title: Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers.
abstract: BACKGROUND
  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.
authors.affiliation: Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padova, 35127, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Franzin
authors.forename: Martina
authors.initials: M
authors.lastname: Piccoli
authors.forename: Elena
authors.initials: E
authors.lastname: Serena
authors.forename: Enrica
authors.initials: E
authors.lastname: Bertin
authors.forename: Luca
authors.initials: L
authors.lastname: Urbani
authors.forename: Camilla
authors.initials: C
authors.lastname: Luni
authors.forename: Valerie
authors.initials: V
authors.lastname: Pasqualetto
authors.forename: Simon
authors.initials: S
authors.lastname: Eaton
authors.forename: Nicola
authors.initials: N
authors.lastname: Elvassore
authors.forename: Paolo
authors.initials: P
authors.lastname: De Coppi
authors.forename: Michela
authors.initials: M
authors.lastname: Pozzobon
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000295:instrumentation*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D061252:Feeder Cells / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D008297:Male; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D015415:Biomarkers; D003470:Culture Media; D017931:DNA Primers
keywords: feeder layer-free cultures; single-cell PCR; stem cells markers; stiffness