Monitoring Differentiation of Human Embryonic Stem Cells Using Real‐Time PCR

Noaksson, Karin; Zoric, Neven; Zeng, Xianmin; Rao, Mahendra S.; Hyllner, Johan; Semb, Henrik; Kubista, Mikael; Sartipy, Peter

doi:10.1634/stemcells.2005-0093

Cited by 63 publications

(56 citation statements)

References 37 publications

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“…We believe that any of the methods we tested is sufficient to monitor the state of hESC, but each method has its advantages and disadvantages. If qPCR is used, a small number of genes is sufficient provided both positive and negative markers are used (present results and [42]). However, the most cost-effective method for the wealth of information obtained may be a focused array that includes many markers such as the genes we have described.…”

Section: Resultsmentioning

confidence: 82%

Assessing Self-Renewal and Differentiation in Human Embryonic Stem Cell Lines

et al. 2005

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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-polymerase 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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Section: Resultsmentioning

confidence: 82%

Assessing Self-Renewal and Differentiation in Human Embryonic Stem Cell Lines

et al. 2005

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“…Since ES cells and PGCs share the expression of many transcription factors and cell surface receptors, we tested the notion that cultured ES cells could be used to examine the function of an oocyte-specific gene promoter. Early PGCs can be distinguished from ES cells and epiblast cells by their differential expression of Oct-4 transgenes containing germ cell-specific regulatory elements [36][37][38][39]. This is possible because the expression of Oct-4 in ES cells, epiblast and PGCs relies on different promoter elements providing a means for identification of these different cell populations.…”

Section: Discussionmentioning

confidence: 99%

The promoter of the oocyte-specific gene, Gdf9, is active in population of cultured mouse embryonic stem cells with an oocyte-like phenotype

Salvador

Silva

Kostetskii

et al. 2008

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The study of germ cell-specific gene regulation in vitro is challenging. Here we report that the promoter of the oocyte-specific gene, Gdf9, is active in a population of cultured murine embryonic stem cells (ES) which have a phenotype resembling ooocytes. The promoter region of the murine Gdf9 coupled to enhanced green fluorescent protein (eGFP) was stably transfected into XX mouse ES cells. eGFP was expressed only in oocytes of chimeric mice generated from the transfected XX ES cells. The transfected ES cells were examined when cultured on feeder layers or as embryoid bodies. Large eGFP-positive cells, surrounded by a structure resembling a zona pellucida appeared transiently in cultures of the ES cells on feeder layers. Surprisingly, they were detectable on days 1 and 2 of culture but virtually absent on day 3. Addition of leukemia inhibitory factor (LIF) to the media significantly increased the number of eGFP-positive cels resembling oocytes. Quantitative real-time PCR demonstrated a parallel increase in Gdf9 and Zp3 mRNA with changes in the abundance of eGFP-positive cells. In embryoid body cultures, eGFP-positive cells appeared transiently and then re-appeared in regional clusters after 30−45 days of culture. These findings demonstrate that a population of cultured murine ES cells contain the transcriptional machinery to drive expression of an oocyte-specific gene, and that those cells phenotypically resemble oocytes.

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“…This quantitative approach is rapid and sensitive and is considered suitable for pharmacological and cytotoxicity screening. Monitoring of ES cell differentiation with quantitative PCR has recently been reported (Noaksson et al, 2005). Secondly, we examined the expression of several genes whose proteins were highly expressed in ES cells compared with EBs.…”

Section: Embryonic Stem (Es) Cells Have Great Potentialmentioning

confidence: 99%

Monitoring of Gene Expression in Differentiation of Embryoid Bodies From Cynomolgus Monkey Embryonic Stem Cells in the Presence of Bisphenol A

et al. 2007

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-An embryonic stem (ES) cell differentiation model would facilitate analysis of developmental processes at the cellular level and the effects of embryotoxic and teratogenic factors in vitro. We explored the use of differentiation of embryoid bodies (EBs) from cynomolgus monkey ES cells for embryotoxicity testing. We determined the mRNA expression of various genes using real-time RT-PCR. Oct-3/4 expression was almost completely suppressed on day 14, suggesting that ES cells reached differentiated status in around 14 days. mRNA expression of E-cadherin, connexin 43, caveolin-1, and argininosuccinate synthetase was reproducibly suppressed during EB differentiation in 7−32% of ES cells in three separate experiments. Although these may not be "general stemness marker genes" such as Oct-3/ 4, they could play a role in readying stem cells for differentiation in response to deletion of signals from feeder cells. Next, we examined the effects of bisphenol A (BPA) on the mRNA expression of several differentiation marker genes for ES cells. That of PAX-6, an ectoderm marker, with 0, 0.1, and 10 μM BPA in 21-day EBs was 3,500%, 6,668%, and 8,394%, respectively, compared with ES cells. The difference between doses of 0 and 10 μM BPA in 21-day EBs was statistically significant (p=0.049). Pax-6 activation in the presence of BPA may interfere with the development of eyes, sensory organs, and certain neural and epidermal tissues usually derived from ectodermal tissues. Differentiation of EBs from cynomolgus monkey ES cells could be a useful model for detecting gene expression changes in response to chemical exposure.

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Monitoring Differentiation of Human Embryonic Stem Cells Using Real‐Time PCR

Cited by 63 publications

References 37 publications

Assessing Self-Renewal and Differentiation in Human Embryonic Stem Cell Lines

Assessing Self-Renewal and Differentiation in Human Embryonic Stem Cell Lines

The promoter of the oocyte-specific gene, Gdf9, is active in population of cultured mouse embryonic stem cells with an oocyte-like phenotype

Monitoring of Gene Expression in Differentiation of Embryoid Bodies From Cynomolgus Monkey Embryonic Stem Cells in the Presence of Bisphenol A

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