Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing

Bao, Xiaoping; Adil, Maroof M.; Muckom, Riya; Zimmermann, J.; Tran, Aurelie; Suhy, Natalie; Xu, Yibo; Sampayo, Rocío; Clark, Douglas S.; Schaffer, David V.

doi:10.1002/stem.3096

Cited by 15 publications

(12 citation statements)

References 46 publications

(61 reference statements)

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“…Obtaining stable transgene expression in hPSCs can be challenging and the promoters used are of great importance due to the high likelihood of transgene silencing in hPSCs (Z. W. Du, Hu, Ayala, Sauer, & Zhang, 2009). To generate a stable hPSC‐FUCCI reporter line, we employed the CRISPR/Cas9‐mediated homology‐directed repair (HDR) and knocked the all‐in‐one FUCCI construct CAG‐Clover‐Geminin‐IRES‐mKO2‐Cdt1 (Figure 1a) into the AAVS1 safe harbor locus, a well‐known site for stable transgene expression (Bao et al, 2019; Smith et al, 2008). After puromycin selection for about 2 weeks, we isolated two Clover fluorescent protein‐expressing single cell‐derived hPSC clones and confirmed transgene presence by PCR genotyping (Figure 1b).…”

Section: Resultsmentioning

confidence: 99%

“…The donor plasmids targeting AAVS1 locus were constructed as previously described (Bao et al, 2019). Briefly, to generate the CAG‐FUCCI plasmid, the Clover‐Geminin (1‐110)‐IRES‐mKO2‐Cdt (30‐120) fragment was amplified from Addgene plasmid #83841 and then cloned into the AAVS1‐Pur‐CAG‐EGFP donor plasmid (Addgene; #80945), replacing the EGFP.…”

Section: Methodsmentioning

confidence: 99%

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Fluorescent indicators for continuous and lineage‐specific reporting of cell‐cycle phases in human pluripotent stem cells

Chang

Hellwarth

Randolph

et al. 2020

Biotech & Bioengineering

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Proper cell‐cycle progression is essential for the self‐renewal and differentiation of human pluripotent stem cells (hPSCs). The fluorescent ubiquitination‐based cell‐cycle indicator (FUCCI) has allowed the dual‐color visualization of the G1 and S/G2/M phases in various dynamic models, but its application in hPSCs is not widely reported. In addition, lineage‐specific FUCCI reporters have not yet been developed to analyze complex tissue‐specific cell‐cycle progression during hPSC differentiation. Desiring a robust tool for spatiotemporal reporting of cell‐cycle events in hPSCs, we employed the CRISPR/Cas9 genome editing tool and successfully knocked the FUCCI reporter into the AAVS1 safe harbor locus of hPSCs for stable and constitutive FUCCI expression, exhibiting reliable cell‐cycle‐dependent fluorescence in both hPSCs and their differentiated progeny. We also established a cardiac‐specific TNNT2‐FUCCI reporter for lineage‐specific cell‐cycle monitoring of cardiomyocyte differentiation from hPSCs. This powerful and modular FUCCI system should provide numerous opportunities for studying human cell‐cycle activity, and enable the identification and investigation of novel regulators for adult tissue regeneration.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Fluorescent indicators for continuous and lineage‐specific reporting of cell‐cycle phases in human pluripotent stem cells

Chang

Hellwarth

Randolph

et al. 2020

Biotech & Bioengineering

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“…unlike immortalized cells) and preserve the phenotype of somatic cells. The most efficient method for generating iPSC is based on retrovirus- or transposon-mediated gene transfer (Cre-loxP or PiggyBac transposition) [ 110 ]. Currently, advances in iPSC technologies have reduced phenotypic variations among iPSC clones and enabled derivation of cells from somatic cells or postmortem tissue [ 111 ].…”

Section: Overview Of Bbb In Vitro Modelsmentioning

confidence: 99%

Modeling blood–brain barrier pathology in cerebrovascular disease in vitro: current and future paradigms

Andjelkovic

Stamatovic

Phillips

et al. 2020

Fluids Barriers CNS

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The complexity of the blood-brain barrier (BBB) and neurovascular unit (NVU) was and still is a challenge to bridge. A highly selective, restrictive and dynamic barrier, formed at the interface of blood and brain, the BBB is a "gatekeeper" and guardian of brain homeostasis and it also acts as a "sensor" of pathological events in blood and brain. The majority of brain and cerebrovascular pathologies are associated with BBB dysfunction, where changes at the BBB can lead to or support disease development. Thus, an ultimate goal of BBB research is to develop competent and highly translational models to understand mechanisms of BBB/NVU pathology and enable discovery and development of therapeutic strategies to improve vascular health and for the efficient delivery of drugs. This review article focuses on the progress being made to model BBB injury in cerebrovascular diseases in vitro.

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“…Observed differences in pluripotency have yet to be related to cellular mechanisms that determine differentiation potential and can be adjusted in cell culture. In addition, assessment of cell differentiation state and population purity mostly relies on the introduction of reporter genes, specific for a required differentiation or maturation state [10],[11],[12].…”

Section: Introductionmentioning

confidence: 99%

Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells

Wesseling-Rozendaal¹,

Holtzer²,

Verhaegh³

et al. 2021

Preprint

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Stem cell research is emerging both as a scientifically and clinically relevant area. One of the current challenges in stem cell research and regenerative medicine is assessment of the pluripotency state of induced pluripotent stem (iPS) cells. Once a stem cell differentiation process is initiated the challenge is how to assess the state of differentiation, and the purity of the differentiated cell population. Stem cell potency and differentiation states are determined by tightly coordinated activity of developmental signaling pathways, such as the Notch, Hedgehog, TGFβ, Wnt, PI3K, MAPK-AP1, and NFκB pathways. Source of the stem cells and culture protocols may influence stem cell phenotype, with potential consequences for pluripotency and in general for experimental reproducibility. Human pluripotent embryonic (hES) and iPS stem cell lines under different culture conditions, organ derived multipotent stem cells, and differentiated cell types, were phenotyped with respect to functional activity of developmental signaling pathways. Methods: We previously reported on the development and validation of a novel assay platform for quantitative measurement of activity of multiple signal transduction pathways (STP) simultaneously in a single sample, based on interpreting a preselected set of target mRNA expression levels. Assays were used to calculate Notch, Hedgehog, TGFβ, Wnt, PI3K, MAPK-AP1, and NFκB signal transduction pathway activity scores for individual cell samples, using publicly available Affymetrix expression microarray data. Results: Culture conditions (e.g. mouse versus human feeder) influenced pluripotent stem cell pathway activity profiles. hES and iPS stem cell lines cultured in the same lab under similar conditions showed minimal variation in pathway activity profile despite different genetic backgrounds, while across different labs larger variations were measured, even for the same stem cell line. Pathway activity scores for PI3K, MAPK, Hedgehog, Notch, TGFβ, and NFκB pathways rapidly decreased upon pluripotent stem cell differentiation, while increasing for the Wnt pathway. Further differentiation to intestinal progenitor cells resulted in higher PI3K, Wnt and Notch pathway activity. In multipotent intestinal crypt stem cells obtained from intestinal mucosa samples, similar Notch and even higher Wnt pathway activity were measured, which disappeared upon differentiation to mucosal cells. Conclusion: Results support the validity of using these STP assays for quantitative phenotyping of stem cells and differentiated derivatives, and enabled definition of a pluripotency profile with high PI3K, MAPK, Hedgehog, TGFβ, Notch, and NFκB, and low Wnt pathway activity scores. Measurement of combined signaling pathway activity scores is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture and differentiation. It enables controlled manipulation of signaling pathway activity using pathway targeting compounds. An envisioned additional utility may lie in quality control for regenerative medicine purposes.

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Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing

Cited by 15 publications

References 46 publications

Fluorescent indicators for continuous and lineage‐specific reporting of cell‐cycle phases in human pluripotent stem cells

Fluorescent indicators for continuous and lineage‐specific reporting of cell‐cycle phases in human pluripotent stem cells

Modeling blood–brain barrier pathology in cerebrovascular disease in vitro: current and future paradigms

Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells

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