In vitro methods to ensure absence of residual undifferentiated human induced pluripotent stem cells intermingled in induced nephron progenitor cells

Tsujimoto, Hiraku; Katagiri, Naoko; Ijiri, Yoshihiro; Sasaki, Ben; Kobayashi, Yoshifumi; Mima, Akira; Ryosaka, Makoto; Furuyama, Kenichiro; Kawaguchi, Yoshiya; Osafune, Kenji

doi:10.1371/journal.pone.0275600

Cited by 7 publications

(3 citation statements)

References 63 publications

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“…Importantly, we did not detect Oct4 + cells in hiPSC-derived subpopulations and detected only low residual expression of mesodermal/endodermal markers or genes involved in hiPSC/ESC pluripotency (e.g. CNMD, DPPA4 and L1TD1) [121][122][123] (Fig. 5D, Suppl.…”

Section: Hipsc-nscs and Hfnscs Include Rg At Different Stages Of Matu...mentioning

confidence: 81%

Human iPSC-derived neural stem cells display a radial glia-like signaturein vitroand favorable long-term safety in transplanted mice

Luciani

Garsia

Beretta

et al. 2023

Preprint

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Human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NSCs) are a promising source for cell therapy approaches to treat neurodegenerative and demyelinating disorders. Despite ongoing efforts to characterize hiPSC-derived cells in vitro and in vivo, we lack comprehensive genome- and transcriptome-wide studies addressing hiPSC-NSC identity and safety, which are critical for establishing accepted criteria for prospective clinical applications. Here, we evaluated the transcriptional and epigenetic signatures of hiPSCs and differentiated hiPSC-NSC progeny, finding that the hiPSC-to-NSC transition results in a complete loss of pluripotency and the acquisition of a radial glia-associated transcriptional signature. Importantly, hiPSC-NSCs share with somatic human fetal NSCs (hfNSCs) the main transcriptional and epigenetic patterns associated with NSC-specific biology. In vivo, long-term observation (up to 10 months) of mice intracerebrally transplanted as neonates with hiPSC-NSCs showed robust engraftment and widespread distribution of human cells in the host brain parenchyma. Engrafted hiPSC-NSCs displayed multilineage potential and preferentially generated glial cells. No hyperproliferation, tumor formation, or expression of pluripotency markers was observed. Finally, we identified a novel role of the Sterol Regulatory Element Binding Transcription Factor 1 (SREBF1) in the regulation of astroglial commitment of hiPSC-NSCs. Overall, these comprehensive in vitro and in vivo analyses provide transcriptional and epigenetic reference datasets to define the maturation stage of NSCs derived from different hiPSC sources, and to clarify the safety profile of hiPSC-NSCs, supporting their continuing development as an alternative to somatic hfNSCs in treating neurodegenerative and demyelinating disorders.

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Section: Hipsc-nscs and Hfnscs Include Rg At Different Stages Of Matu...mentioning

confidence: 81%

Human iPSC-derived neural stem cells display a radial glia-like signaturein vitroand favorable long-term safety in transplanted mice

Luciani

Garsia

Beretta

et al. 2023

Preprint

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“…Processing and differentially expressed gene (DEG) analysis were performed as previously described. ( 28 , 29 ) In brief, the sequenced reads were mapped to the human reference genome GRCh38 v105 using STAR 2.5.1b ( 30 ) with GENCODE v39 gene annotations, and the expected gene counts were calculated and normalized with transcripts per kilobase million (TPM) using RSEM 1.2.23. ( 31 ) DEG analysis was performed using DESeq2 1.34.0 (Bioconductor) as previously described, ( 32 ) and commonly up‐ or downregulated genes among homozygous knockout clones were visualized with Venn diagrams and tables.…”

Section: Methodsmentioning

confidence: 99%

Collagen X Is Dispensable for Hypertrophic Differentiation and Endochondral Ossification of Human iPSC‐Derived Chondrocytes

et al. 2023

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Collagen X is a non‐fibril collagen produced by hypertrophic chondrocytes and was believed to associate with the calcification process of growth plate cartilage. The homozygous loss of Col10a1 gene in mice, however, demonstrated no remarkable effects on growth plate formation or skeletal development. To investigate the role of collagen X in human chondrocytes, we established human induced pluripotent stem cells (hiPSCs) with heterozygous (COL10A1+/−) or homozygous (COL10A1−/−) deletions of COL10A1 gene using the dual sgRNA CRISPR/Cas9 system. Several mutant clones were established and differentiated into hypertrophic chondrocytes by a previously reported 3D induction method. No remarkable differences were observed during the differentiation process between parental and mutant cell lines, which differentiated into cells with features of hypertrophic chondrocytes, indicating that collagen X is dispensable for the hypertrophic differentiation of human chondrocytes in vitro. To investigate the effects of collagen X deficiency in vivo, chondrocyte pellets at the proliferating or prehypertrophic stage were transplanted into immunodeficient mice. Proliferating pellet‐derived tissues demonstrated the zonal distribution of chondrocytes with the transition to bone tissues mimicking growth plates, and the proportion of bone tended to be larger in COL10A1−/− tissues. Prehypertrophic pellet‐derived tissues produced trabecular bone structures with features of endochondral ossification, and there was no clear difference between parental‐ and mutant‐derived tissues. A transcriptome analysis of chondrocyte pellets at the hypertrophic phase showed a lower expression of proliferating‐phase genes and a higher expression of calcification‐phase genes in COL10A1−/− pellets compared with parental cell pellets. These in vitro and in vivo data suggested that collagen X is dispensable for the hypertrophic differentiation and endochondral ossification of human iPSC‐derived chondrocytes, though it may facilitate the differentiation process. Thus, COL10A1−/− iPSC lines are useful for investigating the physiological role of collagen X in chondrocyte differentiation. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

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“…For example, Tsujimoto et al discovered a long non-coding RNA (lncRNA) named MIR302CHG for the precise detection of iPSC. They achieved a sensitivity of 0.0001% in differentiated nephron progenitor populations using spiked-in samples [73]. In addition, Chung et al studied publicly archived datasets of microRNA microarrays and discovered a set of microRNA markers, including miR-302a-5p, miR-302c-3p, miR-302d-5p, miR-518f-5p, and miR-519-3p [74].…”

Section: Pcrmentioning

confidence: 99%

Assessing Tumorigenicity in Stem Cell-Derived Therapeutic Products: A Critical Step in Safeguarding Regenerative Medicine

Wang

2023

Bioengineering

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Stem cells hold promise in regenerative medicine due to their ability to proliferate and differentiate into various cell types. However, their self-renewal and multipotency also raise concerns about their tumorigenicity during and post-therapy. Indeed, multiple studies have reported the presence of stem cell-derived tumors in animal models and clinical administrations. Therefore, the assessment of tumorigenicity is crucial in evaluating the safety of stem cell-derived therapeutic products. Ideally, the assessment needs to be performed rapidly, sensitively, cost-effectively, and scalable. This article reviews various approaches for assessing tumorigenicity, including animal models, soft agar culture, PCR, flow cytometry, and microfluidics. Each method has its advantages and limitations. The selection of the assay depends on the specific needs of the study and the stage of development of the stem cell-derived therapeutic product. Combining multiple assays may provide a more comprehensive evaluation of tumorigenicity. Future developments should focus on the optimization and standardization of microfluidics-based methods, as well as the integration of multiple assays into a single platform for efficient and comprehensive evaluation of tumorigenicity.

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In vitro methods to ensure absence of residual undifferentiated human induced pluripotent stem cells intermingled in induced nephron progenitor cells

Cited by 7 publications

References 63 publications

Human iPSC-derived neural stem cells display a radial glia-like signaturein vitroand favorable long-term safety in transplanted mice

Human iPSC-derived neural stem cells display a radial glia-like signaturein vitroand favorable long-term safety in transplanted mice

Collagen X Is Dispensable for Hypertrophic Differentiation and Endochondral Ossification of Human iPSC‐Derived Chondrocytes

Assessing Tumorigenicity in Stem Cell-Derived Therapeutic Products: A Critical Step in Safeguarding Regenerative Medicine

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