Epithelial Vegfa Specifies a Distinct Endothelial Population in the Mouse Lung

Ellis, Lisandra Vila; Cain, Margo P.; Hutchison, Vera; Flodby, Per; Crandall, Edward D.; Borok, Zea; Zhou, Bin; Ostrin, Edwin J.; Wythe, Joshua D.; Chen, Jichao

doi:10.1016/j.devcel.2020.01.009

Cited by 159 publications

(177 citation statements)

References 62 publications

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“…Better understanding these mechanisms will likely shed significant light on organ development. Reciprocal signaling has been shown to occur between ECs and the surrounding organ microenvironment during development (Kao et al, 2015; Lammert, 2001; Lammert et al, 2003; Lazarus et al, 2011; Matsumoto et al, 2001; Vila Ellis et al, 2020) and ECs can influence development through the supply of membrane-bound or secreted factors, termed “angiocrine factors” (Rafii et al, 2016). Identifying these angiocrine roles of ECs has been challenging to study using in vivo animal models, as the vascular is highly sensitive to modulations in vivo (Ferrara et al, 1996; Shalaby et al, 1995), and it is technically challenging to parse out unique angiocrine roles from metabolic requirements for the vasculature.…”

Section: Discussionmentioning

confidence: 99%

Differentiation of human intestinal organoids with endogenous vascular endothelial cells

Holloway

Czerwinkski

et al. 2020

Preprint

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22Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) generated using directed 23 differentiation lack some cellular populations found in the native organ, including vasculature. Using 24 single cell RNA sequencing (scRNAseq), we have identified a transient population of endothelial cells 25 (ECs) present early in HIO differentiation that are lost over time in culture. Here, we have developed a 26 method to enhance co-differentiation and maintenance of ECs within HIOs (vHIOs). Given that ECs are 27 known to possess organ specific gene expression, morphology and function, we used bulk RNAseq 28 and scRNAseq to interrogate the developing human intestine, lung, and kidney in order to identify 29 organ-enriched EC-gene signatures in these organ systems. By comparing organ-specific gene 30 signatures along with markers validated by fluorescent in situ hybridization to HIO ECs, we find that 31 HIO ECs grown in vitro share the highest similarity with native intestinal ECs relative to kidney and 32 lung. Together, these data show that HIOs can co-differentiate a native EC population that are properly 33 patterned with an intestine-specific EC transcriptional signature in vitro. 34 35 36 37 38 39 40 41 42 43 44 65highly vascularized regions of immunocompromised mice (Cortez et al., 2018; Watson et al., 2014). In 66 these environments, HIOs undergo extensive vascularization by the murine host tissue, and increase in 67 complexity to resemble mature intestinal tissue (Finkbeiner et al., 2015b; Watson et al., 2014). 68However, co-culture approaches or co-differentiating HIOs with a native vasculature prior to in vivo 69 engraftment has not yet been achieved. 71Here, we performed single cell RNA sequencing (scRNAseq) at various timepoints across HIO 72 differentiation in vitro and observed a transient population of endothelial-like cells (ECs) present within 73 HIOs early during differentiation; however, these cells are not maintained during prolonged culture 74 under standard growth conditions. This suggested that early during HIO differentiation, cells within the 75 culture are capable of giving rise to EC-like cells. Based on these observations, we hypothesized that a 76 modified directed differentiation approach would allow the induction and maintenance of a more robust 77 EC population within HIOs (termed vHIO). Our findings demonstrate that modified culture conditions 78 allow a ~13-fold increase in the induction of EC-like cells within HIOs without impacting the other HIO 79 cell populations present (i.e. epithelium, mesenchyme), and support the survival of this population of 80 ECs within HIOs in culture for months. 82Since organ-specific morphology in vascular beds has long been appreciated (Aird, 2007), and 83 organ-specific transcriptional signatures and functions have been described in mouse (Ding et al., 84 2011; Kalucka et al., 2020; Lee et al., 2014; Nolan et al., 2013) and human tissues (Chi et al., 2003; 85 Marcu et al., 2018), we further sought to determine if HIO ECs were properly...

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Section: Discussionmentioning

confidence: 99%

Differentiation of human intestinal organoids with endogenous vascular endothelial cells

Holloway

Czerwinkski

et al. 2020

Preprint

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“…Lung EC were isolated as described (Cheng et al, 2017;Kovacs-Kà sa et al, 2017;Niethamer et al, 2020;Vila Ellis et al, 2020). Briefly, PBS perfused lungs were chopped into small pieces and digested in 2 mg/ml collagenase I (Life technologies) for 1 h at 37 C. Cells suspension were filtered through a 70 mm cell strainer and pelleted by centrifugation at 1500 rpm for 8 minutes.…”

Section: Isolation Of Lung Ec and At2mentioning

confidence: 99%

“…CD45 -CD31 + EC and CD45 -CD31 -nonEC mesenchymal cells were collected. Most of the EC collected should be microvascular ECs (Cheng et al, 2017;Kovacs-Kà sa et al, 2017;Niethamer et al, 2020;Vila Ellis et al, 2020).…”

Section: Isolation Of Lung Ec and At2mentioning

confidence: 99%

Angiocrine Sphingosine-1-Phosphate Activation of S1PR2-YAP Signaling Axis in Alveolar Type II Cells Is Essential for Lung Repair

et al. 2020

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“…Monocle 2.8.0 was used to analyze Seurat clusters in either of progenitor, AT1, and AT2 cells from aggregate control and mutant samples for Sox9 CreER/+ ; Yap1 CKO/CKO ; Taz CKO/CKO and Wnt3a Cre/+ ; Yap1 CKO/CKO ; Taz CKO/CKO or 12 aggregated control samples from E14.5, E16.5, E18.5, P4, P6, P7, P8, P10, P15, P20, 10-week-old, and 15-week-old lungs 11,20,23,52–55 . Cells were ordered using the top 2000 variable genes for control and mutant cells, and 1000 genes for the 12-aggregate sample identified by Seurat to generate pseudotime trajectories.…”

Section: Methodsmentioning

confidence: 99%

Lung lineage transcription factor NKX2-1 epigenetically resolves opposing cell fates in vivo

Little

Lynch

Yan

et al. 2020

Preprint

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Differential use of identical DNA sequences leads to distinct tissue lineages and then multiple cell types within a lineage, an epigenetic process central to progenitor and stem cell biology. The associated genomic changes, especially in native tissues, remain insufficiently understood, and are hereby addressed in the mouse lung, where the same lineage transcription factor NKX2-1 promotes the diametrically opposed alveolar type 1 (AT1) and AT2 cell fates. We show that the cell-type-specific function of NKX2-1 is attributed to its differential chromatin binding that is acquired or retained during development in coordination with partner transcriptional factors. Loss of YAP/TAZ redirects NKX2-1 from its AT1-specific to AT2-specific binding sites, leading to transcriptionally exaggerated AT2 cells when deleted in progenitors or AT1-to-AT2 conversion when deleted after fate commitment. Nkx2-1 mutant AT1 and AT2 cells gain distinct accessible sites including those of the opposite fate while adopting the gastrointestinal fate, suggesting an epigenetic plasticity larger than a transcriptional one. Our genomic analysis of single or purified cells, coupled with precision genetics, provides an epigenetic roadmap of alveolar cell fate and potential, and introduces an experimental benchmark for unraveling the in vivo function of lineage transcription factors.

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Epithelial Vegfa Specifies a Distinct Endothelial Population in the Mouse Lung

Cited by 159 publications

References 62 publications

Differentiation of human intestinal organoids with endogenous vascular endothelial cells

Differentiation of human intestinal organoids with endogenous vascular endothelial cells

Angiocrine Sphingosine-1-Phosphate Activation of S1PR2-YAP Signaling Axis in Alveolar Type II Cells Is Essential for Lung Repair

Lung lineage transcription factor NKX2-1 epigenetically resolves opposing cell fates in vivo

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