Sedimentation field-flow fractionation separation of proliferative and differentiated subpopulations during Ca²⁺-induced differentiation in HaCaT cells

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• 作者：Ludovic Micallef ; Serge Battu ; Aline Pinon ; Jeanne Cook-Moreau ; Philippe J.P. Cardot ; Christiane Delage ; Alain Simon
• 关键词：HaCaT cells ; Calcium-induced differentiation ; Proliferative activity ; Sedimentation field-flow fractionation ; Cell sorting
• 刊名：Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences
• 出版年：2010
• 出版时间：1 May 2010
• 年：2010
• 卷：878
• 期：15-16
• 页码：1051-1058
• 全文大小：618 K

文摘

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 1 mM 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.