Rapid Generation of hPSC-Derived High Endothelial Venule Organoids with In Vivo Ectopic Lymphoid Tissue Capabilities.

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.

Can Kidney Organoid Xenografts Accelerate Therapeutic Development for Genetic Kidney Disorders?

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.

Nanostructured Non-Newtonian Drug Delivery Barrier Prevents Postoperative Intrapericardial Adhesions.

With the increasing volume of cardiovascular surgeries and the rising adoption rate of new methodologies that serve as a bridge to cardiac transplantation and that require multiple surgical interventions, the formation of postoperative intrapericardial adhesions has become a challenging problem that limits future surgical procedures, causes serious complications, and increases medical costs. To prevent this pathology, we developed a nanotechnology-based self-healing drug delivery hydrogel barrier composed of silicate nanodisks and polyethylene glycol with the ability to coat the epicardial surface of the heart without friction and locally deliver dexamethasone, an anti-inflammatory drug. After the fabrication of the hydrogel, mechanical characterization and responses to shear, strain, and recovery were analyzed, confirming its shear-thinning and self-healing properties. This behavior allowed its facile injection (5.75 ± 0.15 to 22.01 ± 0.95 N) and subsequent mechanical recovery. The encapsulation of dexamethasone within the hydrogel system was confirmed by 1H NMR, and controlled release for 5 days was observed. In vitro, limited cellular adhesion to the hydrogel surface was achieved, and its anti-inflammatory properties were confirmed, as downregulation of ICAM-1 and VCAM-1 was observed in TNF-α activated endothelial cells. In vivo, 1 week after administration of the hydrogel to a rabbit model of intrapericardial injury, superior efficacy was observed when compared to a commercial adhesion barrier, as histological and immunohistochemical examination revealed reduced adhesion formation and minimal immune infiltration of CD3+ lymphocytes and CD68+ macrophages, as well as NF-κß downregulation. We presented a novel nanostructured drug delivery hydrogel system with unique mechanical and biological properties that act synergistically to prevent cellular infiltration while providing local immunomodulation to protect the intrapericardial space after a surgical intervention.

Orphan nuclear receptor COUP-TFII enhances myofibroblast glycolysis leading to kidney 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.

Methods for the Study of Renal Fibrosis in Human Pluripotent Stem Cell-Derived Kidney Organoids.

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.

Lymph node fibroblastic reticular cells deposit fibrosis-associated collagen following organ transplantation.

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.

Pathogenic Potential of Hic1-Expressing Cardiac Stromal Progenitors.

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.

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

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.

Interleukin-1ß Activates a MYC-Dependent Metabolic Switch in Kidney Stromal Cells Necessary for Progressive Tubulointerstitial Fibrosis.

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 PGC1α 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-1ß 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-1ß 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-1ß and metabolic switch in fibrosis initiation and progression and highlight IL-1ß and MYC as potential therapeutic targets in tubulointerstitial diseases.

Tissue-resident mesenchymal stromal cells: Implications for tissue-specific antifibrotic therapies.

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.

Pharmacological blockage of fibro/adipogenic progenitor expansion and suppression of regenerative fibrogenesis is associated with impaired skeletal muscle regeneration.

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.

Maintenance of vascular integrity by pericytes is essential for normal kidney function.

Pericytes are tissue-resident mesenchymal progenitor cells anatomically associated with the vasculature that have been shown to participate in tissue regeneration. Here, we tested the hypothesis that kidney pericytes, derived from FoxD1+ mesodermal progenitors during embryogenesis, are necessary for postnatal kidney homeostasis. Diphtheria toxin delivery to FoxD1Cre::RsDTR transgenic mice resulted in selective ablation of >90% of kidney pericytes but not other cell lineages. Abrupt increases in plasma creatinine, blood urea nitrogen, and albuminuria within 96 h indicated acute kidney injury in pericyte-ablated mice. Loss of pericytes led to a rapid accumulation of neutral lipid vacuoles, swollen mitochondria, and apoptosis in tubular epithelial cells. Pericyte ablation led to endothelial cell swelling, reduced expression of vascular homeostasis markers, and peritubular capillary loss. Despite the observed injury, no signs of the acute inflammatory response were observed. Pathway enrichment analysis of genes expressed in kidney pericytes in vivo identified basement membrane proteins, angiogenic factors, and factors regulating vascular tone as major regulators of vascular function. Using novel microphysiological devices, we recapitulated human kidney peritubular capillaries coated with pericytes and showed that pericytes regulate permeability, basement membrane deposition, and microvascular tone. These findings suggest that through the active support of the microvasculature, pericytes are essential to adult kidney homeostasis.

Skeletal muscle-resident MSCs and bone formation.

Recent research has highlighted the importance of bone and muscle interactions during development and regeneration. There still remains, however, a large gap in the current understanding of the cells and mechanisms involved in this interplay. In particular, how muscle-derived cells, specifically mesenchymal stromal cells (MSCs), can impact bone regeneration or lead to pathologic ectopic bone formation is unclear. Here, a review is given of the evidence supporting the contribution of muscle-derived MSC to bone regeneration and suggesting a critical role for the inflammatory milieu. This article is part of a Special Issue entitled "Muscle Bone Interactions".

Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors.

Depending on the inflammatory milieu, injury can result either in a tissue's complete regeneration or in its degeneration and fibrosis, the latter of which could potentially lead to permanent organ failure. Yet how inflammatory cells regulate matrix-producing cells involved in the reparative process is unknown. Here we show that in acutely damaged skeletal muscle, sequential interactions between multipotent mesenchymal progenitors and infiltrating inflammatory cells determine the outcome of the reparative process. We found that infiltrating inflammatory macrophages, through their expression of tumor necrosis factor (TNF), directly induce apoptosis of fibro/adipogenic progenitors (FAPs). In states of chronic damage, however, such as those in mdx mice, macrophages express high levels of transforming growth factor ß1 (TGF-ß1), which prevents the apoptosis of FAPs and induces their differentiation into matrix-producing cells. Treatment with nilotinib, a kinase inhibitor with proposed anti-fibrotic activity, can block the effect of TGF-ß1 and reduce muscle fibrosis in mdx mice. Our findings reveal an unexpected anti-fibrotic role of TNF and suggest that disruption of the precisely timed progression from a TNF-rich to a TGF-ß-rich environment favors fibrotic degeneration of the muscle during chronic injury.

In vivo characterization of neural crest-derived fibro/adipogenic progenitor cells as a likely cellular substrate for craniofacial fibrofatty infiltrating disorders.

The cellular substrate underlying aberrant craniofacial connective tissue accumulation that occurs in disorders such as congenital infiltration of the face (CILF) remain elusive. Here we analyze the in vivo properties of a recently identified population of neural crest-derived CD31-:CD45-:alpha7-:Sca1+:PDGFRa+ fibro/adipogenic progenitors (NCFAPs). In serial transplantation experiments in which NCFAPs were prospectively purified and transplanted into wild type mice, NCFAPs were found to be capable of self-renewal while keeping their adipogenic potential. NCFAPs constitute the main responsive FAP fraction following acute masseter muscle damage, surpassing the number of mesoderm-derived FAPs (MFAPs) during the regenerative response. Lastly, NCFAPs differentiate into adipocytes during muscle regeneration in response to pro-adipogenic systemic cues. Altogether our data indicate that NCFAPs are a population of stem/primitive progenitor cells primarily involved in craniofacial muscle regeneration that can cause tissue degeneration when the damage co-occurs with an obesity inducing diet.

Role of stem/progenitor cells in reparative disorders.

Adult stem cells are activated to proliferate and differentiate during normal tissue homeostasis as well as in disease states and injury. This activation is a vital component in the restoration of function to damaged tissue via either complete or partial regeneration. When regeneration does not fully occur, reparative processes involving an overproduction of stromal components ensure the continuity of tissue at the expense of its normal structure and function, resulting in a "reparative disorder". Adult stem cells from multiple organs have been identified as being involved in this process and their role in tissue repair is being investigated. Evidence for the participation of mesenchymal stromal cells (MSCs) in the tissue repair process across multiple tissues is overwhelming and their role in reparative disorders is clearly demonstrated, as is the involvement of a number of specific signaling pathways. Transforming growth factor beta, bone morphogenic protein and Wnt pathways interact to form a complex signaling network that is critical in regulating the fate choices of both stromal and tissue-specific resident stem cells (TSCs), determining whether functional regeneration or the formation of scar tissue follows an injury. A growing understanding of both TSCs, MSCs and the complex cascade of signals regulating both cell populations have, therefore, emerged as potential therapeutic targets to treat reparative disorders. This review focuses on recent advances on the role of these cells in skeletal muscle, heart and lung tissues.

Functionally convergent white adipogenic progenitors of different lineages participate in a diffused system supporting tissue regeneration.

Pathologies characterized by lipomatous infiltration of craniofacial structures as well as certain forms of lipodystrophies suggest the existence of a distinct adipogenic program in the cephalic region of mammals. Using lineage tracing, we studied the origin of craniofacial adipocytes that accumulate both in cranial fat depots and during ectopic lipomatous infiltration of craniofacial muscles. We found that unlike their counterparts in limb muscle, a significant percentage of cranial adipocytes is derived from the neural crest (NC). In addition, we identified a population of NC-derived Lin(-)/α7(-)/CD34(+)/Sca-1(+) fibro/adipogenic progenitors (NC-FAPs) that resides exclusively in the mesenchyme of cephalic fat and muscle. Comparative analysis of the adipogenic potential, impact on metabolism, and contribution to the regenerative response of NC-FAPs and mesoderm-derived FAPs (M-FAPs) suggests that these cells are functionally indistinguishable. While both NC- and M-FAPs express mesenchymal markers and promyogenic cytokines upon damage-induced activation, NC-FAPs additionally express components of the NC developmental program. Furthermore, we show that craniofacial FAP composition changes with age, with young mice containing FAPs that are almost exclusively of NC origin, while NC-FAPs are progressively replaced by M-FAPs as mice age. Based on these results, we propose that in the adult, ontogenetically distinct FAPs form a diffused system reminiscent of the endothelium, which can originate from multiple developmental intermediates to seed all anatomical locations.