Stem cells
Abstract
The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP–indian hedgehog (Ihh) feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and trace the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with Patched-1 (Ptch1) floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large concentric clonally expanded cell populations within the resting zone (‘patched roses’) and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell-descendants migrated away from the growth plate and eventually transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.
Authors
Shion Orikasa, Yuki Matsushita, Hiroaki Manabe, Michael Fogge, Zachary J. Lee, Koji Mizuhashi, Naoko Sakagami, Wanida Ono, Noriaki Ono
Abstract
We previously established that vascular smooth muscle–derived adventitial progenitor cells (AdvSca1-SM) preferentially differentiate into myofibroblasts and contribute to fibrosis in response to acute vascular injury. However, the role of these progenitor cells in chronic atherosclerosis has not been defined. Using an AdvSca1-SM cell lineage tracing model, scRNA-Seq, flow cytometry, and histological approaches, we confirmed that AdvSca1-SM–derived cells localized throughout the vessel wall and atherosclerotic plaques, where they primarily differentiated into fibroblasts, smooth muscle cells (SMC), or remained in a stem-like state. Krüppel-like factor 4 (Klf4) knockout specifically in AdvSca1-SM cells induced transition to a more collagen-enriched fibroblast phenotype compared with WT mice. Additionally, Klf4 deletion drastically modified the phenotypes of non–AdvSca1-SM–derived cells, resulting in more contractile SMC and atheroprotective macrophages. Functionally, overall plaque burden was not altered with Klf4 deletion, but multiple indices of plaque composition complexity, including necrotic core area, macrophage accumulation, and fibrous cap thickness, were reduced. Collectively, these data support that modulation of AdvSca1-SM cells through KLF4 depletion confers increased protection from the development of potentially unstable atherosclerotic plaques.
Authors
Allison M. Dubner, Sizhao Lu, Austin J. Jolly, Keith A. Strand, Marie F. Mutryn, Tyler Hinthorn, Tysen Noble, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans
Abstract
Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely-used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared to magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACS-purified hiPSC-CMs impacts the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. Global proteomics revealed lactate-purified hiPSC-CMs displayed a differential phenotype over MACS hiPSC-CMs. hiPSC-CMs were then integrated into 3D hiPSC-ECTs and cultured for four weeks. Structurally, there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force and Ca2+ transients measurements revealed similar functional performance between purification methods. High-resolution mass spectrometry (MS)-based quantitative proteomics showed no significant difference in protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable structural, functional, and proteomic features, and suggests lactate purification does not result in an irreversible change in hiPSC-CM phenotype.
Authors
Kalina J. Rossler, Willem J. De Lange, Morgan W. Mann, Timothy J. Aballo, Jake A. Melby, Jianhua Zhang, Gina Kim, Elizabeth F. Bayne, Yanlong Zhu, Emily T. Farrell, Timothy J. Kamp, J. Carter Ralphe, Ying Ge
Abstract
Hair loss is a debilitating condition associated with the depletion of dermal papilla cells (DPCs), which can be replenished by dermal sheath cells (DSCs). Hence, strategies aimed at increasing the population of DPCs and DSCs hold great promise for the treatment of hair loss. In this study, we demonstrated that introducing exogenous DPCs and DSCs (hair follicle mesenchymal stem cells) could effectively migrate and integrate into the dermal papilla and dermal sheath niches, leading to enhanced hair growth and prolonged anagen phases. However, the homing rates of DPCs and DSCs were influenced by various factors, including recipient mouse depilation, cell passage number, cell dose, and immune rejection. Through in vitro and in vivo experiments, we further discovered that the CXCL13/CXCR5 pathway mediated the homing of DPCs and DSCs into hair follicle niches. This study underscores the potential of cell-based therapies for hair loss by targeted delivery of DPCs and DSCs to their respective niches, and sheds light on the intriguing concept that isolated mesenchymal stem cells can home back to their original niche microenvironment.
Authors
Kaitao Li, Fang Liu, Ye He, Qian Qu, Pingping Sun, Lijuan Du, Jin Wang, Ruosi Chen, Yuyang Gan, Danlan Fu, Zhexiang Fan, Bingcheng Liu, Zhiqi Hu, Yong Miao
Abstract
Reducing inflammatory damage and improving alveolar epithelium regeneration are two key approaches to promoting lung repair in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Stimulation of cholinergic-α7nAChR (α7 nicotinic acetylcholine receptor, coded by Chrna7) signaling could dampen lung inflammatory injury. However, whether activation of α7nAChR in alveolar type II (AT2) cells promotes alveolar epithelial injury repair and underlying mechanisms are elusive. Here, we found that α7nAChR was expressed on AT2 cells and was upregulated in response to LPS-induced ALI. Meanwhile, deletion of Chrna7 in AT2 cells impeded lung repair process and worsened lung inflammation in ALI. Using in vivo AT2 lineage-labeled mice and ex vivo AT2-derived alveolar organoids, we demonstrated that activation of α7nAChR expressed on AT2 cells improved alveolar regeneration by promoting AT2 cells to proliferate and subsequently differentiate toward alveolar type I (AT1) cells. Then we screened out the WNT7B signaling pathway by the RNA sequencing analysis of in vivo AT2 lineage-labeled cells, and further confirmed its indispensability for α7nAChR activation-mediated alveolar epithelial proliferation and differentiation. Thus, we have identified an unrecognized pathway that cholinergic-α7nAChR signaling determines alveolar regeneration and repair, which might provide us a novel therapeutic target for combating ALI.
Authors
Xiaoyan Chen, Cuiping Zhang, Tianchang Wei, Jie Chen, Ting Pan, Miao Li, Lu Wang, Juan Song, Cuicui Chen, Yan Zhang, Yuanlin Song, Xiao Su
Abstract
Insulin secretion from pancreatic β cells is essential to the maintenance of glucose homeostasis. Defects in this process result in diabetes. Identifying genetic regulators that impair insulin secretion is crucial for the identification of novel therapeutic targets. Here, we show that reduction of ZNF148 in human islets, and its deletion in stem cell–derived β cells (SC–β cells), enhances insulin secretion. Transcriptomics of ZNF148-deficient SC–β cells identifies increased expression of annexin and S100 genes whose proteins form tetrameric complexes involved in regulation of insulin vesicle trafficking and exocytosis. ZNF148 in SC–β cells prevents translocation of annexin A2 from the nucleus to its functional place at the cell membrane via direct repression of S100A16 expression. These findings point to ZNF148 as a regulator of annexin-S100 complexes in human β cells and suggest that suppression of ZNF148 may provide a novel therapeutic strategy to enhance insulin secretion.
Authors
Eleonora de Klerk, Yini Xiao, Christopher H. Emfinger, Mark P. Keller, David I. Berrios, Valentina Loconte, Axel A. Ekman, Kate L. White, Rebecca L. Cardone, Richard G. Kibbey, Alan D. Attie, Matthias Hebrok
Abstract
Over 30 million people worldwide suffer from untreatable vision loss and blindness associated with childhood-onset and age-related eye diseases caused by photoreceptor (PR), retinal pigment epithelium (RPE), and choriocapillaris (CC) degeneration. Recent work suggests that RPE-based cell therapy may slow down vision loss in late stages of age-related macular degeneration (AMD), a polygenic disease induced by RPE atrophy. However, accelerated development of effective cell therapies is hampered by the lack of large-animal models that allow testing safety and efficacy of clinical doses covering the human macula (20 mm2). We developed a versatile pig model to mimic different types and stages of retinal degeneration. Using an adjustable power micropulse laser, we generated varying degrees of RPE, PR, and CC damage and confirmed the damage by longitudinal analysis of clinically relevant outcomes, including analyses by adaptive optics and optical coherence tomography/angiography, along with automated image analysis. By imparting a tunable yet targeted damage to the porcine CC and visual streak — with a structure similar to the human macula — this model is optimal for testing cell and gene therapies for outer retinal diseases including AMD, retinitis pigmentosa, Stargardt, and choroideremia. The amenability of this model to clinically relevant imaging outcomes will facilitate faster translation to patients.
Authors
Francesca Barone, Juan Amaral, Irina Bunea, Mitra Farnoodian, Rohan Gupta, Rishabh Gupta, Dara Baker, M. Joseph Phillips, Richard J. Blanch, Arvydas Maminishkis, David M. Gamm, Kapil Bharti
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
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
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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