Sedimentation field-flow fractionation separation of proliferative and differentiated subpopulations during Ca2+-induced differentiation in HaCaT cells.
The spontaneously immortalized human keratinocyte cell line HaCaT is widely used as a human keratinocyte model. In a previous comparative study between normal human keratinocytes (NHKs) and HaCaT, we reported that Ca2+ concentrations greater than 1mM induced differentiation in vitro in both cell types, notably characterized by increased expression of differentiation markers keratins 1 (K1), 10 (K10) and involucrin. Surprisingly, cells had a higher proliferative activity than those cultured with low Ca2+ levels. These results raised many questions; in particular concerning the emergence of HaCaT cells subpopulation which would have different differentiation states and/or proliferation rates throughout Ca2+-induced differentiation. To isolate these subpopulations, we used sedimentation field-flow fractionation (SdFFF). Results demonstrated that the most differentiated cells (HC-F1), characterized by the highest expression of keratinocyte differentiation markers, had the lowest proliferative activity. In contrast, less differentiated cells (HC-F2) maintained a higher proliferative activity. SdFFF is a tool to sort differentiated and/or proliferating cells from a total pool previously treated with a Ca2+ concentration inducing differentiation, and can be use to prepare biological models necessary for studying HaCaT cell proliferation after Ca2+-induced differentiation treatment.
Micallef
Ludovic
L
Laboratoire de Chimie Physique, EA 4021 "Biomolécules et Thérapies Anti-tumorales", Université de Limoges, Faculté de Pharmacie, 2 Rue du Dr Marcland, 87025 Limoges Cedex, France.
Battu
Serge
S
Pinon
Aline
A
Cook-Moreau
Jeanne
J
Cardot
Philippe J P
PJ
Delage
Christiane
C
Simon
Alain
A
D000704:Analysis of Variance; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D031521:Fractionation, Field Flow / Q000379:methods*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002118:Calcium