Bioengineering strategies for the fabrication of implantable lymphoid structures mimicking lymph nodes (LNs) and tertiary lymphoid structures (TLS) could amplify the adaptive immune response for therapeutic applications such as cancer immunotherapy. No method to date has resulted in the consistent formation of high endothelial venules (HEVs), which is the specialized vasculature responsible for naïve T cell recruitment and education in both LNs and TLS. Here orthogonal induced differentiation of human pluripotent stem cells carrying a regulatable ETV2 allele is used to rapidly and efficiently induce endothelial differentiation. Assembly of embryoid bodies combining primitive inducible endothelial cells and primary human LN fibroblastic reticular cells results in the formation of HEV-like structures that can aggregate into 3D organoids (HEVOs). Upon transplantation into immunodeficient mice, HEVOs successfully engraft and form lymphatic structures that recruit both antigen-presenting cells and adoptively-transferred lymphocytes, therefore displaying basic TLS capabilities. The results further show that functionally, HEVOs can organize an immune response and promote anti-tumor activity by adoptively-transferred T lymphocytes. Collectively, the experimental approaches represent an innovative and scalable proof-of-concept strategy for the fabrication of bioengineered TLS that can be deployed in vivo to enhance adaptive immune responses.

A number of genetic kidney diseases can now be replicated experimentally, using kidney organoids generated from human pluripotent stem cells. This methodology holds great potential for drug discovery. Under in vitro conditions, however, kidney organoids remain developmentally immature, develop scarce vasculature, and may contain undesired off-target cell types. Those critical deficiencies limit their potential as disease-modeling tools. Orthotopic transplantation under the kidney capsule improves the anatomic maturity and vascularization of kidney organoids, while reducing off-target cell content. The improvements can translate into more accurate representations of disease phenotypes and mechanisms in vivo . Recent studies using kidney organoid xenografts highlighted the unique potential of this novel methodology for elucidating molecular mechanisms driving monogenic kidney disorders and for the development ofnovel pharmacotherapies.

The lack of animal models for some human diseases precludes our understanding of disease mechanisms and our ability to test prospective therapies in vivo. Generation of kidney organoids from Tuberous Sclerosis Complex (TSC) patient-derived-hiPSCs allows us to recapitulate a rare kidney tumor called angiomyolipoma (AML). Organoids derived from TSC2 −/− hiPSCs but not from isogenic TSC2 +/− or TSC2 +/+ hiPSCs share a common transcriptional signature and a myomelanocytic cell phenotype with kidney AMLs, and develop epithelial cysts, replicating two major TSC-associated kidney lesions driven by genetic mechanisms that cannot be consistently recapitulated with transgenic mice. Transplantation of multiple TSC2 −/− renal organoids into the kidneys of immunodeficient rats allows us to model AML in vivo for the study of tumor mechanisms, and to test the efficacy of rapamycin-loaded nanoparticles as an approach to rapidly ablate AMLs. Collectively, our experimental approaches represent an innovative and scalable tissue-bioengineering strategy for modeling rare kidney disease in vivo.

The mechanisms of kidney injury and fibrosis can now be studied using kidney organoids derived from human pluripotent stem cells (hPSCs). Mature kidney organoids contain nephrons and stromal cells with fibrogenic potential, spatially organized in a manner that resembles the anatomy of the kidney. Organoid nephron damage and interstitial fibrosis can be induced under well-controlled experimental conditions in vitro, making this an ideal system for the study of tissue-intrinsic cell signaling and intercellular crosstalk mechanisms in the absence of systemic signals and immune cells that are present in vivo. Here we describe methods for the generation of kidney organoids from a widely used hPSC line, and for the induction and analysis of nephron damage and interstitial fibrosis.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs. This study reveals that upon injury, COUP-TFII is upregulated in myofibroblasts and promotes fibrosis in human fibrotic diseases through augmented glycolysis of the myofibroblasts. This study reveals that upon injury, COUP-TFII is upregulated in myofibroblasts and promotes fibrosis in human fibrotic diseases through augmented glycolysis of the myofibroblasts.

Recent studies demonstrated that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in kidneys of patients with chronic kidney disease, fibrotic lungs of patients with idiopathic pulmonary fibrosis, fibrotic human kidney organoids, and fibrotic mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts was sufficient to enhance glycolysis and increase alpha smooth muscle actin (αSMA) and collagen1 levels. Knockout of COUP-TFII decreased glycolysis and collagen1 levels in fibroblasts. Chip-qPCR assays revealed the binding of COUP-TFII on the promoter of PGC1α, a critical regulator of mitochondrial genesis and oxidative metabolism. Overexpression of COUP-TFII reduced the cellular level of PGC1α. In conclusion, COUP-TFII mediates fibrosis by serving as a key regulator of the shift in cellular metabolism of interstitial pericytes/fibroblasts from oxidative respiration to aerobic glycolysis. The fibrogenic response may share a common pathway in different organ injury and failure. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potential other organ fibrosis.

The cardiac stroma contains multipotent mesenchymal progenitors. However, lineage relationships within cardiac stromal cells are poorly defined. Here, we identified heart-resident PDGFRa(+) SCA-1(+) cells as cardiac fibro/adipogenic progenitors (cFAPs) and show that they respond to ischemic damage by generating fibrogenic cells. Pharmacological blockade of this differentiation step with an anti-fibrotic tyrosine kinase inhibitor decreases post-myocardial infarction (post-MI) remodeling and leads to improvement in cardiac function. In the undamaged heart, activation of cFAPs through lineage-specific deletion of the gene encoding the quiescence-associated factor HIC1 reveals additional pathogenic potential, causing fibrofatty infiltration within the myocardium and driving major pathological features pathognomonic in arrhythmogenic cardiomyopathy (AC). In this regard, cFAPs contribute to multiple pathogenic cell types within cardiac tissue and therapeutic strategies aimed at modifying their activity are expected to have tremendous benefit for the treatment of diverse cardiac diseases.

Although the immune response within draining lymph nodes (DLNs) has been studied for decades, how their stromal compartment contributes to this process remains to be fully explored. Here, we show that donor mast cells were prominent activators of collagen I deposition by fibroblastic reticular cells (FRCs) in DLNs shortly following transplantation. Serial analysis of the DLN indicated that the LN stroma did not return to its baseline microarchitecture following organ rejection and that the DLN contained significant fibrosis following repetitive organ transplants. Using several FRC conditional-knockout mice, we show that induction of senescence in the FRCs of the DLN resulted in massive production of collagen I and a proinflammatory milieu within the DLN. Stimulation of herpes virus entry mediator (HVEM) on FRCs by its ligand LIGHT contributed chiefly to the induction of senescence in FRCs and overproduction of collagen I. Systemic administration of ex vivo-expanded FRCs to mice decreased DLN fibrosis and strengthened the effect of anti-CD40L in prolonging heart allograft survival. These data demonstrate that the transformation of FRCs into proinflammatory myofibroblasts is critically important for the maintenance of a proinflammatory milieu within a fibrotic DLN.

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.

Recent scientific findings support the notion that fibrosis is driven by tissue-specific cellular and molecular mechanisms. Analysis of seemingly equivalent mesenchymal stromal cell (MSC) populations residing in different organs revealed unique properties and lineage capabilities that vary from one anatomical location to another. We review recently characterized tissue-resident MSC populations with a prominent role in fibrosis and highlight therapeutically relevant molecular pathways regulating their activity in chronic disease.

Tuberous sclerosis complex (TSC) is a genetic disorder affecting multiple organ systems observed in approximately 1 in 10,000 live births.1,2 Patients with TSC have a higher risk of developing benign tumors in the brain, kidneys, heart, liver, lungs, and skin, and a variety of neuropsychiatric disorders.3 However, due to the rarity of TSC, its comorbidity landscape has not been fully investigated. To characterize the comorbidities of TSC systematically, we employed a nationwide health insurance claims dataset to analyze the comorbidity landscape of TSC and determined the phenotypical differences between patients with TSC and participants without TSC matched on age and sex.

Background Kidney injury is characterized by persisting inflammation and fibrosis, yet mechanisms by which inflammatory signals drive fibrogenesis remain poorly defined. Methods RNA sequencing of fibrotic kidneys from patients with CKD identified a metabolic gene signature comprising loss of mitochondrial and oxidative phosphorylation gene expression with a concomitant increase in regulators and enzymes of glycolysis under the control of PGC1 α and MYC transcription factors, respectively. We modeled this metabolic switch in vivo , in experimental murine models of kidney injury, and in vitro in human kidney stromal cells (SCs) and human kidney organoids. Results In mice, MYC and the target genes thereof became activated in resident SCs early after kidney injury, suggesting that acute innate immune signals regulate this transcriptional switch. In vitro , stimulation of purified human kidney SCs and human kidney organoids with IL-1 β recapitulated the molecular events observed in vivo , inducing functional metabolic derangement characterized by increased MYC-dependent glycolysis, the latter proving necessary to drive proliferation and matrix production. MYC interacted directly with sequestosome 1/p62, which is involved in proteasomal degradation, and modulation of p62 expression caused inverse effects on MYC expression. IL-1 β stimulated autophagy flux, causing degradation of p62 and accumulation of MYC. Inhibition of the IL-1R signal transducer kinase IRAK4 in vivo or inhibition of MYC in vivo as well as in human kidney organoids in vitro abrogated fibrosis and reduced tubular injury. Conclusions Our findings define a connection between IL-1 β and metabolic switch in fibrosis initiation and progression and highlight IL-1 β and MYC as potential therapeutic targets in tubulointerstitial diseases.

Acute skeletal muscle injury triggers an expansion of fibro/adipogenic progenitors (FAPs) and a transient stage of fibrogenesis characterized by extracellular matrix deposition. While the perpetuation of such phase can lead to permanent tissue scarring, the consequences of its suppression remain to be studied. Using a model of acute muscle damage we were able to determine that pharmacological inhibition of FAP expansion by Nilotinib, a tyrosine kinase inhibitor with potent antifibrotic activity, exerts a detrimental effect on myogenesis during regeneration. We found that Nilotinib inhibits the damage-induced expansion of satellite cells in vivo, but it does not affect in vitro proliferation, suggesting a non cell-autonomous effect. Nilotinib impairs regenerative fibrogenesis by preventing the injury-triggered expansion and differentiation of resident CD45−:CD31−:α7integrin−:Sca1+ mesenchymal FAPs. Our data support the notion that the expansion of FAPs and transient fibrogenesis observed during regeneration play an important trophic role toward tissue-specific stem cells.