Monitoring early differentiation events in human embryonic stem cells by massively parallel signature sequencing and expressed sequence tag scan.
To identify genes that may be involved in the process of human embryonic stem cell (hESC) differentiation, we profiled gene expression by expressed sequenced tag (EST) enumeration and massively parallel signature sequencing (MPSS) using RNA samples from feeder-free cultures of undifferentiated (passages 40-50) and differentiated (day 14) H1, H7, and H9 lines. MPSS and EST scan analysis showed good concordance and identified a large number of genes that changed rapidly as cultures transition from a pluripotent to a differentiated state. These included known and unknown ES cell-specific genes as well as a large number of known genes that were altered as cells differentiate. A subset of genes that were either up- or down-regulated were selected and their differential expression confirmed by a variety of independent methods, including comparison of expression after further differentiation, publicly available databases, and direct assessments by reverse transcriptase (RT)-PCR and immunocytochemistry. The analysis identified markers unique to the hESC and embryoid bodies (hEBs) stage as well as signaling pathways that likely regulate differentiation. The data generated can be used to monitor the state of hESC isolated by different laboratories using independent methods and maintained under differing culture conditions.
Miura
Takumi
T
Gerontology Research Center, Stem Cell Biology Unit/Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Luo
Yongquan
Y
Khrebtukova
Irina
I
Brandenberger
Ralph
R
Zhou
Daixing
D
Thies
R Scott
RS
Vasicek
Tom
T
Young
Holly
H
Lebkowski
Jane
J
Carpenter
Melissa K
MK
Rao
Mahendra S
MS
D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D002874:Chromosome Mapping / Q000379:methods; D002881:Chromosomes, Human, Pair 12; D019992:Databases as Topic; D004622:Embryo, Mammalian / Q000166:cytology*; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D015894:Genome, Human; D006801:Humans; D007150:Immunohistochemistry; D005810:Multigene Family; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology*; D015854:Up-Regulation
D012313:RNA