Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes

Kosanke, Maike; Osetek, Katarzyna; Haase, Alexandra; Wiehlmann, Lutz; Davenport, Colin; Schwarzer, Adrian; Adams, Felix F.; Kleppa, Marc-Jens; Schambach, Axel; Merkert, Sylvia; Wunderlich, Stephanie; Menke, Sandra; Dorda, Marie

doi:10.1016/j.ymthe.2021.04.007

Cited by 13 publications

(15 citation statements)

References 77 publications

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“…Establishment of iPSC lines is time-consuming and requires trained operators but once the PSC cells are obtained they are easy to grow, allowing safe production of many cell doses. [ 60 , 61 , 62 , 64 , 65 , 66 , 67 , 68 , 71 , 74 , 95 ] ESC-NSC Only confirmed in animal models. Turmorigenic risk due to potential PSC residues.…”

Section: Clinical Grade Nsc Therapies: Safety Efficacy Scalability and Quality Control Considerationsmentioning

confidence: 94%

“…There are multiple reports informing on the substantial differences between iPSC and ESC, including different gene expression signatures, residual retention of transcriptional and epigenetic memory of the somatic cell of origin, the existence of lab-specific gene expression differences, early senescence of iPSC progeny, and single-cell heterogeneity of iPSC (reviewed in [ 60 ]). Other logistic and safety issues with iPSC are the low reprogramming efficiency, tumorigenesis related to insertional mutagenesis, and to the fact that reprogramming enriches mutations in oncogenes [ 61 ], and the propensity of cell lines to give rise to derivatives from a specific germ layer [ 62 ]. Nevertheless, many of these reprogramming issues are more theoretical than practical and may not be relevant for iPSC-NSC clinical safety [ 13 ].…”

Section: Nsc Derived From Pluripotent Stem Cellsmentioning

confidence: 99%

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Human Neural Stem Cells for Cell-Based Medicinal Products

Fernández‐Muñoz

García-Delgado

Arribas-Arribas

et al. 2021

Cells

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Neural stem cells represent an attractive tool for the development of regenerative therapies and are being tested in clinical trials for several neurological disorders. Human neural stem cells can be isolated from the central nervous system or can be derived in vitro from pluripotent stem cells. Embryonic sources are ethically controversial and other sources are less well characterized and/or inefficient. Recently, isolation of NSC from the cerebrospinal fluid of patients with spina bifida and with intracerebroventricular hemorrhage has been reported. Direct reprogramming may become another alternative if genetic and phenotypic stability of the reprogrammed cells is ensured. Here, we discuss the advantages and disadvantages of available sources of neural stem cells for the production of cell-based therapies for clinical applications. We review available safety and efficacy clinical data and discuss scalability and quality control considerations for manufacturing clinical grade cell products for successful clinical application.

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Section: Clinical Grade Nsc Therapies: Safety Efficacy Scalability and Quality Control Considerationsmentioning

confidence: 94%

Section: Nsc Derived From Pluripotent Stem Cellsmentioning

confidence: 99%

Human Neural Stem Cells for Cell-Based Medicinal Products

Fernández‐Muñoz

García-Delgado

Arribas-Arribas

et al. 2021

Cells

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“…Furthermore, to investigate presence of a choice of variants at very low AF (≤0.02) in iPSC clones or assess their pre-existence in parental cell populations, a total of 128 variants at 38 different genetic regions were examined individually within their genetic context as described previously ( Kosanke et al., 2021 ) ( supplemental experimental procedures ). The mean error rate and detection limit were calculated individually for every variant, and the presence of variant was confirmed with p = 0.05 against background of errors ( Figure S5 D; Table S2 ).…”

Section: Methodsmentioning

confidence: 99%

“…In contrast to the nuclear genome, selection on mutated mtDNA molecules can act on an intercellular or intracellular level. On the intercellular level, genetically encoded inequalities in cell fitness of parental cells can lead to eliteness of cells to attain iPSC state and their dominance in the reprogramming niche ( Kosanke et al., 2021 ; Shakiba et al., 2019 ). However, high mutational burden or pathogenic mutations in specific mtDNA regions can also hinder reprogramming ( Floros et al., 2018 ; Hung et al., 2016 ; Kang et al., 2016 ; Latorre-Pellicer et al., 2016 ; Wahlestedt et al., 2014 ; Yokota et al., 2015 ).…”

Section: Introductionmentioning

confidence: 99%

iPSC culture expansion selects against putatively actionable mutations in the mitochondrial genome

et al. 2021

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Summary Therapeutic application of induced pluripotent stem cell (iPSC) derivatives requires comprehensive assessment of the integrity of their nuclear and mitochondrial DNA (mtDNA) to exclude oncogenic potential and functional deficits. It is unknown, to which extent mtDNA variants originate from their parental cells or from de novo mutagenesis, and whether dynamics in heteroplasmy levels are caused by inter- and intracellular selection or genetic drift. Sequencing of mtDNA of 26 iPSC clones did not reveal evidence for de novo mutagenesis, or for any selection processes during reprogramming or differentiation. Culture expansion, however, selected against putatively actionable mtDNA mutations. Altogether, our findings point toward a scenario in which intracellular selection of mtDNA variants during culture expansion shapes the mutational landscape of the mitochondrial genome. Our results suggest that intercellular selection and genetic drift exert minor impact and that the bottleneck effect in context of the mtDNA genetic pool might have been overestimated.

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“…Pluripotent stem cell (PSC) technologies come out of age and a number of clinical trials applying embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC)-based cell products are ongoing or in preparation. The tumorigenic potential of PSC-derived cell products, however, either in terms of contaminating undifferentiated cells that form teratoma or due to malignant transformation caused by mutations acquired and enriched during reprogramming 1 or culture expansion 2 is considered as a major safety concern.…”

Section: Introductionmentioning

confidence: 99%

Targeted biallelic integration of an inducible Caspase 9 suicide gene for safer cellular therapies prevents development of drug-resistant escapees in human iPSCs

Wunderlich

Haase

Merkert

et al. 2021

Preprint

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The teratoma forming potential of pluripotent stem cells (PSCs), and genetic aberrations that may lead to tumor formation from PSC derivatives, are considered as a major safety risk for cellular therapies. Introduction of inducible suicide genes as synthetic fail-safe systems has been proposed to minimize these risks. Recent research challenged the usefulness of such systems even for targeted introduction via accurate gene editing approaches. Apparently transgene silencing and elimination of a HTK suicide gene through 'loss-of-heterozygosity' (LoH) led to cell clones that escaped the induced suicide. We have introduced an inducible Caspase9 (iCasp-9) suicide gene into induced pluripotent stem cells (iPSCs), that has already been applied clinically in other settings. The iCASP9 gene coupled to a red nuclear GFP variant under control of the CAG promoter was inserted into the AAVS1 locus, either monoallelic or homozygous on both alleles. Efficient induction of apoptosis in vitro could be induced via treatment of iCASP9 iPSCs with two chemical inducers of dimerization (CID) at different concentrations for 24 hours. While NODSCID mice after transplantation of undifferentiated monoallelic iCASP9 iPSCs under the kidney capsule developed teratomas, CID treatment for three days led to rapid shrinking of such tumor structures. In individual mice, however, that received monoallelic iCASP9 iPSCs, tumor-like human tissue could be detected after CID treatment. Further in vitro experiments confirmed that in very rare subclones monoallelic iCASP9 hiPSCs lost transgene expression and can became resistant to CID induction in vitro with frequencies of ~ 3x10^-8. Analysis of CID-resistant subclones identified either elimination of the transgene, presumably via LoH, or via methylation of the CAG promoter as underlying mechanism. In contrast, we never observed any CID resistant escapees form biallelic iCASP9 iPSC clones, even after treatment of up to 0,5x10^9 iPSCs. This observation further argues for LoH as underlying mechanism of transgene elimination in monoallelic clones and suggests that CAG promoter methylation on both alleles represent independent events. In conclusion, biallelic integration of an iCASP9 safety switch in the AAVS1 locus allows for efficient induction of cellular suicide and may substantially increase the safety level of iPSC-based therapies. We propose that safety levels should be calculated by relating the observed frequencies of clonal escapees to clinically relevant cell numbers, i.e. cell number in tumors of a size that is readily detectable by modern imaging approaches.

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Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes

Cited by 13 publications

References 77 publications

Human Neural Stem Cells for Cell-Based Medicinal Products

Human Neural Stem Cells for Cell-Based Medicinal Products

iPSC culture expansion selects against putatively actionable mutations in the mitochondrial genome

Targeted biallelic integration of an inducible Caspase 9 suicide gene for safer cellular therapies prevents development of drug-resistant escapees in human iPSCs

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