Single-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media

Daniszewski, Maciej; Nguyen, Quan; Chy, Hun S.; Singh, Vikrant; Crombie, Duncan E.; Kulkarni, Tejal; Liang, Helena; Sivakumaran, Priyadharshini; Lidgerwood, Grace E.; Hernández, Damián; Conquest, Alison; Rooney, Louise; Chevalier, Sophie; Andersen, Stacey; Senabouth, Anne; Vickers, James C.; Mackey, David A.; Craig, Jamie E; Laslett, Andrew L.; Hewitt, Alex W.; Powell, Joseph E.; Pébay, Alice

doi:10.1016/j.isci.2018.08.016

Cited by 33 publications

(39 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Further evidence has also demonstrated that OCT3/4 downregulates the downstream gene expression of NANOG, SPP1/ OPN, SOX2, FBXO15, OTX2, and ZFP42/REX1 (Matoba, Niwa et al, 2006). The expression levels of NANOG, ZFP42 and c-MYC in our study were approximately 50 times lower than that of OCT3/4, which is consistent with previous results from sc-RNA-seq iPSC dataset (Daniszewski, Nguyen et al, 2018, Nguyen, Lukowski et al, 2018. Interestingly, NCRM1 and KYOU-DXR0109B, two commercial control lines with high passage number (>30 passages), express relatively high level of NANOG, SOX2 and ZFP42 and reduced levels of OCT3/4, suggesting that extended passaging enhances pluripotency gene expression (Koehler, Tropel et al, 2011).…”

Section: Discussionsupporting

confidence: 92%

Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons

Chen

Abdian

Maussion

et al. 2021

Preprint

View full text Add to dashboard Cite

Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a four-step workflow to assess our newly generated iPSCs for variations and reproducibility relative to each other and iPSCs obtained from external sources. Our benchmarks for evaluating iPSCs include examining iPSC morphology and proliferation in two different media conditions (mTeSR1 and Essential 8) and evaluating their ability to differentiate into each of the three germ layers, with a particular focus on neurons. Genomic stability in the human iPSCs was analyzed by G-band karyotyping and a qPCR-based stability test, and cell-line identity authenticated by Short Tandem Repeat (STR) analysis. Using standardized dual SMAD inhibition methods, all iPSC lines gave rise to neural progenitors that could subsequently be differentiated into cortical neurons. Neural differentiation was analyzed qualitatively by immunocytochemistry and quantitatively by q-PCR for progenitor, neuronal, cortical and glial markers. Taken together, we present a standardized quality control workflow to evaluate variability and reproducibility across and between iPSCs.HighlightsValidation of culture conditions is critical in the expansion and maintenance of an iPSC line.Characterization of pluripotency and genomic stability ensures each line is free of defects at the DNA level, while maintaining its ability to be directed into any of the three germ layers.Forebrain cortical neurons can be generated from all iPSC line tested; however, the morphology and expression pattern of these neurons can vary from line to line.

show abstract

Section: Discussionsupporting

confidence: 92%

Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons

Chen

Abdian

Maussion

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Other studies also report on pluripotency levels of hPSCs in both feeder and feeder-free conditions and observed similar variations in these markers (Carpenter et al 2004;Ghasemi-Dehkordi et al 2015). StemFlex medium has been previously established as an efficient medium for routine culture, comparable to alternative media such as TeSR-E8, in which cells maintain expression of pluripotency markers and characteristic morphology (Daniszewski et al 2018).…”

Section: E8 Direct Protocolmentioning

confidence: 65%

Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells

Aalders

Vreken

Popovic

et al. 2019

In Vitro Cell.Dev.Biol.-Animal

View full text Add to dashboard Cite

Human pluripotent stem cells (hPSCs) are conventionally maintained on mouse embryonic fibroblast (MEF) feeder layers. However, downstream applications, such as directed differentiation protocols, are primarily optimized for feeder-free cultures. Therefore, hPSCs must often be adapted to feeder-free conditions. Here we propose a novel feeder-free adaptation protocol using StemFlex medium, which can be directly applied to thawed hPSC lines. The direct feeder-free adaptation protocol using StemFlex culture medium on Geltrex coating led to robust hPSC cultures in approximately 2 weeks. This approach was tested with three human embryonic stem cell (hESC) lines. All lines were confirmed to be pluripotent, expressing POU5F1, SOX2, and NANOG. No chromosomal imbalances were induced by the feeder-free adaptation. StemFlex medium enabled the efficient adaptation of hPSCs to feeder-free conditions directly after thawing. This protocol is easy to implement in laboratories that perform feeder-free cultures, allowing more convenient adaptation and more robust expansion of cryopreserved hPSCs, even in cases when sample quality is low or unknown.

show abstract

“…This work provides a benchmark for high capacity sequencing platforms applied to highthroughput single cell RNA-seq libraries. iPSC -Consisted of undifferentiated human induced pluripotent stem cells (iPSCs) maintained with StemFlex (ThermoFisher Scientific) that were derived from two unrelated individuals (14).…”

Section: Discussionmentioning

confidence: 99%

“…Additional analyses were conducted on the iPSC and TMWC samples to evaluate the influence of sequencing platform on properties specific to these experiments. Using genotype 310 information from that was generated as described in (14), SNPs were called from the iPSC sample using demuxlet (12). To account for the downsampling of read depth in the MGISEQ-2000 data, only alignments from UMIs detected in the downsampled data were used.…”

Section: Bioinformatic and Computational Analysismentioning

confidence: 99%

Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA-sequencing

Daniszewski

Anderson

Shi

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

AbstractThe libraries generated by high-throughput single cell RNA-sequencing platforms such as the Chromium from 10X Genomics require considerable amounts of sequencing, typically due to the large number of cells. The ability to use this data to address biological questions is directly impacted by the quality of the sequence data. Here we have compared the performance of the Illumina NextSeq 500 and NovaSeq 6000 against the BGI MGISEQ-2000 platform using identical Single Cell 3’ libraries consisting of over 70,000 cells. Our results demonstrate a highly comparable performance between the NovaSeq 6000 and MGISEQ-2000 in sequencing quality, and cell, UMI, and gene detection. However, compared with the NextSeq 500, the MGISEQ-2000 platform performs consistently better, identifying more cells, genes, and UMIs at equalised read depth. We were able to call an additional 1,065,659 SNPs from sequence data generated by the BGI platform, enabling an additional 14% of cells to be assigned to the correct donor from a multiplexed library. However, both the NextSeq 500 and MGISEQ-2000 detected similar frequencies of gRNAs from a pooled CRISPR single cell screen. Our study provides a benchmark for high capacity sequencing platforms applied to high-throughput single cell RNA-seq libraries.

show abstract

Single-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media

Cited by 33 publications

References 45 publications

Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons

Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons

Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells

Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA-sequencing

Contact Info

Product

Resources

About