BM-MSCs (n = 4 patient samples), LPS (n = 4), IL-4 (n = 4), and control treatments (n = 4). iNOS was significantly upregulated in LPS and BM-MSC-CM (BM-CM) treatment. Arginase I levels were significantly upregulated in both CXCL12 and OM-MSC-CM (OM-CM) (mean ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA, Tukey's multiple comparison).(I) Quantification of activin A in microglia media after stimulation with CXCL12 (n = 3), OM-MSC-CM (OM-CM-MG, n = 3 patient samples) and BM-MSC-CM (BM-MSC-MG, n = 3 patient samples) for 24 hr. OM-MSC-CM (OM-CM) and BM-MSC-CM (BM-CM) were used as controls (mean ± SEM).
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fig4: CXCL12 Action May Be via OPC Process Extension and Microglial Polarization(A) CXCR4 expression (green) in oligodendrocytes (OPCs, red O4), microglia (MG) and astrocytes (GFAP, red). White arrows show microglia positive for CXCR4. Scale bar represents 50 μm.(B) CXCR4 and CXCR7 protein levels in microglia (MG) and OPCs (n = 2, both cell types). GAPDH used as loading control.(C) Graphical representation of western blot analysis. Microglia and OPCs show differing expression of various CXCR4 kDa bands. MG preferentially express the 50 kDa band (n = 4), while OPCs predominantly express the 45 kDa band (n = 6) (mean ± SEM, ∗∗p < 0.05, two-way ANOVA, Sidak's multiple comparison).(D) OPC differentiation was quantified using markers for OPCs (NG2), oligodendrocytes (O4), and mature oligodendrocytes (PLP) after CXCL12 treatment (n = 3), and CM from OM-MSCs (n = 3 patient samples) and BM-MSCs (n = 3 patient samples) compared with control (n = 3, mean ± SEM). No difference in marker expression was found.(E) Process extension of OPCs assessed by O4. OM-MSC-CM (OM-CM, n = 3 patient samples), BM-MSC-CM (BM-CM, n = 3 patient samples), and CXCL12 treatment (n = 3) significantly increased the number of complex processes and reduced the number of simple processes formed compared with control media. OM-MSC-CM caused a significant increase in membranous process formation (mean ± SEM, ∗∗∗p < 0.001, two-way ANOVA, Tukey's multiple comparison).(F) Quantification of the PLP-positive cell area of OPCs grown on inert nanofibers and treated with OM-MSC-CM (n = 3, patient samples), BM-MSC-CM (n = 3, patient samples), or CXCL12 (n = 3). There was a significantly greater cell area in the presence of OM-CM and CXCL12 (mean ± SEM, ∗p < 0.05 one-way ANOVA, Tukey multiple comparisons). Example images of OPCs (red PLP, blue DAPI) incubated with inert nanofibers. Scale bar represents 100 μm.(G and H) Graphical representation of western blot analysis (H) of arginase I and iNOS after treatment with CXCL12 (n = 4), CM from OM-MSCs (n = 4 patient samples) and BM-MSCs (n = 4 patient samples), LPS (n = 4), IL-4 (n = 4), and control treatments (n = 4). iNOS was significantly upregulated in LPS and BM-MSC-CM (BM-CM) treatment. Arginase I levels were significantly upregulated in both CXCL12 and OM-MSC-CM (OM-CM) (mean ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA, Tukey's multiple comparison).(I) Quantification of activin A in microglia media after stimulation with CXCL12 (n = 3), OM-MSC-CM (OM-CM-MG, n = 3 patient samples) and BM-MSC-CM (BM-MSC-MG, n = 3 patient samples) for 24 hr. OM-MSC-CM (OM-CM) and BM-MSC-CM (BM-CM) were used as controls (mean ± SEM).

Mentions: CXCL12 is known to mediate its effect via CXCR4 and CXCR7, therefore cellular expression may help determine the mode of action. Oligodendrocyte precursor cells (OPCs) and microglia were both found to express CXCR4, however no expression was found on astrocytes (n = 3, all cell types; Figures 4A and 4B). Western blotting of CXCR4 revealed OPCs (n = 6) and microglia (n = 4) to have at least three distinct isoforms, however, both had differential expression of each (Figures 4B and 4C). It was found that the most abundant isoform expressed within microglia was the 50 kDa isoform, while OPCs were found to predominantly have the 45 kDa isoform. Total protein quantification of all CXCR4 isoforms was found not to be significantly different between microglia (1.72 ± 0.31 a.u.) versus OPCs (1.33 ± 0.35 a.u.). CXCR7 expression was only barely detectable by western blot and not immunocytochemistry on both OPCs and microglia (Figure 4B). Therefore the pro-myelinating capabilities of CXCL12 could be mediated via its action on OPCs or microglia, or both. Purified OPCs were treated with CXCL12, OM-, or BM-MSC-CM (from three different patient samples for both) and labeled with markers of OPC differentiation (n = 3; Figure 4D). There were no differences in immunoreactivity of NG2 (early OPC marker), O4 (middle/late OPC marker), or PLP (late myelinating OPC marker). However, CXCL12 significantly changed OPC morphological appearance from a predominantly simple (bipolar) to a more complex (multi-branched) morphology, similar to that found with OM- or BM-MSC-CM treatment (Figure 4E; p < 0.001). OM-MSC-CM resulted in significantly more OPCs exhibiting a membranous morphology when compared with either control or BM-MSC-CM (p < 0.001 for both), and although the result of OM-MSC-CM treatment looked to be greater, it was not significantly different to CXCL12 treatment. Overall this suggests that CXCL12 can mediate morphological OPC differentiation via its direct action on the OPC itself. This hypothesis was confirmed by examining the effect of CXCL12, OM-, and BM-MSC-CM on purified OPCs incubated with inert nanofibers (Figure 4F). In these experiments, PLP-positive OPCs had significantly greater cell areas ensheathing axons in both CXCL12 and OM-MSC-CM compared with controls (n = 3 each treatment and patient samples). BM-MSC-CM also caused an increase in PLP-positive cell area, however this was found not to be significantly different from control due to the variability among samples (n = 3 patient samples). This suggests that both CXCL12 and OM-MSC-CM promoted process extension and wrapping in the absence of axonal signals to a greater extent than BM-MSC-CM.


Comparative miRNA-Based Fingerprinting Reveals Biological Differences in Human Olfactory Mucosa- and Bone-Marrow-Derived Mesenchymal Stromal Cells.

Lindsay SL, Johnstone SA, McGrath MA, Mallinson D, Barnett SC - Stem Cell Reports (2016)

CXCL12 Action May Be via OPC Process Extension and Microglial Polarization(A) CXCR4 expression (green) in oligodendrocytes (OPCs, red O4), microglia (MG) and astrocytes (GFAP, red). White arrows show microglia positive for CXCR4. Scale bar represents 50 μm.(B) CXCR4 and CXCR7 protein levels in microglia (MG) and OPCs (n = 2, both cell types). GAPDH used as loading control.(C) Graphical representation of western blot analysis. Microglia and OPCs show differing expression of various CXCR4 kDa bands. MG preferentially express the 50 kDa band (n = 4), while OPCs predominantly express the 45 kDa band (n = 6) (mean ± SEM, ∗∗p < 0.05, two-way ANOVA, Sidak's multiple comparison).(D) OPC differentiation was quantified using markers for OPCs (NG2), oligodendrocytes (O4), and mature oligodendrocytes (PLP) after CXCL12 treatment (n = 3), and CM from OM-MSCs (n = 3 patient samples) and BM-MSCs (n = 3 patient samples) compared with control (n = 3, mean ± SEM). No difference in marker expression was found.(E) Process extension of OPCs assessed by O4. OM-MSC-CM (OM-CM, n = 3 patient samples), BM-MSC-CM (BM-CM, n = 3 patient samples), and CXCL12 treatment (n = 3) significantly increased the number of complex processes and reduced the number of simple processes formed compared with control media. OM-MSC-CM caused a significant increase in membranous process formation (mean ± SEM, ∗∗∗p < 0.001, two-way ANOVA, Tukey's multiple comparison).(F) Quantification of the PLP-positive cell area of OPCs grown on inert nanofibers and treated with OM-MSC-CM (n = 3, patient samples), BM-MSC-CM (n = 3, patient samples), or CXCL12 (n = 3). There was a significantly greater cell area in the presence of OM-CM and CXCL12 (mean ± SEM, ∗p < 0.05 one-way ANOVA, Tukey multiple comparisons). Example images of OPCs (red PLP, blue DAPI) incubated with inert nanofibers. Scale bar represents 100 μm.(G and H) Graphical representation of western blot analysis (H) of arginase I and iNOS after treatment with CXCL12 (n = 4), CM from OM-MSCs (n = 4 patient samples) and BM-MSCs (n = 4 patient samples), LPS (n = 4), IL-4 (n = 4), and control treatments (n = 4). iNOS was significantly upregulated in LPS and BM-MSC-CM (BM-CM) treatment. Arginase I levels were significantly upregulated in both CXCL12 and OM-MSC-CM (OM-CM) (mean ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA, Tukey's multiple comparison).(I) Quantification of activin A in microglia media after stimulation with CXCL12 (n = 3), OM-MSC-CM (OM-CM-MG, n = 3 patient samples) and BM-MSC-CM (BM-MSC-MG, n = 3 patient samples) for 24 hr. OM-MSC-CM (OM-CM) and BM-MSC-CM (BM-CM) were used as controls (mean ± SEM).
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fig4: CXCL12 Action May Be via OPC Process Extension and Microglial Polarization(A) CXCR4 expression (green) in oligodendrocytes (OPCs, red O4), microglia (MG) and astrocytes (GFAP, red). White arrows show microglia positive for CXCR4. Scale bar represents 50 μm.(B) CXCR4 and CXCR7 protein levels in microglia (MG) and OPCs (n = 2, both cell types). GAPDH used as loading control.(C) Graphical representation of western blot analysis. Microglia and OPCs show differing expression of various CXCR4 kDa bands. MG preferentially express the 50 kDa band (n = 4), while OPCs predominantly express the 45 kDa band (n = 6) (mean ± SEM, ∗∗p < 0.05, two-way ANOVA, Sidak's multiple comparison).(D) OPC differentiation was quantified using markers for OPCs (NG2), oligodendrocytes (O4), and mature oligodendrocytes (PLP) after CXCL12 treatment (n = 3), and CM from OM-MSCs (n = 3 patient samples) and BM-MSCs (n = 3 patient samples) compared with control (n = 3, mean ± SEM). No difference in marker expression was found.(E) Process extension of OPCs assessed by O4. OM-MSC-CM (OM-CM, n = 3 patient samples), BM-MSC-CM (BM-CM, n = 3 patient samples), and CXCL12 treatment (n = 3) significantly increased the number of complex processes and reduced the number of simple processes formed compared with control media. OM-MSC-CM caused a significant increase in membranous process formation (mean ± SEM, ∗∗∗p < 0.001, two-way ANOVA, Tukey's multiple comparison).(F) Quantification of the PLP-positive cell area of OPCs grown on inert nanofibers and treated with OM-MSC-CM (n = 3, patient samples), BM-MSC-CM (n = 3, patient samples), or CXCL12 (n = 3). There was a significantly greater cell area in the presence of OM-CM and CXCL12 (mean ± SEM, ∗p < 0.05 one-way ANOVA, Tukey multiple comparisons). Example images of OPCs (red PLP, blue DAPI) incubated with inert nanofibers. Scale bar represents 100 μm.(G and H) Graphical representation of western blot analysis (H) of arginase I and iNOS after treatment with CXCL12 (n = 4), CM from OM-MSCs (n = 4 patient samples) and BM-MSCs (n = 4 patient samples), LPS (n = 4), IL-4 (n = 4), and control treatments (n = 4). iNOS was significantly upregulated in LPS and BM-MSC-CM (BM-CM) treatment. Arginase I levels were significantly upregulated in both CXCL12 and OM-MSC-CM (OM-CM) (mean ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA, Tukey's multiple comparison).(I) Quantification of activin A in microglia media after stimulation with CXCL12 (n = 3), OM-MSC-CM (OM-CM-MG, n = 3 patient samples) and BM-MSC-CM (BM-MSC-MG, n = 3 patient samples) for 24 hr. OM-MSC-CM (OM-CM) and BM-MSC-CM (BM-CM) were used as controls (mean ± SEM).
Mentions: CXCL12 is known to mediate its effect via CXCR4 and CXCR7, therefore cellular expression may help determine the mode of action. Oligodendrocyte precursor cells (OPCs) and microglia were both found to express CXCR4, however no expression was found on astrocytes (n = 3, all cell types; Figures 4A and 4B). Western blotting of CXCR4 revealed OPCs (n = 6) and microglia (n = 4) to have at least three distinct isoforms, however, both had differential expression of each (Figures 4B and 4C). It was found that the most abundant isoform expressed within microglia was the 50 kDa isoform, while OPCs were found to predominantly have the 45 kDa isoform. Total protein quantification of all CXCR4 isoforms was found not to be significantly different between microglia (1.72 ± 0.31 a.u.) versus OPCs (1.33 ± 0.35 a.u.). CXCR7 expression was only barely detectable by western blot and not immunocytochemistry on both OPCs and microglia (Figure 4B). Therefore the pro-myelinating capabilities of CXCL12 could be mediated via its action on OPCs or microglia, or both. Purified OPCs were treated with CXCL12, OM-, or BM-MSC-CM (from three different patient samples for both) and labeled with markers of OPC differentiation (n = 3; Figure 4D). There were no differences in immunoreactivity of NG2 (early OPC marker), O4 (middle/late OPC marker), or PLP (late myelinating OPC marker). However, CXCL12 significantly changed OPC morphological appearance from a predominantly simple (bipolar) to a more complex (multi-branched) morphology, similar to that found with OM- or BM-MSC-CM treatment (Figure 4E; p < 0.001). OM-MSC-CM resulted in significantly more OPCs exhibiting a membranous morphology when compared with either control or BM-MSC-CM (p < 0.001 for both), and although the result of OM-MSC-CM treatment looked to be greater, it was not significantly different to CXCL12 treatment. Overall this suggests that CXCL12 can mediate morphological OPC differentiation via its direct action on the OPC itself. This hypothesis was confirmed by examining the effect of CXCL12, OM-, and BM-MSC-CM on purified OPCs incubated with inert nanofibers (Figure 4F). In these experiments, PLP-positive OPCs had significantly greater cell areas ensheathing axons in both CXCL12 and OM-MSC-CM compared with controls (n = 3 each treatment and patient samples). BM-MSC-CM also caused an increase in PLP-positive cell area, however this was found not to be significantly different from control due to the variability among samples (n = 3 patient samples). This suggests that both CXCL12 and OM-MSC-CM promoted process extension and wrapping in the absence of axonal signals to a greater extent than BM-MSC-CM.

Bottom Line: Previously we reported that nestin-positive human mesenchymal stromal cells (MSCs) derived from the olfactory mucosa (OM) enhanced CNS myelination in vitro to a greater extent than bone-marrow-derived MSCs (BM-MSCs). miRNA-based fingerprinting revealed the two MSCs were 64% homologous, with 26 miRNAs differentially expressed.The lower expression of miR-140-5p in OM-MSCs correlated with higher secretion of CXCL12 compared with BM-MSCs.Nestin-positive OM-MSCs could therefore offer a cell transplantation alternative for CNS repair, should these biological behaviors be translated in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection, Inflammation and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Sir Graeme Davies Building, 120 University Place, Glasgow G12 8TA, UK.

No MeSH data available.


Related in: MedlinePlus