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a Laboratory of Neurosciences, National Institute on Aging, Department of Health and Human Services (DHHS), Baltimore, Maryland, USA;
b Cellular Neurobiology Branch, National Institute on Drug Abuse, DHHS, Baltimore, Maryland, USA;
c Program in Stem Cells and Regeneration, The Burnham Institute, La Jolla, California, USA;
d Invitrogen, Carlsbad, California, USA;
e Buck Insititute, Novato, California, USA
Key Words. Human embryonic stem cell • Embryoid body • Differentiation
Correspondence: Xianmin Zeng, Ph.D., Buck Institute for Age Research, 8001 Redwood Blvd., Novato, California 94945, USA. Telephone: 415-209-2211; Fax: 415-209-2230; e-mail: xzeng{at}buckinstitute.org and Mahendra S. Rao, M.D., Ph.D., Invitrogen, 1600 Faraday Dr., Carlsbad, California 92008, USA. Telephone: 410-852-1788; e-mail: mahendra.rao{at}invitrogen.com
Received on March 30, 2005;
accepted for publication on November 14, 2005.
Like other cell populations, undifferentiated human embryonic stem cells (hESCs) express a characteristic set of proteins and mRNA that is unique to the cells regardless of culture conditions, number of passages, and methods of propagation. We sought to identify a small set of markers that would serve as a reliable indicator of the balance of undifferentiated and differentiated cells in hESC populations. Markers of undifferentiated cells should be rapidly downregulated as the cells differentiate to form embryoid bodies (EBs), whereas markers that are absent or low during the undifferentiated state but that are induced as hESCs differentiate could be used to assess the presence of differentiated cells in the cultures. In this paper, we describe a list of markers that reliably distinguish undifferentiated and differentiated cells. An initial list of approximately 150 genes was generated by scanning published massively parallel signature sequencing, expressed sequence tag scan, and microarray datasets. From this list, a subset of 109 genes was selected that included 55 candidate markers of undifferentiated cells, 46 markers of hESC derivatives, four germ cell markers, and four trophoblast markers. Expression of these candidate marker genes was analyzed in undifferentiated hESCs and differentiating EB populations in four different lines by immunocytochemistry, reverse transcription–polymer-ase chain reaction (RT-PCR), microarray analysis, and quantitative RT-PCR (qPCR). We show that qPCR, with as few as 12 selected genes, can reliably distinguish differentiated cells from undifferentiated hESC populations.
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