Generation of Human PSC-Derived Kidney Organoids with Patterned Nephron Segments and a De Novo Vascular Network

Low, Jian Hui; Пин, Ли; Chew, Elaine Guo Yan; Zhou, Bo; Suzuki, Keiichiro; Zhang, Tian; Lian, Michelle Mulan; Li, Meng; Aizawa, Emi; Esteban, Concepción Rodrı́guez; Yong, Kylie Su Mei; Chen, Yi‐Ping Phoebe; Campistol, Josep M.; Fang, Mingliang; Khor, Chiea Chuen; Foo, Jia Nee; Belmonte, Juan Carlos Izpisúa; Xia, Yun

doi:10.1016/j.stem.2019.06.009

Cited by 249 publications

(274 citation statements)

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“…While organoids are genetically tractable, complex in vitro human systems, they do not entirely recapitulate the full complement of cell types or complex physiology found in native tissues (Holloway et al, 2019). Several groups have been working to improve organoids, and approaches include implementing strategies to increase organoid complexity and maturation to more accurately mimic the native tissue (Fujii et al, 2018; Low et al, 2019; Mansour et al, 2018; Ouchi et al, 2019). In the case of cell types lacking in HIOs, such as ectoderm-derived enteric neurons, co-culture techniques using enteric neuron precursors (vagal neural crest cells) have been developed, facilitating the integration of a functional enteric nervous system that can stimulate motility following transplantation (Schlieve et al, 2017; Workman et al, 2016).…”

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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“…Our approach is applicable to any inherited disease, both WGS and RNA-seq techniques are commercially available, gold standards are being established and the analysis tools are readily accessible 47, 48, 49–55, 81, 82 . Ex vivo organoid models are being developed for a multitude of tissues including brain 83, 84 , kidney 85 , liver 86, 87 and lung 88 .…”

Section: Discussionmentioning

confidence: 99%

A combined RNA-seq and whole genome sequencing approach for identification of non-coding pathogenic variants in single families

Bronstein

Capowski

Mehrotra

et al. 2019

Preprint

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Inherited retinal degenerations (IRDs) are at the focus of current genetic therapeutic advancements. For a genetic treatment such as gene therapy to be successful an accurate genetic diagnostic is required. Genetic diagnostics relies on the assessment of the probability that a given DNA variant is pathogenic. Non-coding variants present a unique challenge for such assessments as compared to coding variants. For one, non-coding variants are present at much higher number in the genome than coding variants. In addition, our understanding of the rules that govern the non-coding regions of the genome is less complete than our understanding of the coding regions. Methods that allow for both the identification of candidate non-coding pathogenic variants and their functional validation may help overcome these caveats allowing for a greater number of patients to benefit from advancements in genetic therapeutics. We present here an unbiased approach combining whole genome sequencing (WGS) with patient induced pluripotent stem cell (iPSC) derived retinal organoids (ROs) transcriptome analysis. With this approach we identified and functionally validated a novel pathogenic non-coding variant in a small family with a previously unresolved genetic diagnosis.

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“…Modifications to the differentiation protocols are often necessary in order to optimize cell type content—namely organoid composition—and tissue patterning. For example, alterations in FGF9 or CHIR99201 concentration, or relative duration of the incubation with either factor can greatly affect nephron composition, and patterning in hPSC‐derived kidney organoids . Therefore, changes introduced to a differentiation protocol must be carefully documented in order to ensure the reproducibility of the method.…”

Section: Current Limitations Of Hpsc‐derived Organoid Models and Missmentioning

confidence: 99%

“…For example, alterations in FGF9 or CHIR99201 concentration, or relative duration of the incubation with either factor can greatly affect nephron composition, and patterning in hPSC-derived kidney organoids. 38,89 Therefore, changes introduced to a differentiation protocol must be carefully documented in order to ensure the reproducibility of the method. Another important factor to take into account is organoid tissue maturity.…”

Section: Current Limitations Of Hpsc-derived Organoid Models and MImentioning

confidence: 99%

Recapitulating human tissue damage, repair, and fibrosis with human pluripotent stem cell-derived organoids

Sobral-Reyes

Lemos

2019

Stem Cells

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As new applications for human pluripotent stem cell‐derived organoids in drug screenings and tissue replacement therapies emerge, there is a need to examine the mechanisms of tissue injury and repair recently reported for various organoid models. In most cases, organoids contain the main cell types and tissues present in human organs, spatially arranged in a manner that largely resembles the architecture of the organ. Depending on the differentiation protocol used, variations may exist in cell type ratios relative to the organ of reference, and certain tissues, including some parenchymal components and the endothelium, might be poorly represented, or lacking altogether. Despite those caveats, recent studies have shown that organoid tissue injury recapitulates major events and histopathological features of damaged human tissues. In particular, major mechanisms of parenchyma cell damage and interstitial fibrosis can be reproduced with remarkable faithfulness. Although further validation remains to be done in order to establish the relevance of using organoid for either mechanistic studies or drug assays, this technology is becoming a promising tool for the study of human tissue homeostasis, injury, and repair.

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Generation of Human PSC-Derived Kidney Organoids with Patterned Nephron Segments and a De Novo Vascular Network

Cited by 249 publications

References 69 publications

Differentiation of human intestinal organoids with endogenous vascular endothelial cells

Differentiation of human intestinal organoids with endogenous vascular endothelial cells

A combined RNA-seq and whole genome sequencing approach for identification of non-coding pathogenic variants in single families

Recapitulating human tissue damage, repair, and fibrosis with human pluripotent stem cell-derived organoids

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