Optimal-Transport Analysis of Single-Cell Gene Expression Identifies Developmental Trajectories in Reprogramming

Schiebinger, Geoffrey; Shu, Jian; Tabaka, Marcin; Cleary, Brian; Subramanian, Vidya; Solomon, Aryeh; Gould, Joshua; Liu, Siyan; Lin, Stacie; Berube, Peter; Lee, Lia; Chen, Jenny; Brumbaugh, Justin; Rigollet, Philippe; Hochedlinger, Konrad; Jaenisch, Rudolf; Regev, Aviv; Lander, Eric S.

doi:10.1016/j.cell.2019.02.026

Cited by 133 publications

(192 citation statements)

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“…Indeed, mouse primed iPSC can be obtained using medium containing FGF and activin (Han et al, 2011), similar to culture conditions for propagation of conventional hPSC (Vallier et al, 2005). Induction of naïve pluripotency is relatively robust in the mouse system and is increasingly well characterized at the molecular level Schiebinger et al, 2019;Stadhouders et al, 2018). Reprogramming of human fibroblasts to naïve iPSC has only recently been reported, however, and appears variable and inefficient (Kilens et al, 2018;Liu et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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: Introductionmentioning

confidence: 99%

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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“…While reports of direct reprogramming were first documented many years ago, the tools available to dissect the precise molecular mechanism of these processes were limited. Advances in single cell RNA sequencing have created avenues to identify the path a single cell can take to its endpoint, and identify the molecular determinants of these trajectories (Cacchiarelli et al, 2018;Schiebinger et al, 2019). Simultaneous advances in machine learning have improved the information gleaned from scRNA-seq data, as well as the ability to correlate changes between gene expression and chromatin remodeling (Cao et al, 2018;Deng et al, 2019;Lopez et al, 2018;Way and Greene, 2018;Welch et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Unique Transcription Factor Functions Regulate Epigenetic and Transcriptional Dynamics During Cardiac Reprogramming

Thomas

et al. 2019

Preprint

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Direct lineage conversion, whereby a somatic cell assumes a new cellular identity, can be driven by ectopic expression of combinations of lineage-enriched transcription factors. To determine the molecular mechanisms by which expression of Gata4, Mef2c, and Tbx5 (GMT) induces direct reprogramming from a cardiac fibroblast toward an induced cardiomyocyte, we performed a comprehensive transcriptomic and epigenomic interrogation of the reprogramming process. Single cell RNA sequencing indicated that a reprogramming trajectory was acquired within 48 hours of GMT introduction, did not require cell division, and was limited mainly by successful expression of GMT.Evaluation of chromatin accessibility by ATAC-seq supported the expression dynamics and revealed widespread chromatin remodeling at early stages of the reprogramming process. Chromatin immunoprecipitation followed by sequencing of each factor alone or in combinations revealed that GMT bind DNA individually and in combination, and that ectopic expression of either Mef2c or Tbx5 is sufficient in some contexts to increase accessibility. We also find evidence for cooperative facilitation and refinement of each factor's binding in a combinatorial setting. A random-forest classifier that integrated the observed gene expression dynamics with regions of dynamic chromatin accessibility suggested Tbx5 binding is a primary driver of gene expression changes and revealed additional transcription factor motifs co-segregating with reprogramming factor motifs, suggesting new factors that may be involved in the reprogramming process. These results begin to explain the mechanisms by which transcription factors normally expressed in multiple germ layers can function combinatorially to direct lineage conversion.3

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“…Existing tools can enable the reconstruction of differentiation trajectories and therefore be used as a proxy to study developmental processes (Qiu et al, 2017;Schiebinger et al, 2019;Trapnell et al, 2014). To infer pseudotemporal cellular trajectories, SoptSC orders cells from the original cell-cell graph by calculating a collection of distances between an initial cell and all other surrounding cells.…”

Section: Cellular Entropy and Rna Velocity Predict The Likelihood Ofmentioning

confidence: 99%

Single cell transcriptomics of human epidermis reveals basal stem cell transition states

Wang¹,

Drummond²,

Guerrero‐Juarez³

et al. 2019

Preprint

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How stem cells give rise to human interfollicular epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find at least four spatially distinct stem cell populations that decorate the top and bottom of rete ridge architecture and hold transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling through co-variance of cognate ligand-receptor pairs indicate that the basal cell populations distinctly serve as critical signaling hubs that maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest the "transitional" basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed "transitional" basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity. Joost et al., 2018). Despite these studies, epidermal stem cell heterogeneity of human IFE remains unresolved. To address this issue, we interrogated epidermal cell heterogeneity within human neonatal foreskin epidermis using droplet-enabled scRNA-seq and identify four spatially distinct basal stem cell subpopulations. Interrogation of the "transitional" basal subpopulations that spatially occupy both the basal and suprabasal layers indicate their essential role in epidermal homeostasis. Our findings argue against a single population of progenitor cells and suggest a more complex model of multiple epidermal stem cell transitions that maintain epidermal homeostasis. RESULTS Single cell transcriptome analysis reveals robust cellular heterogeneity in human neonatal epidermis.To define the cellular heterogeneity of human IFE, we isolated viable, single cells from discarded and de-identified human neonatal foreskin epidermis and subjected them to droplet-enabled scRNA-seq to resolve their individual transcriptomes ( Figure 1A; Supplementary Figure 1; Supplementary Table 1; n = 5). We chose foreskin epidermis because it is composed of mostly IFE and contains few rudimentary skin appendages, such as hair follicles and sweat glands (Tuncali et al., 2005). We processed a total of 17,553 cells and performed quality control analysis on individual libraries using Seurat (Supplementary Figure 2) (Macosko et al., 2015). We used Similarity matrix-based OPtimization for Single Cell (SoptSC) to bioinformatically parse and analyze our data . The SoptSC algorithm is based on a cell-cell similarity matrix that can coherently perform many inference tasks -including unsupervised clustering, pseudotemporal ordering, cell lineage inference, cell-cell communication, and network inference.We used library three as a repr...

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Optimal-Transport Analysis of Single-Cell Gene Expression Identifies Developmental Trajectories in Reprogramming

Cited by 133 publications

References 0 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

Unique Transcription Factor Functions Regulate Epigenetic and Transcriptional Dynamics During Cardiac Reprogramming

Single cell transcriptomics of human epidermis reveals basal stem cell transition states

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