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Peripheral blood derived mononuclear cells enhance the migration and chondrogenic differentiation of multipotent mesenchymal stromal cells.

Hopper N, Wardale J, Howard D, Brooks R, Rushton N, Henson F - Stem Cells Int (2015)

Bottom Line: In scratch assay the wound closure was 55% higher after 3 hours in the PBMC stimulated test group (P = 0.002), migration rate was 9 times faster (P = 0.008), and total MSC migration was 25 times higher after 24 hours (P = 0.014).Analysis of MSCs by PCR array demonstrated that PBMCs induced the upregulation of genes associated with chondrogenic differentiation over 15-fold.In conclusion, PBMCs increase both MSC migration and differentiation suggesting that they are an ideal candidate for inclusion in regenerative medicine therapies aimed at cartilage repair.

View Article: PubMed Central - PubMed

Affiliation: Division of Trauma and Orthopaedic Surgery, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge BC2 0QQ, UK.

ABSTRACT
A major challenge in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into damaged areas and strategies to promote this should be developed. The aim of this study was to evaluate the effect of peripheral blood derived mononuclear cell (PBMC) stimulation on mesenchymal stromal cells (MSCs) derived from the infrapatellar fat pad of human OA knee. Cell migration was measured using an xCELLigence electronic migration chamber system in combination with scratch assays. Gene expression was quantified with stem cell PCR arrays and validated using quantitative real-time PCR (rtPCR). In both migration assays PBMCs increased MSC migration by comparison to control. In scratch assay the wound closure was 55% higher after 3 hours in the PBMC stimulated test group (P = 0.002), migration rate was 9 times faster (P = 0.008), and total MSC migration was 25 times higher after 24 hours (P = 0.014). Analysis of MSCs by PCR array demonstrated that PBMCs induced the upregulation of genes associated with chondrogenic differentiation over 15-fold. In conclusion, PBMCs increase both MSC migration and differentiation suggesting that they are an ideal candidate for inclusion in regenerative medicine therapies aimed at cartilage repair.

No MeSH data available.


Related in: MedlinePlus

Change in mRNA levels after 24 h PBMC stimulation with a cut-off value of 4. (a) A scatter plot showing up- and downregulated genes and core genes with no change (n = 1). (b) A heat map visualization of 2log2-fold change of the 84 genes in the stem cell PCR array (red: upregulated and green: downregulated). Grey shows the genes that were undetermined (no Ct value with a cut-off value of 35). (c) Real-time PCR validation of 5 key chondrogenic genes (n = 4) normalized to B2M housekeeping gene mRNA levels.
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fig3: Change in mRNA levels after 24 h PBMC stimulation with a cut-off value of 4. (a) A scatter plot showing up- and downregulated genes and core genes with no change (n = 1). (b) A heat map visualization of 2log2-fold change of the 84 genes in the stem cell PCR array (red: upregulated and green: downregulated). Grey shows the genes that were undetermined (no Ct value with a cut-off value of 35). (c) Real-time PCR validation of 5 key chondrogenic genes (n = 4) normalized to B2M housekeeping gene mRNA levels.

Mentions: In total the expressions of 158 genes related to stem cell differentiation, growth, pluripotency, self-renewal, and the mesenchymal lineage commitment pathways were measured using a commercial array. In total mRNA levels of 52 genes involved in MSC differentiation and growth were upregulated by the PBMC stimulation as compared to the unstimulated test group. Stemness marker mRNA levels for FGF2, INS, LIF, SOX2, TERT, WNT3A, and ZFP42 were upregulated (>15-fold) by PBMC stimulation. The mRNA levels of 19 genes specific to MSCs (ALCAM, ANPEP, BMP2, CASP3, CD44, ENG, ERBB2 (HER2), FUT4, FZD9, ITGA6, ITGAV, KDR, MCAM, NGFR, NT5E, PDGFRB, PROM1, THY1, and VCAM1) were also upregulated in PBMC stimulated MSCs. A partial effect was seen on the mRNA levels of 11 genes specific to osteogenesis, where 7 of them (BMP2, BMP6, FGF10, HNF1A, KDR, RUNX2, and TBX5) were upregulated by 24-hour PBMC stimulation. Messenger-RNA levels for 8 genes coding ABCB1, BMP2, BMP6, GDF5, GDF6, GDF7, ITGAX, and SOX9 proteins specific to chondrogenic differentiation were upregulated by over 15-fold following PBMC stimulation. Real-time PCR was used to further investigate five key chondrogenic genes and the results confirm that mRNA levels for all these genes were upregulated by the PBMCs (Figure 3(c): BMP2, BMP6 (P = 0.049), GDF5, GDF6 (P = 0.028), and Sox9 (P = 0.043)).


Peripheral blood derived mononuclear cells enhance the migration and chondrogenic differentiation of multipotent mesenchymal stromal cells.

Hopper N, Wardale J, Howard D, Brooks R, Rushton N, Henson F - Stem Cells Int (2015)

Change in mRNA levels after 24 h PBMC stimulation with a cut-off value of 4. (a) A scatter plot showing up- and downregulated genes and core genes with no change (n = 1). (b) A heat map visualization of 2log2-fold change of the 84 genes in the stem cell PCR array (red: upregulated and green: downregulated). Grey shows the genes that were undetermined (no Ct value with a cut-off value of 35). (c) Real-time PCR validation of 5 key chondrogenic genes (n = 4) normalized to B2M housekeeping gene mRNA levels.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4309296&req=5

fig3: Change in mRNA levels after 24 h PBMC stimulation with a cut-off value of 4. (a) A scatter plot showing up- and downregulated genes and core genes with no change (n = 1). (b) A heat map visualization of 2log2-fold change of the 84 genes in the stem cell PCR array (red: upregulated and green: downregulated). Grey shows the genes that were undetermined (no Ct value with a cut-off value of 35). (c) Real-time PCR validation of 5 key chondrogenic genes (n = 4) normalized to B2M housekeeping gene mRNA levels.
Mentions: In total the expressions of 158 genes related to stem cell differentiation, growth, pluripotency, self-renewal, and the mesenchymal lineage commitment pathways were measured using a commercial array. In total mRNA levels of 52 genes involved in MSC differentiation and growth were upregulated by the PBMC stimulation as compared to the unstimulated test group. Stemness marker mRNA levels for FGF2, INS, LIF, SOX2, TERT, WNT3A, and ZFP42 were upregulated (>15-fold) by PBMC stimulation. The mRNA levels of 19 genes specific to MSCs (ALCAM, ANPEP, BMP2, CASP3, CD44, ENG, ERBB2 (HER2), FUT4, FZD9, ITGA6, ITGAV, KDR, MCAM, NGFR, NT5E, PDGFRB, PROM1, THY1, and VCAM1) were also upregulated in PBMC stimulated MSCs. A partial effect was seen on the mRNA levels of 11 genes specific to osteogenesis, where 7 of them (BMP2, BMP6, FGF10, HNF1A, KDR, RUNX2, and TBX5) were upregulated by 24-hour PBMC stimulation. Messenger-RNA levels for 8 genes coding ABCB1, BMP2, BMP6, GDF5, GDF6, GDF7, ITGAX, and SOX9 proteins specific to chondrogenic differentiation were upregulated by over 15-fold following PBMC stimulation. Real-time PCR was used to further investigate five key chondrogenic genes and the results confirm that mRNA levels for all these genes were upregulated by the PBMCs (Figure 3(c): BMP2, BMP6 (P = 0.049), GDF5, GDF6 (P = 0.028), and Sox9 (P = 0.043)).

Bottom Line: In scratch assay the wound closure was 55% higher after 3 hours in the PBMC stimulated test group (P = 0.002), migration rate was 9 times faster (P = 0.008), and total MSC migration was 25 times higher after 24 hours (P = 0.014).Analysis of MSCs by PCR array demonstrated that PBMCs induced the upregulation of genes associated with chondrogenic differentiation over 15-fold.In conclusion, PBMCs increase both MSC migration and differentiation suggesting that they are an ideal candidate for inclusion in regenerative medicine therapies aimed at cartilage repair.

View Article: PubMed Central - PubMed

Affiliation: Division of Trauma and Orthopaedic Surgery, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge BC2 0QQ, UK.

ABSTRACT
A major challenge in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into damaged areas and strategies to promote this should be developed. The aim of this study was to evaluate the effect of peripheral blood derived mononuclear cell (PBMC) stimulation on mesenchymal stromal cells (MSCs) derived from the infrapatellar fat pad of human OA knee. Cell migration was measured using an xCELLigence electronic migration chamber system in combination with scratch assays. Gene expression was quantified with stem cell PCR arrays and validated using quantitative real-time PCR (rtPCR). In both migration assays PBMCs increased MSC migration by comparison to control. In scratch assay the wound closure was 55% higher after 3 hours in the PBMC stimulated test group (P = 0.002), migration rate was 9 times faster (P = 0.008), and total MSC migration was 25 times higher after 24 hours (P = 0.014). Analysis of MSCs by PCR array demonstrated that PBMCs induced the upregulation of genes associated with chondrogenic differentiation over 15-fold. In conclusion, PBMCs increase both MSC migration and differentiation suggesting that they are an ideal candidate for inclusion in regenerative medicine therapies aimed at cartilage repair.

No MeSH data available.


Related in: MedlinePlus