Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers

• Published in: Biology of the cell 2013-12, Vol.105 (12), p.549-560
• Main Authors: Franzin, Chiara, Piccoli, Martina, Serena, Elena, Bertin, Enrica, Urbani, Luca, Luni, Camilla, Pasqualetto, Valerie, Eaton, Simon, Elvassore, Nicola, De Coppi, Paolo, Pozzobon, Michela
• Format: Article
• Language: English
• Subjects: Animals; Biomarkers - metabolism; Cell Culture Techniques - instrumentation; Cell Differentiation; Cells, Cultured; Culture Media - chemistry; Culture Media - metabolism; DNA Primers - genetics; DNA Primers - metabolism; Embryonic Stem Cells - cytology; Embryonic Stem Cells - metabolism; Feeder Cells - metabolism; feeder layer-free cultures; Fibroblasts - cytology; Fibroblasts - metabolism; Life Sciences; Male; Mice; Polymerase Chain Reaction - methods; Single-Cell Analysis - methods; single-cell PCR; stem cells markers; stiffness
• ISSN: 0248-4900; 1768-322X
• DOI: 10.1111/boc.201300034

Background Information In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single‐cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. Results Compared with population analysis, using a single‐cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co‐expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. Conclusions This single‐cell assay allows for the assessment of the stemness maintenance at a single‐cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential. Research article Here, we designed and tested a single‐cell multiplex PCR primers set for murine pluripotency markers, and applied it to murine embryonic stem cells cultured on (i) mouse embryonic fibroblasts (MEF) feeder layer, (ii) Matrigel coating, and (iii) Matrigel micropatterned hydrogel. The study highlighted the benefit of a single‐cell approach compared with population analysis, and showed that while feeder layer seems to remain the best condition for stemness preservation, micropatterned hydrogel could represent a good alternative in defining an optimal feeder‐free culture protocol.