-
Nature Protocols Aug 2023Developing models of human kidney tissue in vitro is an important challenge in regenerative nephrology research, given the paucity of novel and effective therapies in... (Review)
Review
Developing models of human kidney tissue in vitro is an important challenge in regenerative nephrology research, given the paucity of novel and effective therapies in kidney disease. However, the de novo generation of kidney tissues from human pluripotent stem cells (hPSCs) is challenging owing to the structural and functional complexity of the organ, as well its developmental origin from two distinct embryologic populations: the metanephric mesenchyme and the ureteric bud (UB). Directed differentiation strategies have been developed to generate kidney organoids containing nephron-like structures; we recently reported an efficient and practical method to generate UB tissues. Here, we describe a detailed step-by-step protocol for differentiation of hPSCs into three-dimensonal UB organoids that exhibit complex morphological development and the capacity to differentiate into functional collecting duct tissues. Over 3 d, hPSCs are induced into PAX2GATA3 pronephric (anterior) intermediate mesoderm fates in monolayer cultures at high efficiency. The cells are aggregated into three-dimensional spheroids, which then assemble and organize into nephric duct-like tissue over 4 d. When embedded into an extracellular matrix, the spheroids grow into UB organoids that recapitulate fetal branching morphogenesis for 1 week of culture. When switched to permissive conditions, the UB organoids spontaneously differentiate to form collecting duct principal cells. This approach provides robust and reproducible methods that can be readily adopted by users with basic experience in hPSC and organoid differentiation to generate UB tissues, which may be used to investigate human kidney development, model disease processes and catalyze further efforts in engineering functional kidney tissue.
Topics: Humans; Kidney; Organoids; Pluripotent Stem Cells; Cell Differentiation; Tissue Engineering
PubMed: 37460630
DOI: 10.1038/s41596-023-00847-2 -
Advanced Science (Weinheim,... Jul 2023Evidence suggests a unique association between bone aging and neurodegenerative/cerebrovascular disorders. However, the mechanisms underlying bone-brain interplay remain...
Evidence suggests a unique association between bone aging and neurodegenerative/cerebrovascular disorders. However, the mechanisms underlying bone-brain interplay remain elusive. Here platelet-derived growth factor-BB (PDGF-BB) produced by preosteoclasts in bone is reported to promote age-associated hippocampal vascular impairment. Aberrantly elevated circulating PDGF-BB in aged mice and high-fat diet (HFD)-challenged mice correlates with capillary reduction, pericyte loss, and increased blood-brain barrier (BBB) permeability in their hippocampus. Preosteoclast-specific Pdgfb transgenic mice with markedly high plasma PDGF-BB concentration faithfully recapitulate the age-associated hippocampal BBB impairment and cognitive decline. Conversely, preosteoclast-specific Pdgfb knockout mice have attenuated hippocampal BBB impairment in aged mice or HFD-challenged mice. Persistent exposure of brain pericytes to high concentrations of PDGF-BB upregulates matrix metalloproteinase 14 (MMP14), which promotes ectodomain shedding of PDGF receptor β (PDGFRβ) from pericyte surface. MMP inhibitor treatment alleviates hippocampal pericyte loss and capillary reduction in the conditional Pdgfb transgenic mice and antagonizes BBB leakage in aged mice. The findings establish the role of bone-derived PDGF-BB in mediating hippocampal BBB disruption and identify the ligand-induced PDGFRβ shedding as a feedback mechanism for age-associated PDGFRβ downregulation and the consequent pericyte loss.
Topics: Animals; Mice; Becaplermin; Hippocampus; Mice, Knockout; Mice, Transgenic; Pericytes; Proto-Oncogene Proteins c-sis; Receptor, Platelet-Derived Growth Factor beta
PubMed: 37102631
DOI: 10.1002/advs.202206938 -
Cell Reports Aug 2023Circular RNAs are generated by backsplicing and control cellular signaling and phenotypes. Pericytes stabilize capillary structures and play important roles in the...
Circular RNAs are generated by backsplicing and control cellular signaling and phenotypes. Pericytes stabilize capillary structures and play important roles in the formation and maintenance of blood vessels. Here, we characterize hypoxia-regulated circular RNAs (circRNAs) in human pericytes and show that the circular RNA of procollagen-lysine,2-oxoglutarate 5-dioxygenase-2 (circPLOD2) is induced by hypoxia and regulates pericyte functions. Silencing of circPLOD2 affects pericytes and increases proliferation, migration, and secretion of soluble angiogenic proteins, thereby enhancing endothelial migration and network capability. Transcriptional and epigenomic profiling of circPLOD2-depleted cells reveals widespread changes in gene expression and identifies the transcription factor krüppel-like factor 4 (KLF4) as a key effector of the circPLOD2-mediated changes. KLF4 depletion mimics circPLOD2 silencing, whereas KLF4 overexpression reverses the effects of circPLOD2 depletion on proliferation and endothelial-pericyte interactions. Together, these data reveal an important function of circPLOD2 in controlling pericyte proliferation and capillary formation and show that the circPLOD2-mediated regulation of KLF4 significantly contributes to the transcriptional response to hypoxia.
Topics: Humans; Hypoxia; Pericytes; RNA, Circular
PubMed: 37481725
DOI: 10.1016/j.celrep.2023.112824 -
BioRxiv : the Preprint Server For... Jul 2023Implantation of the human embryo commences a critical developmental stage that comprises profound morphogenetic alteration of embryonic and extra-embryonic tissues, axis...
Implantation of the human embryo commences a critical developmental stage that comprises profound morphogenetic alteration of embryonic and extra-embryonic tissues, axis formation, and gastrulation events. Our mechanistic knowledge of this window of human life remains limited due to restricted access to samples for both technical and ethical reasons. Additionally, human stem cell models of early post-implantation development with both embryonic and extra-embryonic tissue morphogenesis are lacking. Here, we present iDiscoid, produced from human induced pluripotent stem cells via an engineered a synthetic gene circuit. iDiscoids exhibit reciprocal co-development of human embryonic tissue and engineered extra-embryonic niche in a model of human post-implantation. They exhibit unanticipated self-organization and tissue boundary formation that recapitulates yolk sac-like tissue specification with extra-embryonic mesoderm and hematopoietic characteristics, the formation of bilaminar disc-like embryonic morphology, the development of an amniotic-like cavity, and acquisition of an anterior-like hypoblast pole and posterior-like axis. iDiscoids offer an easy-to-use, high-throughput, reproducible, and scalable platform to probe multifaceted aspects of human early post-implantation development. Thus, they have the potential to provide a tractable human model for drug testing, developmental toxicology, and disease modeling.
PubMed: 37398391
DOI: 10.1101/2023.06.15.545118 -
The Journal of Experimental Medicine Feb 2024In dorsal root ganglia (DRG), macrophages reside close to sensory neurons and have largely been explored in the context of pain, nerve injury, and repair. However, we...
In dorsal root ganglia (DRG), macrophages reside close to sensory neurons and have largely been explored in the context of pain, nerve injury, and repair. However, we discovered that most DRG macrophages interact with and monitor the vasculature by sampling macromolecules from the blood. Characterization of the DRG vasculature revealed a specialized endothelial bed that transformed in molecular, structural, and permeability properties along the arteriovenous axis and was covered by macrophage-interacting pericytes and fibroblasts. Macrophage phagocytosis spatially aligned with peak endothelial permeability, a process regulated by enhanced caveolar transcytosis in endothelial cells. Profiling the DRG immune landscape revealed two subsets of perivascular macrophages with distinct transcriptome, turnover, and function. CD163+ macrophages self-maintained locally, specifically participated in vasculature monitoring, displayed distinct responses during peripheral inflammation, and were conserved in mouse and man. Our work provides a molecular explanation for the permeability of the blood-DRG barrier and identifies an unappreciated role of macrophages as integral components of the DRG-neurovascular unit.
Topics: Humans; Ganglia, Spinal; Endothelial Cells; Macrophages; Pericytes; Permeability
PubMed: 38117255
DOI: 10.1084/jem.20230675 -
Cancer Genomics & Proteomics 2023Angioleiomyoma is a benign tumor, occurs at any age, and arises most frequently in the lower extremities. Genetic information on angioleiomyomas is restricted to six...
BACKGROUND/AIM
Angioleiomyoma is a benign tumor, occurs at any age, and arises most frequently in the lower extremities. Genetic information on angioleiomyomas is restricted to six reported abnormal karyotypes, losses in chromosome 22 and gains in Xq found by comparative genomic hybridization, and mutation analysis of notch receptor 2 (NOTCH2), NOTCH3, platelet-derived growth factor receptor beta (PDGFRB), and mediator complex subunit 12 (MED12) in a few tumors. Herein, we report the genetic findings in another three angioleiomyomas.
MATERIALS AND METHODS
The tumors were examined using G-banding and karyotyping, RNA sequencing, reverse transcription-polymerase chain reaction, Sanger sequencing, and expression analysis.
RESULTS
The first tumor carried a t(4;5)(p12;q32) translocation resulting in fusion of the cardiac mesoderm enhancer-associated non-coding RNA (CARMN in 5q32) with the TXK tyrosine kinase gene (TXK in 4p12) leading to overexpression of TXK. To our knowledge, this is the first time that a recurrent chromosome translocation and its resulting fusion gene have been described in angioleiomyomas. The second tumor carried a four-way translocation, t(X;3;4;16)(q22;p11;q11;p13) which fused the myosin heavy chain 11 gene (MYH11 in 16p13) with intergenic sequences from Xq22 that mapped a few kilobase pairs distal to the insulin receptor substrate 4 gene (IRS4), resulting in enhanced IRS4 expression. The third angioleiomyoma carried another rearrangement of chromosome band Xq22, t(X;9)(q22;q32), as the sole cytogenetic aberration, but no material was available for further molecular investigation.
CONCLUSION
Our data, together with previously reported abnormal karyotypes in angioleiomyomas, show the presence of two recurrent genetic pathways in this tumor type: The first is characterized by presence of the translocation t(4;5)(p12;q32), which generates a CARMN::TXK chimera. The second is recurrent rearrangement of Xq22 resulting in overexpression of IRS4.
Topics: Humans; Angiomyoma; Comparative Genomic Hybridization; Chromosome Aberrations; Translocation, Genetic; Transcription Factors; Abnormal Karyotype
PubMed: 37889065
DOI: 10.21873/cgp.20405 -
ELife Nov 2023In vitro culture systems that structurally model human myogenesis and promote PAX7 myogenic progenitor maturation have not been established. Here we report that human...
In vitro culture systems that structurally model human myogenesis and promote PAX7 myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single-cell comparison to human fetal and adult myogenic progenitor /satellite cells reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (//) and dormant (//) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis.
Topics: Humans; Muscle, Skeletal; Cell Differentiation; Fetus; Satellite Cells, Skeletal Muscle; Muscle Development; PAX7 Transcription Factor
PubMed: 37963071
DOI: 10.7554/eLife.87081