Use of CFDA-SE for evaluating the in vitro proliferation pattern of human mesenchymal stem cells.
BACKGROUND
Mesenchymal stem cells (MSC) are multipotent progenitors retaining the capability to undergo multilineage differentiation, mostly towards all the mesodermal cellular lineages. MSC growing under standard conditions are composed of two main subpopulations with a characteristic distribution in the morphologic flow cytometric scatter: RS (recycling stem) cells (small, agranular) and m (mature) MSC (large, moderately granular cells).


METHODS
MSC obtained from BM of healthy donors and expanded in culture were characterized by evaluating both the expression of conventional markers and differentiation potential. We used CFSE, a lipophilic dye that is taken up by cell membranes, to investigate separately the proliferative activity of RS cells and mMSC subsets.


RESULTS
With flow cytometric analysis, RS cells and mMSC showed nearly the same immunophenotypic pattern, even if a significantly smaller percentage of RS cells expressed some of the classic mesenchymal Ag. The RS cell fraction was confirmed to have a higher proliferative potential and such a feature was particularly evident under certain culture conditions.


CONCLUSIONS
CFSE has been shown as a reliable method for studying the proliferative activity of MSC subpopulations identified by flow cytometric analysis. The acquisition parameter strategy is crucial for the accuracy of the analysis.
Urbani
S
S
Hematology Unit, Careggi Hospital, Viale Morgagni 85, 50134 Florence, Italy.
Caporale
R
R
Lombardini
L
L
Bosi
A
A
Saccardi
R
R
D017667:Adipocytes / Q000166:cytology; D015703:Antigens, CD / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology; D002452:Cell Count; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation; D002470:Cell Survival; D019902:Chondrocytes / Q000166:cytology; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006680:HLA Antigens / Q000032:analysis; D006801:Humans; D007700:Kinetics; D059630:Mesenchymal Stem Cells / Q000737:chemistry / Q000166:cytology*; D010006:Osteoblasts / Q000166:cytology; D013388:Succinimides / Q000737:chemistry*
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D015703:Antigens, CD; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006680:HLA Antigens; D013388:Succinimides