Stable Expression of hrGFP by Mouse Embryonic Stem Cells: Promoter Activity in the Undifferentiated State and During Dopaminergic Neural Differentiation

Zeng, Xianmin; Chen, Jia; Sanchez, Joseph F.; Coggiano, Mark A.; Dillon‐Carter, Ora; Petersen, Josiah; Freed, William J.

doi:10.1634/stemcells.21-6-647

Cited by 65 publications

(53 citation statements)

References 25 publications

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“…Expression of stem cell and neuronal markers was examined by immunocytochemistry, and staining procedures were as described previously [21]. Briefly, the ES cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100.…”

Section: Immunocytochemistrymentioning

confidence: 99%

Dopaminergic Differentiation of Human Embryonic Stem Cells

et al. 2004

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In this manuscript we report that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells. After 3 weeks of differentiation, approximately 87% of hES colonies contained tyrosine hydroxylase (TH)–positive cells, and a high percentage of the cells in most of the colonies expressed TH. Differentiation was inhibited by exposure to BMP4 or serum. TH‐positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for dopamine‐β‐hydroxylase, a marker of noradrenergic neurons. PA6‐induced neurons were able to release dopamine and 3,4‐dihydroxphe‐hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K+. When transplanted into 6‐hydroxydopamine–treated animals, hES‐derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH‐positive cells were present but in very small numbers compared with the high frequency of TH‐positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons.

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Section: Immunocytochemistrymentioning

confidence: 99%

Dopaminergic Differentiation of Human Embryonic Stem Cells

et al. 2004

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“…Immunocytochemistry and staining procedures were as described previously [32]. Briefly, hESCs grown either on MEF feeder cells or under feeder-free conditions and 7-day and 14-day EBs either attached or floated and were fixed with 2% paraformaldehyde for 30 minutes.…”

Section: Immunocytochemistrymentioning

confidence: 99%

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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“…(ii) the specifics of construct and driving promoters [36][37][38][39][40][41] , as well as (iii) the transfection method employed and the resulting integration sites [42][43][44][45] . Single-cell RT-PCR analysis as well as post hoc immunohistochemistry could be used to exclude subpopulation analysis and to demonstrate additional features of recorded cells.…”

Section: Problem Possible Reason Solutionmentioning

confidence: 99%