A Panel of Tests to Standardize the Characterization of Human Embryonic Stem Cells

Pal, Rajarshi; Mandal, Arundhati; Rao, Harinarayana S.; Rao, Mahendra S.; Khanna, Aparna

doi:10.2217/17460751.2.2.179

Cited by 11 publications

(5 citation statements)

References 38 publications

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“…Combining the profiling of microRNAs and mRNAs, correlation of gene and microRNA expression predicts regulatory interaction of microRNA and mRNAs involved in both maintenance of pluripotency and differentiation [145]. Based on this, microRNA expression pattern in human embryonic stem cells is included in the panel of tests suggested for qualification of human embryonic stem cells [146]. Consistent with the observation for individual microRNAs, mouse embryonic stem (ES) knockout cells lacking Dicer [25], required for cleavage of microRNA precursors, or DGCR8 [37], an RNA-binding protein essential for processing microRNAs, exhibit a failure to undergo differentiation and completely prevent mouse ESC from differentiating into embryoid bodies, suggesting that microRNAs are required to inhibit ES self-renewal.…”

Section: Micrornas In Embryonic and Adult Stem Cellsmentioning

confidence: 99%

Concise Review: MicroRNA Expression in Multipotent Mesenchymal Stromal Cells

Lakshmipathy¹,

Hart

2007

Stem Cells

118

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Mesenchymal stem cells, or multipotent mesenchymal stromal cells (MSC), isolated from various adult tissue sources have the capacities to self-renew and to differentiate into multiple lineages. Both of these processes are tightly regulated by genetic and epigenetic mechanisms. Emerging evidence indicates that the class of single-stranded noncoding RNAs known as microRNAs also plays a critical role in this process. First described in nematodes and plants, microRNAs have been shown to modulate major regulatory mechanisms in eukaryotic cells involved in a broad array of cellular functions. Studies with various types of embryonic as well as adult stem cells indicate an intricate network of microRNAs regulating key transcription factors and other genes, which in turn determine cell fate. In addition, expression of unique microRNAs in specific cell types serves as a useful diagnostic marker to define a particular cell type. MicroRNAs are also found to be regulated by extracellular signaling pathways that are important for differentiation into specific tissues, suggesting that they play a role in specifying tissue identity. In this review, we describe the importance of microRNAs in stem cells, focusing on our current understanding of microRNAs in MSC and their derivatives.

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Section: Micrornas In Embryonic and Adult Stem Cellsmentioning

confidence: 99%

Concise Review: MicroRNA Expression in Multipotent Mesenchymal Stromal Cells

Lakshmipathy¹,

Hart

2007

Stem Cells

118

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“…The expression of OCT4 is commonly used as a measure of ESC pluripotency [11,12], while CDX2 is a marker for trophoblast stem cells (TSC) [13]. NANOG is a second ICM-specific transcription factor identified in ESC [14,15].…”

Section: Introductionmentioning

confidence: 99%

Inner Cell Mass Localization of NANOG Precedes OCT3/4 in Rhesus Monkey Blastocysts

Harvey

Armant

Bavister

et al. 2009

Stem Cells and Development

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The mechanism by which the inner cell mass (ICM) and trophectoderm (TE) become specified is poorly understood. Considerable species variation is evident in the expression of lineage-specific and embryonic stem cell (ESC) regulatory markers. We sought to investigate localization patterns of these markers in rhesus macaque compact morulae and blastocysts. NANOG protein was restricted to the ICM of blastocysts. In contrast to a previous report, the expression of CDX2 was detected in the primate blastocyst, localized specifically to the TE. Unlike the mouse embryo, OCT4 protein was detected using two different antibodies in both the ICM and TE. The ubiquitous pattern of OCT4 expression is consistent with observations in human, cow, and pig embryos. Significantly, lack of restricted OCT4 protein, and ICM localization of NANOG in primate blastocysts, suggests that NANOG may determine inner cell mass fate more specifically during primate development or may be less susceptible to culture artifacts. These results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of NANOG to constitute a complex interactive network. This difference may also underlie observations that regulatory mechanisms in ESC differ between mice and primates.

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“…In our earlier report, we demonstrated overexpression of multiple genes in an undifferentiated and differentiated population of hESCs by uniplex PCR, q-PCR, and a microarray technique. 20,21 The present study refines the set of stemness genes to identify those that are rapidly and unambiguously downregulated as ESC undergoes lineage commitment. The selected genes represent a battery of biomarkers to distinguish differentiated cells from pluripotent ESCs.…”

Section: Discussionmentioning

confidence: 81%

“…HUES-7 and 9 were grown routinely on mitomycin C–inactivated (10 µg/mL; Sigma, St. Louis, MO) MEF feeder layers as described in our previous report. 21 Manual passaging was preferred over the enzymatic method to guarantee the best quality of hESCs for downstream characterization. Briefly, manual passaging was performed by mechanical dissociation of undifferentiated hESC colonies into small clumps of about 100 to 200 cells using the sharp edge of a flame-pulled Pasteur pipette under the stereomicroscope (Olympus; SZX16).…”

Section: Methodsmentioning

confidence: 99%

“…Earlier we had proposed a characterization scheme of hESC, including RT-PCR, immunochemistry, karyotype, human leukocyte antigen (HLA) and short tandem repeat (STR) analyses, telomerase assay, teratoma formation in severe combined immunodeficient (SCID) mice, real-time PCR, and focused cDNA microarray, miRNA, and mitochondrial DNA analysis. 20,21 The objective of the present study was to characterize hESCs on a molecular level and to determine the quality and degree of variability among pluripotent cells and their early progenies (EB). Here, we illustrate an approach, based on single-reaction multiplex RT-PCR, for semiquantitative evaluation of hESC proliferation during ex vivo expansion.…”

Section: Introductionmentioning

confidence: 99%

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Application of Multiplex PCR for Characterization of Human Embryonic Stem Cells (hESCs) and Its Differentiated Progenies

Mamidi¹,

Pal

Bhonde³

et al. 2010

SLAS Discovery

Self Cite

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Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.

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A Panel of Tests to Standardize the Characterization of Human Embryonic Stem Cells

Cited by 11 publications

References 38 publications

Concise Review: MicroRNA Expression in Multipotent Mesenchymal Stromal Cells

Concise Review: MicroRNA Expression in Multipotent Mesenchymal Stromal Cells

Inner Cell Mass Localization of NANOG Precedes OCT3/4 in Rhesus Monkey Blastocysts

Application of Multiplex PCR for Characterization of Human Embryonic Stem Cells (hESCs) and Its Differentiated Progenies

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