Assessing self-renewal and differentiation in hESC lines

¹ Laboratory of Neurosciences, National Institute on Aging, Baltimore, Maryland
² Cellular Neurobiology Branch, National Institute on Drug Abuse, DHHS, Baltimore, Maryland
³ Burnham Institute, La Jolla, California
⁴ Invitrogen Corp., Carlsbad, California
⁵ Buck Institute for Age Research, Novato, California

^* To whom correspondence should be addressed. E-mail: xzeng{at}buckinstitute.org.

	Abstract

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 have 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 down-regulated as the cells differentiate to form embryoid bodies (EBs), while markers that are absent or low during the undifferentiated state but are induced as hESCs differentiate could be used to assess the presence of differentiated cells in the cultures. In this manuscript we describe a list of markers that reliably distinguish undifferentiated and differentiated cells. An initial list of approximately one hundred and fifty genes was generated by scanning published MPSS, EST 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, 4 germ cell markers and 4 trophoblast markers. Expression of these candidate marker genes was analyzed in undifferentiated hESCs and differentiating EB populations in four different lines by immunocytochemistry, 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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