Direct generation of human naive induced pluripotent stem cells from somatic cells in microfluidics

Giulitti, Stefano; Pellegrini, Marco; Zorzan, Irene; Martini, Paolo; Gagliano, Onelia; Mutarelli, Margherita; Ziller, Michael J.; Cacchiarelli, Davide; Romualdi, Chiara; Elvassore, Nicola; Martello, Graziano

doi:10.1038/s41556-018-0254-5

Cited by 79 publications

(119 citation statements)

References 60 publications

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“…We found that supplementation with XAV markedly improves the efficiency of reprogramming to the naïve state, in line with observations during resetting of conventional PSC (Guo et al, 2017). This may be a key difference from previous reports that found low efficiency of naive reprogramming using media that typically included the GSK3 inhibitor CH (Giulitti et al, 2019;Kilens et al, 2018;Liu et al, 2017). Our analysis shows that the presence of CH inhibits reprogramming to naïve status.…”

Section: Discussionmentioning

confidence: 65%

“…Transgene-free naïve iPSC have also been produced using chemically modified RNAs, but the efficiency of this approach was reported to depend on cell confinement in a microfluidic chamber (Giulitti et al, 2019), which restricts general application. In contrast, our results demonstrate that reprogramming to the naïve state can be highly efficient using unmodified RNAs in standard cell culture conditions.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover the reprogrammed cells obtained were heterogeneous with poorly characterized differentiation behavior. Very recently, reprogramming to the human naïve state was achieved using modified mRNA vectors applied in a microfluidic apparatus (Giulitti et al, 2019). In that study the authors report that serial transfection with chemically modified mRNAs over at least 7 days within a microfluidic chamber is important for induction of naïve cells.…”

Section: Introductionmentioning

confidence: 99%

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Efficient RNA-mediated reprogramming of human somatic cells to naïve pluripotency facilitated by tankyrase inhibition

Bredenkamp

Yang

Clarke

et al. 2019

Preprint

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In contrast to conventional human pluripotent stem cells (hPSC) that are related to post-implantation embryo stages, naïve hPSC exhibit features of pre-implantation epiblast. Naïve hPSC are established by resetting conventional hPSC, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naïve pluripotency. We find that tankyrase inhibition promotes RNA-mediated induction of naïve pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naïve hPSC can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naïve hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naïve epiblast identity. This system for efficient, facile, and reliable induction of transgene free naïve hPSC offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naïve versus conventional hPSC.

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Section: Discussionmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Efficient RNA-mediated reprogramming of human somatic cells to naïve pluripotency facilitated by tankyrase inhibition

Bredenkamp

Yang

Clarke

et al. 2019

Preprint

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“…Unfortunately, the existing nonintegrating methods, such as small molecules, mRNA, or episomal transfection, yield reprogramming efficiencies lower than viral transduction. Yet, we foresee that the development of miniaturized microfluidic devices enabling massive scale-down and parallelization of iNSC generation will further reduce the production costs [95] and promote a widespread clinical use.…”

Section: Discussionmentioning

confidence: 99%

Take the shortcut – direct conversion of somatic cells into induced neural stem cells and their biomedical applications

et al. 2019

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Second-generation reprogramming of somatic cells directly into the cell type of interest avoids induction of pluripotency and subsequent cumbersome differentiation procedures. Several recent studies have reported direct conversion of human somatic cells into stably proliferating induced neural stem cells (iNSCs). Importantly, iNSCs are easier, faster, and more cost-efficient to generate than induced pluripotent stem cells (iPSCs), and also have a higher level of clinical safety. Stably, self-renewing iNSCs can be derived from different cellular sources, such as skin fibroblasts and peripheral blood mononuclear cells, and readily differentiate into neuronal and glial lineages that are indistinguishable from their iPSC-derived counterparts or from NSCs isolated from primary tissues. This review focuses on the derivation and characterization of iNSCs and their biomedical applications. We first outline different approaches to generate iNSCs and then discuss the underlying molecular mechanisms. Finally, we summarize the preclinical validation of iNSCs to highlight that these cells are promising targets for disease modeling, autologous cell therapy, and precision medicine. Take the shortcut: from iPSC to iNSCForced expression of lineage-specific transcription factors (TFs) has been shown to reprogram the developmental potential of various somatic cell types e.g. by inducing pluripotency [1]. The seminal breakthrough of fibroblast reprogramming into iPSCs offers a novel experimental tool to generate patient-specific cells for developmental studies and diverse biomedical applications, such as disease modeling and cell therapy. Since then, efficiency and robustness of reprogramming protocols have been substantially improved, but the derivation of patient-specific iPSCs remains a lengthy and cumbersome procedure. Moreover, clinical applications require subsequent redifferentiation of iPSCs into the cell type of interest, which is inefficient and risky because transplanted undifferentiated iPSCs are tumorigenic. In the shadow of iPSC-type reprogramming, second-generation reprogramming paradigms have been developed to bypass the pluripotency state Abbreviations

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“…Bi-allelic expression of imprinted genes will indicate LOI. Bi-allelic expression of X-linked genes may indicate reactivation of the X chromosome, as expected in the early embryo 18 or in naive pluripotent stem cells 12,13,19,20 , X chromosome erosion, as observed after extensive culture of pluripotent cells 21 , or simply escape of single genes from the XCI mechanisms, as recently documented in somatic cells 22,23 .…”

Section: Mainmentioning

confidence: 71%

Allelic expression analysis of Imprinted and X-linked genes from bulk and single-cell transcriptomes

Martini

Sales

Perrera

et al. 2020

Preprint

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Genomic imprinting and X chromosome inactivation (XCI) are two prototypical epigenetic mechanisms whereby a set of genes is expressed monoallelically in order to fine tune their expression levels. Defects in genomic imprinting have been observed in several neurodevelopmental disorders, in a wide range of tumors and in induced pluripotent stem cells (iPSCs). Single Nucleotide Variations (SNVs) are readily detectable by RNA-sequencing allowing determination of whether imprinted or X-linked genes are aberrantly expressed from both alleles, although standardised analysis methods are still missing. We have developed a tool, named BrewerIX, that provides comprehensive information about allelic expression of a large, manually-curated set of imprinted and X-linked genes. BrewerIX does not require programming skills, runs on a standard personal computer, and can analyse both bulk and single-cell transcriptomes of human and mouse cells directly from raw sequencing data.BrewerIX confirmed and extended previous observations regarding the aberrant expression of imprinted genes in pluripotent cells, in the early embryo and in breast cancer cells and identified new genes escaping XCI in human somatic cells. We believe BrewerIX will be useful for the study of genomic imprinting and XCI during development and reprogramming, and for detecting aberrations in cancer and iPSCs. Due to its ease of use to non-computational biologists, its implementation could become standard practice during sample assessment, thus raising robustness and reproducibility of future studies.

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Direct generation of human naive induced pluripotent stem cells from somatic cells in microfluidics

Cited by 79 publications

References 60 publications

Efficient RNA-mediated reprogramming of human somatic cells to naïve pluripotency facilitated by tankyrase inhibition

Efficient RNA-mediated reprogramming of human somatic cells to naïve pluripotency facilitated by tankyrase inhibition

Take the shortcut – direct conversion of somatic cells into induced neural stem cells and their biomedical applications

Allelic expression analysis of Imprinted and X-linked genes from bulk and single-cell transcriptomes

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