An in situ hybridization-based screen for heterogeneously expressed genes in mouse ES cells

Abstract

We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out highthroughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures. KeywordsES cells; EC cells; pluripotent stem cells; heterogeneous gene expression; homogeneous gene expression; Zscan4; Pou5f1; Oct4; Oct3/4; Krt8; EndoA; Whsc2; Nelfa; Rhox9; Zfp42; Rex1; Rest; Zfp42; Rex1; Rest; Atf4; Pa2g4; E2f2; Nanog; Dppa3; Pgc7; Stella; Esrrb; Fscn1 &Corresponding author: Minoru S.H. Ko, M.D., Ph.D., Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, Triad Technology Center, suite 3000, 333 Cassell Drive, Baltimore, MD 21224, USA, Phone: (410) 558-8359; Fax: (410) 558-8331, E-mail: kom@mail.nih.gov. # Current Address: Center for Regenerative Biology, University of Connecticut, Storrs, Connecticut, 06269 $ Current Address: Stem Cell and Drug Discovery Institute, Kyoto 600-8813, Japan @ Current Address: Nagoya University Graduate School of Medicine, Aichi 466-8550, Japan * Equal contribution Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Falco et al., 2007). We suspect the existence of other transcription-regulating genes that show similar expression patterns. NIH Public AccessThe goals of this study are to find additional evidence for heterogeneous cell populations within undifferentiated ES cell cultures by identifying such genes and characterizing their expression patte...

Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation

“…Consis- Figure S1D), suggesting that Zscan4f accelerated the reprogramming process. We also cloned another analogous gene, Zscan4c, which is transcribed predominantly in ESCs [23,36], and showed that it has a similar activity during iPS cell generation (Supplementary information, Figure S1E). Furthermore, endogenous Zscan4 was expressed at a low level in MEFs before and after introduction of OSKM or OSKMZ (Supplementary information, Figure S1F).…”

A possible role of Reproductive homeobox 6 in primordial germ cell differentiation

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Self-Renewal, Pluripotency and Tumorigenesis in Pluripotent Stem Cells Revisited

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