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Stem cells

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EPIREGULIN creates a developmental niche for spatially organized human intestinal enteroids
Charlie J. Childs, … , Michael P. Verzi, Jason R. Spence
Charlie J. Childs, … , Michael P. Verzi, Jason R. Spence
Published February 23, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.165566.
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EPIREGULIN creates a developmental niche for spatially organized human intestinal enteroids

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Abstract

Epithelial organoids derived from intestinal tissue, called ‘enteroids’, recapitulate many aspects of the organ in vitro, and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identify an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells, feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown and EREG-grown enteroids show that EGF-enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine-like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

Authors

Charlie J. Childs, Emily M. Holloway, Caden W. Sweet, Yu-Hwai Tsai, Angeline Wu, Abigail Vallie, Madeline K. Eiken, Meghan M. Capeling, Rachel K. Zwick, Brisa Palikuqi, Coralie Trentesaux, Joshua H. Wu, Oscar Pellon-Cardenas, Charles J. Zhang, Ian A. Glass, Claudia Loebel, Qianhui Yu, J. Gray Camp, Jonathan Z. Sexton, Ophir D. Klein, Michael P. Verzi, Jason R. Spence

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Spatiotemporal mapping of immune and stem cell dysregulation after volumetric muscle loss
Jacqueline A. Larouche, … , Eric Buras, Carlos A. Aguilar
Jacqueline A. Larouche, … , Eric Buras, Carlos A. Aguilar
Published February 23, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.162835.
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Spatiotemporal mapping of immune and stem cell dysregulation after volumetric muscle loss

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Abstract

Volumetric muscle loss (VML) is an acute trauma that results in persistent inflammation, supplantation of muscle tissue with fibrotic scarring, and decreased muscle function. The cell types, nature of cellular communication, and tissue locations that drive the aberrant VML response have remained elusive. Herein, we used spatial transcriptomics on a mouse model of VML and observed VML engenders a unique spatial pro-fibrotic pattern driven by crosstalk between fibrotic and inflammatory macrophages and mesenchymal derived cells. The dysregulated response impinged on muscle stem cell mediated repair, and targeting this circuit resulted in increased regeneration and reductions in inflammation and fibrosis. Collectively, these results enhance our understanding of the cellular crosstalk that drives aberrant regeneration and provides further insight into possible avenues for fibrotic therapy exploration.

Authors

Jacqueline A. Larouche, Emily C. Wallace, Bonnie D. Spence, Eric Buras, Carlos A. Aguilar

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The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment
Zhaoqiang Zhang, … , Yanfeng Wu, Huiyong Shen
Zhaoqiang Zhang, … , Yanfeng Wu, Huiyong Shen
Published February 16, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.162436.
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The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment

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Abstract

Mesenchymal stem cells (MSCs) possess strong immunoregulatory functions, one aspect of which is recruiting monocytes from peripheral vessels to local tissue by secreting MCP1. However, the regulatory mechanisms of MCP1 secretion in MSCs are still unclear. Recently, N6-methyladenosine (m6A) modification was reported to be involved in the functional regulation of MSCs. In this study, we demonstrated that methyltransferase-like 16 (METTL16) negatively regulated MCP1 expression in MSCs through m6A modification. Specifically, the expression of METTL16 in MSCs decreased gradually and was negatively correlated with the expression of MCP1 after coculture with monocytes. Knocking down METTL16 markedly enhanced MCP1 expression and the ability to recruit monocytes. Mechanistically, knocking down METTL16 decreased MCP1 mRNA degradation, which was mediated by the m6A reader YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). We further revealed that YTHDF2 specifically recognized m6A sites on MCP1 mRNA in the CDS region and thus negatively regulated MCP1 expression. Moreover, an in vivo assay showed that MSCs transfected with METTL16 siRNA showed a stronger ability to recruit monocytes. These findings reveal a potential mechanism by which the m6A methylase METTL16 regulates MCP1 expression through YTHDF2-mediated mRNA degradation and suggest a potential strategy to manipulate MCP1 expression in MSCs.

Authors

Zhaoqiang Zhang, Zhongyu Xie, Jiajie Lin, Zehang Sun, Zhikun Li, Wenhui Yu, Yipeng Zeng, Guiwen Ye, Jinteng Li, Feng Ye, Zepeng Su, Yunshu Che, Peitao Xu, Chenying Zeng, Peng Wang, Yanfeng Wu, Huiyong Shen

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Sorting-nexin-10 sustains platelet-derived growth factor receptor signaling in glioblastoma stem cells via endosomal protein sorting
Ryan C. Gimple, … , Sameer Agnihotri, Jeremy N. Rich
Ryan C. Gimple, … , Sameer Agnihotri, Jeremy N. Rich
Published February 16, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.158077.
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Sorting-nexin-10 sustains platelet-derived growth factor receptor signaling in glioblastoma stem cells via endosomal protein sorting

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Abstract

Glioblastoma is the most malignant primary brain tumor for which the prognosis remains dismal even with aggressive surgical, medical, and radiation therapies. Glioblastoma stem cells (GSCs) promote therapeutic resistance and cellular heterogeneity due to their self-renewal properties and capacity for plasticity. To understand the molecular processes essential for maintaining GSCs, we performed an integrative analysis comparing active enhancer landscapes, transcriptional profiles, and functional genomics profiles of GSCs and non-neoplastic neural stem cells (NSCs). We identified sorting nexin 10 (SNX10), an endosomal protein sorting factor, as selectively expressed in GSCs compared to NSCs and essential for GSC survival. Targeting SNX10 impaired GSC viability and proliferation, induced apoptosis, and reduced self-renewal capacity. Mechanistically, GSCs utilized endosomal protein sorting to promote platelet-derived growth factor receptor β (PDGFRβ) proliferative and stem cell signaling pathways through post-transcriptional regulation of the PDGFR tyrosine kinase. Targeting SNX10 expression extended survival of orthotopic xenograft-bearing mice, and high SNX10 expression correlated with poor glioblastoma patient prognosis, suggesting its potential clinical importance. Thus, our study reveals an essential connection between endosomal protein sorting and oncogenic receptor tyrosine kinase signaling and suggests that targeting endosomal sorting may represent a promising therapeutic approach for glioblastoma treatment.

Authors

Ryan C. Gimple, Guoxin Zhang, Shuai Wang, Tengfei Huang, Jina Lee, Suchet Taori, Deguan Lv, Deobrat Dixit, Matthew E. Halbert, Andrew R. Morton, Reilly L. Kidwell, Zhen Dong, Briana C. Prager, Leo Kim, Zhixin Qiu, Linjie Zhao, Qi Xie, Qiulian Wu, Sameer Agnihotri, Jeremy N. Rich

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Hypoxia enhances IPF mesenchymal progenitor cell fibrogenicity via the lactate/GPR81/HIF1α pathway
Libang Yang, … , Peter B. Bitterman, Craig A. Henke
Libang Yang, … , Peter B. Bitterman, Craig A. Henke
Published January 19, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.163820.
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Hypoxia enhances IPF mesenchymal progenitor cell fibrogenicity via the lactate/GPR81/HIF1α pathway

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Abstract

Hypoxia is a sentinel feature of IPF. The IPF microenvironment contains high lactate levels and hypoxia enhances cellular lactate production. Lactate, acting through the GPR81 lactate receptor, serves as a signal molecule regulating cellular processes. We previously identified intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients that drive fibrosis. However, whether hypoxia enhances IPF MPC fibrogenicity is unclear. We hypothesized that hypoxia increases IPF MPC fibrogenicity via lactate and its cognate receptor GPR81. Here we show that hypoxia promotes IPF MPC self-renewal. The mechanism involves hypoxia-mediated enhancement of LDHA function and lactate production and release. Hypoxia also increases HIF1α levels, which in turn augments the expression of GPR81. Exogenous lactate operating through GPR81 promotes IPF MPC self-renewal. IHC analysis of IPF lung tissue demonstrate IPF MPCs expressing GPR81 and hypoxic markers on the periphery of the fibroblastic focus. We show that hypoxia enhances IPF MPC fibrogenicity in vivo. We demonstrate that knock-down of GPR81 inhibits hypoxia-induced IPF MPC self-renewal in vitro and attenuates hypoxia-induced IPF MPC fibrogenicity in vivo. Our data demonstrate that hypoxia creates a feed-forward loop that augments IPF MPC fibrogenicity via the lactate/GPR81/HIF1α pathway.

Authors

Libang Yang, Adam Gilbertsen, Hong Xia, Alexey Benyumov, Karen A. Smith, Jeremy A. Herrera, Emilian Racila, Peter B. Bitterman, Craig A. Henke

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Biliary epithelial cells are facultative liver stem cells during liver regeneration in adult zebrafish
Isaac M. Oderberg, Wolfram Goessling
Isaac M. Oderberg, Wolfram Goessling
Published January 10, 2023
Citation Information: JCI Insight. 2023;8(1):e163929. https://doi.org/10.1172/jci.insight.163929.
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Biliary epithelial cells are facultative liver stem cells during liver regeneration in adult zebrafish

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Abstract

The liver is a highly regenerative organ, yet the presence of a dedicated stem cell population remains controversial. Here, we interrogate a severe hepatocyte injury model in adult zebrafish to define that regeneration involves a stem cell population. After near-total hepatocyte ablation, single-cell transcriptomic and high-resolution imaging analyses throughout the entire regenerative timeline reveal that biliary epithelial cells undergo transcriptional and morphological changes to become hepatocytes. As a population, biliary epithelial cells give rise to both hepatocytes and biliary epithelial cells. Biliary epithelial cells proliferate and dedifferentiate to express hepatoblast transcription factors prior to hepatocyte differentiation. This process is characterized by increased MAPK, PI3K, and mTOR signaling, and chemical inhibition of these pathways impairs biliary epithelial cell proliferation and fate conversion. We conclude that, upon severe hepatocyte ablation in the adult liver, biliary epithelial cells act as facultative liver stem cells in an EGFR-PI3K-mTOR–dependent manner.

Authors

Isaac M. Oderberg, Wolfram Goessling

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Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells
Vitaly Ievlev, … , John F. Engelhardt, Kalpaj R. Parekh
Vitaly Ievlev, … , John F. Engelhardt, Kalpaj R. Parekh
Published December 13, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.162041.
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Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells

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Abstract

Keratin expression dynamically changes in airway basal cells (BCs) following acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In Bronchiolitis Obliterans (BO) following lung transplantation, BC clonogenicity declines which is associated with a switch from Keratin15 (Krt15) to Keratin14 (Krt14). We investigated the roles of these keratins using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs failed to differentiate into club and ciliated cells, but had enhanced clonogenicity. By contrast, Krt15-KO did not alter BC differentiation, but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo following injury. Krt14, but not Krt15, bound the tumor suppressor Stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, while dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts that of Krt14-KO and resembles the phenotype in BO with decreased clonogenicity, increased Krt14 and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior which is relevant in chronic disease states like BO.

Authors

Vitaly Ievlev, Thomas J. Lynch, Kyle W. Freischlag, Caitlyn B. Gries, Anit Shah, Albert C. Pai, Bethany A. Ahlers, Soo Yeun Park, John F. Engelhardt, Kalpaj R. Parekh

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Culture impact on the transcriptomic programs of primary and iPSC-derived human alveolar type 2 cells
Konstantinos-Dionysios Alysandratos, … , Carla F. Kim, Darrell N. Kotton
Konstantinos-Dionysios Alysandratos, … , Carla F. Kim, Darrell N. Kotton
Published December 1, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.158937.
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Culture impact on the transcriptomic programs of primary and iPSC-derived human alveolar type 2 cells

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Abstract

Dysfunction of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, is implicated in pulmonary disease pathogenesis, highlighting the importance of human in vitro models. However, AEC2-like cells in culture have yet to be directly compared to their in vivo counterparts at single cell resolution. Here, we perform head-to-head comparisons between the transcriptomes of fresh primary (1o) adult human AEC2s, their cultured progeny, and human induced pluripotent stem cell-derived AEC2s (iAEC2s). We find each population occupies a distinct transcriptomic space with cultured AEC2s (1o and iAEC2s) exhibiting similarities to and differences from freshly purified 1o cells. Across each cell type, we find an inverse relationship between proliferative and maturation states, with pre-culture 1o AEC2s being most quiescent/mature and iAEC2s being most proliferative/least mature. Cultures of either type of human AEC2 do not generate detectable alveolar type 1 cells in these defined conditions; however, a subset of iAEC2s co-cultured with fibroblasts acquires a “transitional cell state” described in mice and humans to arise during fibrosis or following injury. Hence, we provide direct comparisons of the transcriptomic programs of 1o and engineered AEC2s, two in vitro models that can be harnessed to study human lung health and disease.

Authors

Konstantinos-Dionysios Alysandratos, Carolina Garcia-de-Alba, Changfu Yao, Patrizia Pessina, Jessie Huang, Carlos Villacorta-Martin, Olivia T. Hix, Kasey Minakin, Claire L. Burgess, Pushpinder Bawa, Aditi Murthy, Bindu Konda, Michael F. Beers, Barry R. Stripp, Carla F. Kim, Darrell N. Kotton

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An RPS19-edited model for Diamond-Blackfan anemia reveals TP53-dependent impairment of hematopoietic stem cell activity
Senthil Velan Bhoopalan, … , Marcin W. Wlodarski, Mitchell J. Weiss
Senthil Velan Bhoopalan, … , Marcin W. Wlodarski, Mitchell J. Weiss
Published November 22, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.161810.
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An RPS19-edited model for Diamond-Blackfan anemia reveals TP53-dependent impairment of hematopoietic stem cell activity

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Abstract

Diamond–Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multi-lineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/− HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome-editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.

Authors

Senthil Velan Bhoopalan, Jonathan S. Yen, Thiyagaraj Mayuranathan, Kalin D. Mayberry, Yu Yao, Maria Angeles Lillo Osuna, Yoonjeong Jang, Janaka S.S. Liyange, Lionel Blanc, Steven R. Ellis, Marcin W. Wlodarski, Mitchell J. Weiss

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In vivo base editing by a single intravenous vector injection for treatment of hemoglobinopathies
Chang Li, … , Evangelia Yannaki, André Lieber
Chang Li, … , Evangelia Yannaki, André Lieber
Published August 25, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.162939.
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In vivo base editing by a single intravenous vector injection for treatment of hemoglobinopathies

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Abstract

Individuals with beta-thalassemia or Sickle Cell Disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% HbF appear to be symptom-free. Here, we used a non-integrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113A>G HPFH mutation in the gamma-globin promoters in “healthy” CD46/β-YAC mice carrying the human β-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only subcutaneous and intravenous injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/β-YAC mice resulted in >60% -113A>G conversion with 30% γ-globin of human beta globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-seq. In vitro, in HSCs from beta-thalassemia and Sickle Cell Disease patients, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.

Authors

Chang Li, Aphrodite Georgakopoulou, Gregory A. Newby, Kelcee A. Everette, Evangelos Nizamis, Kiriaki Paschoudi, Efthymia Vlachaki, Sucheol Gil, Anna K. Anderson, Theodore Koob, Lishan Huang, Hongjie Wang, Hans-Peter Kiem, David R. Liu, Evangelia Yannaki, André Lieber

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