Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research

Shibamiya, Aya; Schulze, E.; Krauß, Dana; Augustin, Christa; Reinsch, Marina; Schulze, Mirja L; Steuck, Simone; Mearini, Giulia; Mannhardt, Ingra; Schulze, Thomas G.; Klampe, Birgit; Werner, Tessa; Saleem, Umber; Knaust, Anika E.; Laufer, Sandra D.; Neuber, Christiane; Lemme, Marta; Behrens, Charlotta Sophie; Loos, Malte; Weinberger, Florian; Fuchs, Sigrid; Eschenhagen, Thomas; Hansen, Arne; Ulmer, Bärbel

doi:10.1002/cpsc.127

Cited by 14 publications

(14 citation statements)

References 51 publications

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“…Finally, improved viability by the use of boron has been described for mesenchymal stem cells [79]. Until there is a general agreement on the optimal method for stem cell or AML cell preservation, it will be important to include detailed descriptions of the methods for cryopreservation and thawing in future biobank studies, and only to include/compare samples that are prepared according to the same methodological protocols [80,81].…”

Section: Discussionmentioning

confidence: 99%

Proteomic Characterization of Spontaneous Stress-Induced In Vitro Apoptosis of Human Acute Myeloid Leukemia Cells; Focus on Patient Heterogeneity and Endoplasmic Reticulum Stress

Aasebø

Brenner

Hernandez-Valladares

et al. 2021

Hemato

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In vitro culture is widely used for characterization of primary human acute myeloid leukemia (AML) cells, but even when using optimized handling and culture conditions the AML cells show spontaneous in vitro apoptosis with a gradual decrease in cell viability during culture. The extent of this stress-induced apoptosis varies between patients, and a high degree of apoptosis is associated with high pre-culture BCL2 levels together with low levels of BAX and Heat Shock Proteins 30 and 90. We compared the global proteomic profiles during ongoing in vitro apoptosis for patients with high and low AML cell viability (i.e., less extensive versus extensive spontaneous apoptosis) after 48 h of culture. We identified 7902 proteins, but only 276 proteins differed significantly between patients with high (i.e., >25% viable cells; 192 upregulated and 84 downregulated peptides) and low viability after in vitro culture. Protein interaction network analysis based on these 276 protein identified three protein networks that included 18 proteins; most of these proteins were localized to the endoplasmic reticulum and several of them are involved in or are altered during the process of endoplasmic reticulum stress/unfolded protein stress response. To conclude, primary AML cells are heterogeneous with regard to degree of apoptosis in response to cellular stress, and this difference in regulation of apoptosis is associated with differences in the induction of and/or response to the unfolded protein stress response.

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Section: Discussionmentioning

confidence: 99%

Proteomic Characterization of Spontaneous Stress-Induced In Vitro Apoptosis of Human Acute Myeloid Leukemia Cells; Focus on Patient Heterogeneity and Endoplasmic Reticulum Stress

Aasebø

Brenner

Hernandez-Valladares

et al. 2021

Hemato

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“…The medium was triturated exactly twice (again with force: first top of the well, then bottom of the well) to loosen colonies from well surface. The exact volume of the calculated split ratio of the cell suspension described here does not, strictly speaking, yield a homogeneous batch of hiPSCs as not all hiPSCs of all wells were pooled and then equally redistributed before freezing (e.g., as described in Wagner and Welch, 2010;Liu and Chen, 2014;Shibamiya et al, 2020). However, in our opinion such a procedure is not feasible for all research laboratories, as often the infrastructure for such a large-scale freezing process is not available.…”

Section: Culture and Splittingmentioning

confidence: 99%

Academic application of Good Cell Culture Practice for induced pluripotent stem cells

Tigges

Bielec

Brockerhoff

et al. 2021

ALTEX

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entist's or even their own experiments, respectively (Baker and Penny, 2016;Miyakawa, 2020). Among the factors contributing to this reproducibility crisis are selective reporting, low statistical power, or poor analysis and experimental design (Baker and Penny, 2016). In addition, poor starting material -especially for hiPSC research -can be a severe source of irreproducibility (Stacey et al., 2013;Pamies et al., 2017). Therefore, already in 2013 "an urgent need" to establish routine screening methods for the characterization of quality-controlled stem cells was identified (Stacey et al., 2013;Crook et al., 2017).While there is guidance available for Good In Vitro Methods Practices in general (OECD, 2018) or stem cell-based Good Cell Culture Practice specifically (Pamies et al., 2017(Pamies et al., , 2018(Pamies et al., , 2020, giving detailed insights into the broad subject of quality assurance (QA) and quality control (QC) of in vitro (stem cell-based) methods, these leave the average academic researcher with a plethora of QC assays, discussing pros and cons that might or

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“…When working with hiPSCs it is important to consider possible limitations of the model such as unstable genome integrity, storage of hiPSC lines, immaturity and reproducibility when using hiPSC-CMs (for reviews, see 34,48,49 ). To comply with best cell culture practices we applied regular karyotyping, genotyping, and established master cell banks 50 of each hiPSC line, resulting in the validation of previous disease modeling data obtained in the ACTN2het line, 6 and the description of a novel disease feature in both mutant ACTN2 lines. Another limitation is that we cannot fully claim that ACTN2 aggregate formation is detrimental, contributing to disease progression, or rather beneficial, to avoid sarcomere incorporation of the mutant protein.…”

Section: Study Limitationsmentioning

confidence: 99%

ACTN2mutant causes proteopathy in human iPSC-derived cardiomyocytes

Zech

Prondzynski

Singh

et al. 2021

Preprint

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RationaleGenetic variants in ACTN2, encoding α-actinin 2 (ACTN2), are associated with several forms of (cardio)myopathy in the heterozygous state, and can cause progressive, severe cardiomyopathy in the homozygous state. We previously reported a heterozygous missense (c.740C>T) ACTN2 variant, associated with hypertrophic cardiomyopathy (HCM), which induced an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).ObjectiveTo evaluate the impact of heterozygosity and homozygosity of the c.740C>T ACTN2 variant on cardiomyocyte structure and function.Methods and ResultsNext to the previously established hiPSC line carrying the heterozygous ACTN2 variant (ACTN2het), we created a homozygous line (ACTN2hom) with CRISPR/Cas9. Differentiation into cardiomyocytes revealed myofibrillar disarray, increased cell area and volume, multinucleation, and ACTN2 aggregates in mutant hiPSC-CMs. Live cell imaging in hiPSC-CMs showed an inverse correlation between sarcomeric incorporation and aggregation of exogenously expressed mutant ACTN2. RNA-seq and proteomic analyses showed alteration of several canonical pathways involved in metabolism, oxidative and cellular stress, and proteostasis, including the ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) in mutant hiPSC-CMs. Detailed evaluation of the UPS and ALP, including a high-content screening with a mWasabi-mTagRFP-hLC3 tandem construct, revealed a global activation of these systems in mutant hiPSC-CMs. The ACTN2 missense variant caused allele dose-dependent and -independent effects on several cardiomyocyte features. ACTN2hom exhibited markedly lower levels of sarcomere-associated proteins, and hypocontractility in engineered heart tissues, resembling a dilated cardiomyopathy phenotype.ConclusionThis study showed allele dose-independent effects of the ACTN2 variant on multinucleation, ACTN2 aggregation, and UPS activation, and allele dose-dependent effects of the variant on cellular hypertrophy, myofibrillar disarray, and ALP activation. Activation of the proteolytic systems is likely to cope with ACTN2 aggregation. Our data indicates proteopathy as an additional cellular feature caused by the c.740C>T ACTN2 variant, which may contribute to human ACTN2-associated inherited cardiomyopathy.

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Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research

Cited by 14 publications

References 51 publications

Proteomic Characterization of Spontaneous Stress-Induced In Vitro Apoptosis of Human Acute Myeloid Leukemia Cells; Focus on Patient Heterogeneity and Endoplasmic Reticulum Stress

Proteomic Characterization of Spontaneous Stress-Induced In Vitro Apoptosis of Human Acute Myeloid Leukemia Cells; Focus on Patient Heterogeneity and Endoplasmic Reticulum Stress

Academic application of Good Cell Culture Practice for induced pluripotent stem cells

ACTN2mutant causes proteopathy in human iPSC-derived cardiomyocytes

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