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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.

Show MeSH
Morphological aspects of MSCs and proliferation rates. a) MSCs sorted for CD146 by fluorescence cytometry (DMEM-FACS and ES-FACS) or magnetically (DMEM-MACS and ES-MACS) showed no morphological differences to the control MSCs cultured in DMEM-LG or ES medium. Light microscopy, magnification x100. b) Results displayed are the means and SD of the growth index per day divided by the growth index of control cells for each respective donor. *p < 0.05.
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Fig2: Morphological aspects of MSCs and proliferation rates. a) MSCs sorted for CD146 by fluorescence cytometry (DMEM-FACS and ES-FACS) or magnetically (DMEM-MACS and ES-MACS) showed no morphological differences to the control MSCs cultured in DMEM-LG or ES medium. Light microscopy, magnification x100. b) Results displayed are the means and SD of the growth index per day divided by the growth index of control cells for each respective donor. *p < 0.05.

Mentions: No morphological differences were observed between sorted and unsorted cells and between the two media conditions (Figure 2). No significant differences concerning proliferation were observed between the two media conditions. The mean relative growth index per day was higher in FACS-separated cells and lower in MACS-separated cells in P2, resulting in a significantly higher proliferation of FACS vs. MACS-sorted cells (Figure 2, p = 0.022). MACS-sorted cells closed up to FACS-sorted MSCs in P3 (Figure 2, comparison MACS/FACS: p = 0.388).Figure 2


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)

Morphological aspects of MSCs and proliferation rates. a) MSCs sorted for CD146 by fluorescence cytometry (DMEM-FACS and ES-FACS) or magnetically (DMEM-MACS and ES-MACS) showed no morphological differences to the control MSCs cultured in DMEM-LG or ES medium. Light microscopy, magnification x100. b) Results displayed are the means and SD of the growth index per day divided by the growth index of control cells for each respective donor. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Morphological aspects of MSCs and proliferation rates. a) MSCs sorted for CD146 by fluorescence cytometry (DMEM-FACS and ES-FACS) or magnetically (DMEM-MACS and ES-MACS) showed no morphological differences to the control MSCs cultured in DMEM-LG or ES medium. Light microscopy, magnification x100. b) Results displayed are the means and SD of the growth index per day divided by the growth index of control cells for each respective donor. *p < 0.05.
Mentions: No morphological differences were observed between sorted and unsorted cells and between the two media conditions (Figure 2). No significant differences concerning proliferation were observed between the two media conditions. The mean relative growth index per day was higher in FACS-separated cells and lower in MACS-separated cells in P2, resulting in a significantly higher proliferation of FACS vs. MACS-sorted cells (Figure 2, p = 0.022). MACS-sorted cells closed up to FACS-sorted MSCs in P3 (Figure 2, comparison MACS/FACS: p = 0.388).Figure 2

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