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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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Related in: MedlinePlus

Osteogenic, adipogenic and chondrogenic differentiation results. a) Alizarin red contents per protein at days 3, 7, 14 and 21 are displayed in the right diagram, ALP contents per protein per ml and min at days 3, 7, 14 and 21 are displayed in the left diagram. No significant differences between fluorescence or magnetic sorting and the control MSCs could be detected. b) No differences regarding adipogenic differentiation of MSCs cultured in DMEM or ES medium and sorted for CD146 by fluorescence cytometry (DMEM-FACS and ES-FACS) or magnetically (DMEM-MACS and ES-MACS) could be detected. Light microscopy, magnification x100. c) Mean GAG/DNA content of control MSCs, FACS and MACS sorted cells after 21 days of chondrogenic differentiation is displayed. *p < 0.05.
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Fig4: Osteogenic, adipogenic and chondrogenic differentiation results. a) Alizarin red contents per protein at days 3, 7, 14 and 21 are displayed in the right diagram, ALP contents per protein per ml and min at days 3, 7, 14 and 21 are displayed in the left diagram. No significant differences between fluorescence or magnetic sorting and the control MSCs could be detected. b) No differences regarding adipogenic differentiation of MSCs cultured in DMEM or ES medium and sorted for CD146 by fluorescence cytometry (DMEM-FACS and ES-FACS) or magnetically (DMEM-MACS and ES-MACS) could be detected. Light microscopy, magnification x100. c) Mean GAG/DNA content of control MSCs, FACS and MACS sorted cells after 21 days of chondrogenic differentiation is displayed. *p < 0.05.

Mentions: The greatest heterogeneity of the MSC preparations was observed for CD146, which was highly donor-dependent (expression ranging from 48.69% to 89.43% in P0, Figure 4). While no significant differences between the passages could be detected, CD146 expression was higher when MSCs were cultured in DMEM compared to ES medium in P0 and P2 (Table 1, Figure 3, P0 and P2: p = 0.028).Figure 4


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)

Osteogenic, adipogenic and chondrogenic differentiation results. a) Alizarin red contents per protein at days 3, 7, 14 and 21 are displayed in the right diagram, ALP contents per protein per ml and min at days 3, 7, 14 and 21 are displayed in the left diagram. No significant differences between fluorescence or magnetic sorting and the control MSCs could be detected. b) No differences regarding adipogenic differentiation of MSCs cultured in DMEM or ES medium and sorted for CD146 by fluorescence cytometry (DMEM-FACS and ES-FACS) or magnetically (DMEM-MACS and ES-MACS) could be detected. Light microscopy, magnification x100. c) Mean GAG/DNA content of control MSCs, FACS and MACS sorted cells after 21 days of chondrogenic differentiation is displayed. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Osteogenic, adipogenic and chondrogenic differentiation results. a) Alizarin red contents per protein at days 3, 7, 14 and 21 are displayed in the right diagram, ALP contents per protein per ml and min at days 3, 7, 14 and 21 are displayed in the left diagram. No significant differences between fluorescence or magnetic sorting and the control MSCs could be detected. b) No differences regarding adipogenic differentiation of MSCs cultured in DMEM or ES medium and sorted for CD146 by fluorescence cytometry (DMEM-FACS and ES-FACS) or magnetically (DMEM-MACS and ES-MACS) could be detected. Light microscopy, magnification x100. c) Mean GAG/DNA content of control MSCs, FACS and MACS sorted cells after 21 days of chondrogenic differentiation is displayed. *p < 0.05.
Mentions: The greatest heterogeneity of the MSC preparations was observed for CD146, which was highly donor-dependent (expression ranging from 48.69% to 89.43% in P0, Figure 4). While no significant differences between the passages could be detected, CD146 expression was higher when MSCs were cultured in DMEM compared to ES medium in P0 and P2 (Table 1, Figure 3, P0 and P2: p = 0.028).Figure 4

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
Related in: MedlinePlus