Genotoxic Effects of Culture Media on Human Pluripotent Stem Cells

Bangalore, Megha Prakash; Adhikarla, Syama; Mukherjee, Odity; Panicker, Mitradas M.

doi:10.1038/srep42222

Cited by 15 publications

(7 citation statements)

References 55 publications

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“…Therefore, the cultivation of experimental mouse teratomas for 7 days yielded proliferating teratomas with cells that retained pluripotency typical for ESCs derived from the inner cell mass, and GCNIS precursors of TGCTs that are thought to be germ cells of impaired differentiation [15,16,51]. The high concentration of the ROS marker 8-OHdG found in teratomas could be due to the very nature of in vitro cultivation, as it may have genotoxic [52] or even epigenetically regulated developmental consequences [53], which should be investigated further.…”

Section: Histopathological and Molecular Analysis Of The Experimental Teratoma In Vitro Systemmentioning

confidence: 99%

Teratoma Growth Retardation by HDACi Treatment of the Tumor Embryonal Source

Krasić

Škara

Ulamec

et al. 2020

Cancers

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Among testicular germ cell tumors, teratomas may often be very aggressive and therapy-resistant. Our aim was to investigate the impact of histone deacetylase inhibitors (HDACi) on the in vitro growth of experimental mouse teratoma by treating their embryonic source, the embryo-proper, composed only of the three germ layers. The growth of teratomas was measured for seven days, and histopathological analysis, IHC/morphometry quantification, gene enrichment analysis, and qPCR analysis on a selected panel of pluripotency and early differentiation genes followed. For the first time, within teratomas, we histopathologically assessed the undifferentiated component containing cancer stem cell-like cells (CSCLCs) and differentiated components containing numerous lymphocytes. Mitotic indices were higher than apoptotic indices in both components. Both HDACi treatments of the embryos-proper significantly reduced teratoma growth, although this could be related neither to apoptosis nor proliferation. Trichostatin A increased the amount of CSCLCs, and upregulated the mRNA expression of pluripotency/stemness genes as well as differentiation genes, e.g., T and Eomes. Valproate decreased the amount of CSCLCs, and downregulated the expressions of pluripotency/stemness and differentiation genes. In conclusion, both HDACi treatments diminished the inherent tumorigenic growth potential of the tumor embryonal source, although Trichostatin A did not diminish the potentially dangerous expression of cancer-related genes and the amount of CSCLC.

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Section: Histopathological and Molecular Analysis Of The Experimental Teratoma In Vitro Systemmentioning

confidence: 99%

Teratoma Growth Retardation by HDACi Treatment of the Tumor Embryonal Source

Krasić

Škara

Ulamec

et al. 2020

Cancers

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“…Indeed, whereas iPSCs in our study were feeder-free and cultured in Essential 8 (E8) media, those in the abovementioned study [ 14 ] were cultured on mouse embryonic fibroblasts (MEFs) in 20% Knockout Serum Replacement (KSR) media. Direct comparison of media choice (E8 vs. KSR) revealed that hPSCs cultured in E8 possessed nuclear/nucleolar differences, elevated ROS and DNA damage, and higher mitochondrial membrane potential when compared to their KSR-cultured counterparts [ 28 ]. Moreover, it is possible that the absence of MEF-secreted supportive factors in feeder-free cultures might render iPSCs more susceptible to toxicity.…”

Section: Discussionmentioning

confidence: 99%

A Non-Toxic Concentration of Telomerase Inhibitor BIBR1532 Fails to Reduce TERT Expression in a Feeder-Free Induced Pluripotent Stem Cell Model of Human Motor Neurogenesis

Pandya

Crerar

Mitchell

et al. 2021

IJMS

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Several studies have shown that human induced pluripotent stem cell (iPSC)-derivatives are essentially fetal in terms of their maturational status. Inducing ageing in iPSC-motor neuron (MN) models of amyotrophic lateral sclerosis (ALS) has the potential to capture pathology with higher fidelity and consequently improve translational success. We show here that the telomerase inhibitor BIBR1532, hypothesised to recapitulate the telomere attrition hallmark of ageing in iPSC-MNs, was in fact cytotoxic to feeder-free iPSCs when used at doses previously shown to be effective in iPSCs grown on a layer of mouse embryonic fibroblasts. Toxicity in feeder-free cultures was not rescued by co-treatment with Rho Kinase (ROCK) inhibitor (Y-27632). Moreover, the highest concentration of BIBR1532 compatible with continued iPSC culture proved insufficient to induce detectable telomerase inhibition. Our data suggest that direct toxicity by BIBR1532 is the most likely cause of iPSC death observed, and that culture methods may influence enhanced toxicity. Therefore, recapitulation of ageing hallmarks in iPSC-MNs, which might reveal novel and relevant human disease targets in ALS, is not achievable in feeder-free culture through the use of this small molecule telomerase inhibitor.

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“…Lentiviruses were collected and HFs were infected 24 and 48 h after transfection. A day after the last transduction round, HFs were seeded over a feeder layer of mitotically inactivated SNL cells in gelatin pre-coated wells (Thermo Fisher Scientific, Waltham, MA, United States) and medium was changed to iPSC medium composed of F12 medium (Sigma-Aldrich, St. Louis, MO, United States) supplemented with 10% Knockout serum replacement (KSR, Gibco Laboratories, Gaithersburg, MD, United States) to avoid any genotoxic effect ( Rodríguez-Pizà et al, 2010 ; Prakash Bangalore et al, 2017 ), 5% no essential amino acids (NEAA, Gibco Laboratories, Gaithersburg, MD, United States) 1% 2-Mercaptoethanol, 5% penicillin/streptomycin and bFGF (10 ng/ml). Medium was changed every day until small iPSC-like colonies appeared.…”

Section: Methodsmentioning

confidence: 99%

A Human Gonadal Cell Model From Induced Pluripotent Stem Cells

2018

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Sertoli cells are main players in the male gonads development and their study may shed light on 46,XY disorders of sex development (DSD). Mature primary Sertoli cells are incapable of proliferating in prolonged in vitro cultures and the available Sertoli cell models have several limitations since they derive from mouse or human cancer tissues. We differentiated human fibroblasts (HFs)-derived induced pluripotent stem cells into Sertoli-like cells (SLC) and, in order to characterize this new Sertoli cell model, we performed gene expression analyses by NextGeneration Sequencing techniques. This approach revealed that our putative SLC have reduced expression of pluripotency markers and expressed Sertoli cell markers such as SRY-Related HMG-Box 9 (SOX9), vimentin (VIM), and claudin-11 (CLDN-11). More in detail, the transcriptional profile analysis suggested that these cells are in an early stage of Sertoli cells maturation. Harnessing the power of induced pluripotent stem cells, we were able to generate SLC that show genetic and functional similarities to human Sertoli cells (HSerCs). SLC could become an excellent source of patient-specific Sertoli cells that could be of paramount benefit for both basic research and personalized medicine in sex development and reproductive medicine.

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Genotoxic Effects of Culture Media on Human Pluripotent Stem Cells

Cited by 15 publications

References 55 publications

Teratoma Growth Retardation by HDACi Treatment of the Tumor Embryonal Source

Teratoma Growth Retardation by HDACi Treatment of the Tumor Embryonal Source

A Non-Toxic Concentration of Telomerase Inhibitor BIBR1532 Fails to Reduce TERT Expression in a Feeder-Free Induced Pluripotent Stem Cell Model of Human Motor Neurogenesis

A Human Gonadal Cell Model From Induced Pluripotent Stem Cells

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