Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF). The aim of this study was to test the therapeutic effects of MSC-extracellular vesicles/exosomes (MEx) in a bleomycin-induced pulmonary fibrosis model and investigate putative mechanisms of action. Exosomes were isolated from media conditioned by human bone marrow MSCs. Adult mice (C57BL/6 strain) were challenged with endotracheal instillation of bleomycin and treated with MEx concurrently or for reversal models, at day 7 or 21. Experimental groups were assessed at day 7 and/or at day 14 or 28. Bleomycin-challenged mice presented with severe septal thickening and prominent fibrosis, and this was effectively prevented or reversed by a single dose of MEx. Furthermore, MEx therapy modulated whole lung macrophage phenotype and shifted the proportion of lung ‘proinflammatory’ classical monocytes, non-classical monocytes and alveolar macrophages to favor the monocyte/macrophage profiles of untreated-control mice. A parallel immunomodulatory effect was demonstrated in the bone marrow. Notably, transplantation of MEx-preconditioned bone marrow-derived monocytes alleviated core features of pulmonary fibrosis and lung inflammation. Proteomic analysis further revealed a signature enriched in non-inflammatory monocyte genes following MEx therapy supporting the immuno-regulatory, anti-inflammatory effect of MEx.We conclude that a bolus dose of MEx prevents and reverts core features of bleomycin-induced pulmonary fibrosis, and that the beneficial actions of MEx may be mediated via systemic modulation of monocyte phenotypes.
Nahal Mansouri, Gareth R. Willis, Angeles Fernandez-Gonzalez, Monica Reis, Sina Nassiri, Alex Mitsialis, Stella Kourembanas
The control of voluntary skeletal muscle contraction relies on action potentials, which send signals from the motor neuron through the neuromuscular junction (NMJ). Although dysfunction of the NMJ causes various neuromuscular diseases, a reliable in vitro system for disease modeling is currently unavailable. Here, we present a potentially novel 2-step, self-organizing approach for generating in vitro human NMJs from human induced pluripotent stem cells. Our simple and robust approach results in a complex NMJ structure that includes functional connectivity, recapitulating in vivo synapse formation. We used these in vitro NMJs to model the pathological features of spinal muscular atrophy, revealing the developmental and functional defects of NMJ formation and NMJ-dependent muscular contraction. Our differentiation system is therefore useful for investigating and understanding the physiology and pathology of human NMJs.
Chuang-Yu Lin, Michiko Yoshida, Li-Tzu Li, Akihiro Ikenaka, Shiori Oshima, Kazuhiro Nakagawa, Hidetoshi Sakurai, Eriko Matsui, Tatsutoshi Nakahata, Megumu K. Saito
The complex process of platelet formation originates with the hematopoietic stem cell, which differentiates through the myeloid lineage, matures, and releases proplatelets into the BM sinusoids. How formed platelets maintain a low basal activation state in the circulation remains unknown. We identify Lepr+ stromal cells lining the BM sinusoids as important contributors to sustaining low platelet activation. Ablation of murine Lepr+ cells led to a decreased number of platelets in the circulation with an increased activation state. We developed a potentially novel culture system for supporting platelet formation in vitro using a unique population of CD51+PDGFRα+ perivascular cells, derived from human umbilical cord tissue, which display numerous mesenchymal stem cell (MSC) properties. Megakaryocytes cocultured with MSCs had altered LAT and Rap1b gene expression, yielding platelets that are functional with low basal activation levels, a critical consideration for developing a transfusion product. Identification of a regulatory cell that maintains low baseline platelet activation during thrombopoiesis opens up new avenues for improving blood product production ex vivo.
Avital Mendelson, Ana Nicolle Strat, Weili Bao, Peter Rosston, Georgia Fallon, Sophie Ohrn, Hui Zhong, Cheryl Lobo, Xiuli An, Karina Yazdanbakhsh
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease with diverse etiologies. Therefore, the identification of common disease mechanisms and therapeutics targeting these mechanisms could dramatically improve clinical outcomes. To this end, we developed induced motor neuron (iMN) models from C9ORF72 and sporadic ALS (sALS) patients to identify targets that are effective against these types of cases, which together comprise ~90% of patients. We find that iMNs from C9ORF72 and several sporadic ALS patients share two common defects – impaired autophagosome formation and the aberrant accumulation of glutamate receptors. Moreover, we show that an anticoagulation-deficient form of activated protein C, 3K3A-APC, rescues these defects in both C9ORF72 and sporadic ALS iMNs. As a result, 3K3A-APC treatment lowers C9ORF72 dipeptide repeat protein (DPR) levels, restores nuclear TDP-43 localization, and rescues the survival of both C9ORF72 and sporadic ALS iMNs. Importantly, 3K3A-APC also lowers glutamate receptor levels and rescues proteostasis in vivo in C9ORF72 gain- and loss-of-function mouse models. Thus, motor neurons from C9ORF72 and at least a subset of sporadic ALS patients share common, early defects in autophagosome formation and glutamate receptor homeostasis and a single therapeutic approach may be efficacious against these disease processes.
Yingxiao Shi, Shu-Ting Hung, Gabriel Rocha, Shaoyu Lin, Gabriel R. Linares, Kim A. Staats, Carina Seah, Yaoming Wang, Michael Chickering, Jesse Lai, Tohru Sugawara, Abhay P. Sagare, Berislav V. Zlokovic, Justin K. Ichida
The lung is a relatively quiescent organ during homeostasis, but has a remarkable capacity for repair after injury. Alveolar epithelial type I cells (AEC1s) line airspaces and mediate gas exchange. After injury, they are regenerated by differentiation from their progenitors — alveolar epithelial type II cells (AEC2s) — which also secrete surfactant to maintain surface tension and alveolar patency. While recent studies showed that the maintenance of AEC2 stemness is Wnt dependent, the molecular mechanisms underlying AEC2-AEC1 differentiation in adult lung repair are still incompletely understood. Here we show that WWTR1 (TAZ) plays a crucial role in AEC differentiation. Using an in vitro organoid culture system, we found that tankyrase inhibition can efficiently block AEC2-AEC1 differentiation, and this effect was due to the inhibition of TAZ. In a bleomycin induced lung injury model, conditional deletion of TAZ in AEC2s dramatically reduced AEC1 regeneration during recovery, leading to exacerbated alveolar lesions and fibrosis. In patients with idiopathic pulmonary fibrosis (IPF), decreased blood levels of RAGE, a biomarker of AEC1 health, were associated with more rapid disease progression. Our findings implicate TAZ as a critical factor involved in AEC2 to AEC1 differentiation, and hence the maintenance of alveolar integrity after injury.
Tianhe Sun, Zhiyu Huang, Hua Zhang, Clara Posner, Guiquan Jia, Thirumalai R. Ramalingam, Min Xu, Hans D. Brightbill, Jackson G. Egen, Anwesha Dey, Joseph R. Arron
Dystrophin deficiency leads to progressive muscle degeneration in Duchenne muscular dystrophy (DMD) patients. No known cure exists, and standard care relies on the use of antiinflammatory steroids, which are associated with side effects that complicate long-term use. Here, we report that a single intravenous dose of clinical-stage cardiac stromal cells, called cardiosphere-derived cells (CDCs), improves the dystrophic phenotype in mdx mice. CDCs augment cardiac and skeletal muscle function, partially reverse established heart damage, and boost the regenerative capacity of skeletal muscle. We further demonstrate that CDCs work by secreting exosomes, which normalize gene expression at the transcriptome level, and alter cell signaling and biological processes in mdx hearts and skeletal muscle. The work reported here motivated the ongoing HOPE-2 clinical trial of systemic CDC delivery to DMD patients, and identifies exosomes as next-generation cell-free therapeutic candidates for DMD.
Russell G. Rogers, Mario Fournier, Lizbeth Sanchez, Ahmed G. Ibrahim, Mark A. Aminzadeh, Michael I. Lewis, Eduardo Marbán
Glioblastoma represent universally lethal cancers, containing stem cell-like glioblastoma stem cells (GSCs). While neural stem cells (NSCs) are usually quiescent, single-cell studies suggest that proliferating glioblastoma cells reside in the GSC population. Interrogating in silico glioma databases for epigenetic regulators that correlate with cell cycle regulation, we identified the chromatin remodeler, HELLS, as a potential target in glioblastoma. GSCs preferentially expressed HELLS compared to their differentiated tumor progeny and non-malignant brain cells. Targeting HELLS disrupted GSC proliferation, survival, and self-renewal with induction of replication stress and DNA damage. Investigating potential molecular mechanisms downstream of HELLS revealed that HELLS interacted with the core oncogenic transcription factors, E2F3 and MYC, to regulate gene expression critical to GSC proliferation and maintenance. Supporting the interaction, HELLS expression strongly correlated with targets of E2F3 and MYC transcriptional activity in glioblastoma patients. Potential clinical significance of HELLS was reinforced by improved survival of tumor-bearing mice upon targeting HELLS and poor prognosis of glioma patients with elevated HELLS expression. Collectively, targeting HELLS may permit the functional disruption of the relatively undruggable MYC and E2F3 transcription factors and serve as a novel therapeutic paradigm for glioblastoma.
Guoxin Zhang, Zhen Dong, Briana C. Prager, Leo J. Y. Kim, Qiulian Wu, Ryan C. Gimple, Xiuxing Wang, Shideng Bao, Petra Hamerlik, Jeremy N. Rich
Myotonic dystrophy (DM) is the most common autosomal dominant muscular dystrophy and encompasses both skeletal muscle and cardiac complications. Myotonic dystrophy is nucleotide repeat expansion disorder in which type 1 (DM1) is due to a trinucleotide repeat expansion on chromosome 19 and type 2 (DM2) arises from a tetranucleotide repeat expansion on chromosome 3. Developing representative models of myotonic dystrophy in animals has been challenging due to instability of nucleotide repeat expansions, especially for DM2 which is characterized by nucleotide repeat expansions often greater than 5000 copies. To investigate mechanisms of human DM, we generated cellular models of DM1 and DM2. We used regulated MyoD expression to reprogram urine-derived cells into myotubes. In this myogenic cell model, we found impaired dystrophin expression, MBNL foci, and aberrant splicing in DM1 but not in DM2 cells. We generated induced pluripotent stem cells (iPSC) from healthy controls, DM1 and DM2 subjects and differentiated these into cardiomyocytes. DM1 and DM2 cells displayed an increase in RNA foci concomitant with cellular differentiation. IPSC-derived cardiomyocytes from DM1 but not DM2 had aberrant splicing of known target genes and MBNL sequestration. High resolution imaging revealed tight association between MBNL clusters and RNA FISH foci in DM1. Ca2+ transients differed between DM1 and DM2 IPSC-derived cardiomyocytes and each differed from healthy control cells. RNA-sequencing from DM1 and DM2 iPSC-derived cardiomyocytes revealed distinct misregulation of gene expression as well as differential aberrant splicing patterns. Together these data support that DM1 and DM2, despite some shared clinical and molecular features, have distinct pathological signatures.
Ellis Y. Kim, David Y. Barefield, Andy H. Vo, Anthony M. Gacita, Emma J. Schuster, Eugene J. Wyatt, Janel L. Davis, Biqin Dong, Cheng Sun, Patrick Page, Lisa Dellefave-Castillo, Alexis Demonbreun, Hao F. Zhang, Elizabeth M. McNally
Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT). DCs play critical roles in GVHD induction. Modulating autophagy represents a promising therapeutic strategy for the treatment of immunological diseases. Complement receptors C3aR/C5aR expressed on DCs regulate immune responses by translating extracellular signals into intracellular activity. In the current study, we found that C3aR/C5aR deficiency enhanced ceramide-dependent lethal mitophagy (CDLM) in DCs. Cotransfer of host-type C3aR–/–/C5aR–/– DCs in the recipients significantly improved GVHD outcome after allogeneic HCT, primarily through enhancing CDLM in DCs. C3aR/C5aR deficiency in the host hematopoietic compartment significantly reduced GVHD severity via impairing Th1 differentiation and donor T cell glycolytic activity while enhancing Treg generation. Prophylactic treatment with C3aR/C5aR antagonists effectively alleviated GVHD while maintaining the graft-versus-leukemia (GVL) effect. Altogether, we demonstrate that inhibiting C3aR/C5aR induces lethal mitophagy in DCs, which represents a potential therapeutic approach to control GVHD while preserving the GVL effect.
Hung Nguyen, Sandeepkumar Kuril, David Bastian, Jisun Kim, Mengmeng Zhang, Silvia G. Vaena, Mohammed Dany, Min Dai, Jessica Lauren Heinrichs, Anusara Daenthanasanmak, Supinya Iamsawat, Steven Schutt, Jianing Fu, Yongxia Wu, David P. Fairlie, Carl Atkinson, Besim Ogretmen, Stephen Tomlinson, Xue-Zhong Yu
In high-grade serous ovarian cancer (OC), chemotherapy eliminates the majority of tumor cells, leaving behind residual tumors enriched in OC stem cells (OCSC). OCSC, defined as aldehyde dehydrogenase–positive (ALDH+), persist and contribute to tumor relapse. Inflammatory cytokine IL-6 is elevated in residual tumors after platinum treatment, and we hypothesized that IL-6 plays a critical role in platinum-induced OCSC enrichment. We demonstrate that IL-6 regulates stemness features of OCSC driven by ALDH1A1 expression and activity. We show that platinum induces IL-6 secretion by cancer-associated fibroblasts in the tumor microenvironment, promoting OCSC enrichment in residual tumors after chemotherapy. By activating STAT3 and upregulating ALDH1A1 expression, IL-6 treatment converted non-OCSC to OCSC. Having previously shown altered DNA methylation in OCSC, we show here that IL-6 induces DNA methyltransferase 1 (DNMT1) expression and the hypomethylating agent (HMA) guadecitabine induced differentiation of OCSC and reduced — but did not completely eradicate — OCSC. IL-6 neutralizing antibody (IL-6-Nab) combined with HMA fully eradicated OCSC, and the combination blocked IL-6/IL6-R/pSTAT3–mediated ALDH1A1 expression and eliminated OCSC in residual tumors that persisted in vivo after chemotherapy. We conclude that IL-6 signaling blockade combined with an HMA can eliminate OCSC after platinum treatment, supporting this strategy to prevent tumor recurrence after standard chemotherapy.
Yinu Wang, Xingyue Zong, Sumegha Mitra, Anirban Kumar Mitra, Daniela Matei, Kenneth P. Nephew
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