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Fluorescence activated enrichment of CD146+ cells during expansion of human bone-marrow derived mesenchymal stromal cells augments proliferation and GAG/DNA content in chondrogenic media.

Hagmann S, Frank S, Gotterbarm T, Dreher T, Eckstein V, Moradi B - BMC Musculoskelet Disord (2014)

Bottom Line: Despite a high variability of CD146 expression among the donors, fluorescence sorting significantly increased the number of CD146+ cells compared to control MSCs, while magnetic sorting led to a lesser enrichment.However, FACS-sorted cells showed significantly increased GAG/DNA content after chondrogenic differentiation compared to control MSCs.The underlying mechanism of increased GAG/DNA content after enrichment during expansion remains unclear, but may be linked to increased proliferation rates in these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic and Trauma Surgery, University Hospital Heidelberg, Schlierbacher Landstrasse 200a, 69118 Heidelberg, Germany. babakmoradi@gmx.de.

ABSTRACT

Background: While numerous subpopulations of BM-MSCs have been identified, the relevance of these findings regarding the functional properties remains mostly unclear. With regards to attempts of enhancing differentiation results by preselecting certain MSC subtypes, we have evaluated the efficiency of CD146 purification during expansion, and evaluated whether these measures enhanced MSC differentiation results.

Methods: Human MSCs were derived from bone marrow of six donors and cultured in two different culture media. After P1, MSCs were purified by either magnetic or fluorescence sorting for CD146, with unsorted cells as controls. Growth characteristics and typical MSC surface markers were assessed from P0 to P3. After P3, chondrogenic, osteogenic and adipogenic differentiation potential were assessed.

Results: Despite a high variability of CD146 expression among the donors, fluorescence sorting significantly increased the number of CD146+ cells compared to control MSCs, while magnetic sorting led to a lesser enrichment. Osteogenic and adipogenic differentiation potential was not affected by the sorting process. However, FACS-sorted cells showed significantly increased GAG/DNA content after chondrogenic differentiation compared to control MSCs.

Conclusion: FACS sorting of CD146+ cells was more efficient than magnetic sorting. The underlying mechanism of increased GAG/DNA content after enrichment during expansion remains unclear, but may be linked to increased proliferation rates in these cells.

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Gating strategy for flow cytometry analysis of MSCs. FSC/SSC: forward scatter/sideward scatter. Isotype AB: isotype control antibody. The bar defines the border of 99.5% of the isotype control fluorescence signal. In this example, 99.46% of the MSCs express CD73.
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Fig1: Gating strategy for flow cytometry analysis of MSCs. FSC/SSC: forward scatter/sideward scatter. Isotype AB: isotype control antibody. The bar defines the border of 99.5% of the isotype control fluorescence signal. In this example, 99.46% of the MSCs express CD73.

Mentions: Confirmation of purification for CD146 was performed by flow cytometry in all groups after separation. For cell saving reasons, flow cytometry was performed for the entirety of the antibodies listed below in P0, P2 and P3, and in the control MSCs after P1. MSCs were suspended in PBS with 0.5% FCS and 2 mM EDTA and labelled with the following mouse anti-human antibodies: CD14 FITC, CD34 PE, CD45 APC-Cy™7, CD90 FITC, CD73 PE, CD105 PerCP-Cy™5 and CD146 PE (all BD Biosciences, Heidelberg, Germany). Cell viability was assessed using a 7AAD Viability Staining Solution (eBioscience, Frankfurt, Germany). Flow cytometry was conducted with a MACS Quant™ analyser and the MACS Quantify 2.1 software (Miltenyi Biotec, Bergisch Gladbach, Germany). Isotype matched antibodies were employed for background fluorescence detection. Positive fluorescence was defined as any event occurring above a gate defined by containing 99.5% of the events measured for background fluorescence in a histogram plot. The gating strategy is demonstrated in Figure 1.Figure 1


Fluorescence activated enrichment of CD146+ cells during expansion of human bone-marrow derived mesenchymal stromal cells augments proliferation and GAG/DNA content in chondrogenic media.

Hagmann S, Frank S, Gotterbarm T, Dreher T, Eckstein V, Moradi B - BMC Musculoskelet Disord (2014)

Gating strategy for flow cytometry analysis of MSCs. FSC/SSC: forward scatter/sideward scatter. Isotype AB: isotype control antibody. The bar defines the border of 99.5% of the isotype control fluorescence signal. In this example, 99.46% of the MSCs express CD73.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4196082&req=5

Fig1: Gating strategy for flow cytometry analysis of MSCs. FSC/SSC: forward scatter/sideward scatter. Isotype AB: isotype control antibody. The bar defines the border of 99.5% of the isotype control fluorescence signal. In this example, 99.46% of the MSCs express CD73.
Mentions: Confirmation of purification for CD146 was performed by flow cytometry in all groups after separation. For cell saving reasons, flow cytometry was performed for the entirety of the antibodies listed below in P0, P2 and P3, and in the control MSCs after P1. MSCs were suspended in PBS with 0.5% FCS and 2 mM EDTA and labelled with the following mouse anti-human antibodies: CD14 FITC, CD34 PE, CD45 APC-Cy™7, CD90 FITC, CD73 PE, CD105 PerCP-Cy™5 and CD146 PE (all BD Biosciences, Heidelberg, Germany). Cell viability was assessed using a 7AAD Viability Staining Solution (eBioscience, Frankfurt, Germany). Flow cytometry was conducted with a MACS Quant™ analyser and the MACS Quantify 2.1 software (Miltenyi Biotec, Bergisch Gladbach, Germany). Isotype matched antibodies were employed for background fluorescence detection. Positive fluorescence was defined as any event occurring above a gate defined by containing 99.5% of the events measured for background fluorescence in a histogram plot. The gating strategy is demonstrated in Figure 1.Figure 1

Bottom Line: Despite a high variability of CD146 expression among the donors, fluorescence sorting significantly increased the number of CD146+ cells compared to control MSCs, while magnetic sorting led to a lesser enrichment.However, FACS-sorted cells showed significantly increased GAG/DNA content after chondrogenic differentiation compared to control MSCs.The underlying mechanism of increased GAG/DNA content after enrichment during expansion remains unclear, but may be linked to increased proliferation rates in these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic and Trauma Surgery, University Hospital Heidelberg, Schlierbacher Landstrasse 200a, 69118 Heidelberg, Germany. babakmoradi@gmx.de.

ABSTRACT

Background: While numerous subpopulations of BM-MSCs have been identified, the relevance of these findings regarding the functional properties remains mostly unclear. With regards to attempts of enhancing differentiation results by preselecting certain MSC subtypes, we have evaluated the efficiency of CD146 purification during expansion, and evaluated whether these measures enhanced MSC differentiation results.

Methods: Human MSCs were derived from bone marrow of six donors and cultured in two different culture media. After P1, MSCs were purified by either magnetic or fluorescence sorting for CD146, with unsorted cells as controls. Growth characteristics and typical MSC surface markers were assessed from P0 to P3. After P3, chondrogenic, osteogenic and adipogenic differentiation potential were assessed.

Results: Despite a high variability of CD146 expression among the donors, fluorescence sorting significantly increased the number of CD146+ cells compared to control MSCs, while magnetic sorting led to a lesser enrichment. Osteogenic and adipogenic differentiation potential was not affected by the sorting process. However, FACS-sorted cells showed significantly increased GAG/DNA content after chondrogenic differentiation compared to control MSCs.

Conclusion: FACS sorting of CD146+ cells was more efficient than magnetic sorting. The underlying mechanism of increased GAG/DNA content after enrichment during expansion remains unclear, but may be linked to increased proliferation rates in these cells.

Show MeSH