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
Sudden death is the most common mode of exodus in patients with heart failure and preserved ejection fraction (HFpEF). Cardiosphere-derived cells (CDCs) reduce inflammation and fibrosis in a rat model of HFpEF, improving diastolic function and prolonging survival. We tested the hypothesis that CDCs decrease ventricular arrhythmias (VAs) and thereby possibly contribute to prolonged survival. Dahl salt-sensitive rats were fed a high-salt diet to induce HFpEF. Allogeneic rat CDCs (or phosphate-buffered saline as placebo) were injected in rats with echo-verified HFpEF. CDC-injected HFpEF rats were less prone to VA induction by programmed electrical stimulation. Action potential duration (APD) was shortened, and APD homogeneity was increased by CDC injection. Transient outward potassium current density was upregulated in cardiomyocytes from CDC rats relative to placebo, as were the underlying transcript (Kcnd3) and protein (Kv4.3) levels. Fibrosis was attenuated in CDC-treated hearts, and survival was increased. Sudden death risk also trended down, albeit nonsignificantly. CDC therapy decreased VA in HFpEF rats by shortening APD, improving APD homogeneity, and decreasing fibrosis. Unlike other stem/progenitor cells, which often exacerbate arrhythmias, CDCs reverse electrical remodeling and suppress arrhythmogenesis in HFpEF.
Jae Hyung Cho, Peter J. Kilfoil, Rui Zhang, Ryan E. Solymani, Catherine Bresee, Elliot M. Kang, Kristin Luther, Russell G. Rogers, Geoffrey de Couto, Joshua I. Goldhaber, Eduardo Marbán, Eugenio Cingolani
The maintenance of effective immunity over time is dependent on the capacity of hematopoietic stem cells (HSCs) to sustain the pool of immunocompetent mature cells. Decline of immune competence with old age may stem from HSC defects, including reduced self-renewal potential and impaired lymphopoiesis, as suggested in murine models. To obtain further insights into aging-related alteration of hematopoiesis, we performed a comprehensive study of blood hematopoietic progenitor cells (HPCs) from older humans. In the elderly, HPCs present active oxidative phosphorylation and are pressed to enter cell cycling. However, p53-p21 and p15 cell senescence pathways, associated with telomerase activity deficiency, strong telomere attrition, and oxidative stress, are engaged, thus limiting cell cycling. Moreover, survival of old HPCs is impacted by pyroptosis, an inflammatory form of programmed cell death. Lastly, telomerase activity deficiency and telomere length attrition of old HPCs may be passed on to progeny cells such as naive T lymphocytes, further highlighting the poor hematopoietic potential of the elderly. This pre-senescent profile is characteristic of the multiple intrinsic and extrinsic factors affecting HPCs in elderly individuals and represents a major obstacle in terms of immune reconstitution and efficacy with advanced age.
Tinhinane Fali, Véronique Fabre-Mersseman, Takuya Yamamoto, Charles Bayard, Laura Papagno, Solène Fastenackels, Rima Zoorab, Richard A. Koup, Jacques Boddaert, Delphine Sauce, Victor Appay
Generation of homogeneous populations of subtype-specific cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) and their comprehensive phenotyping is crucial for a better understanding of the subtype-related disease mechanisms and as tools for the development of chamber-specific drugs. The goals of this study were to apply a simple and efficient method for differentiation of iPSCs into defined functional CM subtypes in feeder-free conditions and to obtain a comprehensive understanding of the molecular, cell biological, and functional properties of atrial and ventricular iPSC-CMs on both the single-cell and engineered heart muscle (EHM) level. By a stage-specific activation of retinoic acid signaling in monolayer-based and well-defined culture, we showed that cardiac progenitors can be directed towards a highly homogeneous population of atrial CMs. By combining the transcriptome and proteome profiling of the iPSC-CM subtypes with functional characterizations via optical action potential and calcium imaging, and with contractile analyses in EHM, we demonstrated that atrial and ventricular iPSC-CMs and -EHM highly correspond to the atrial and ventricular heart muscle, respectively. This study provides a comprehensive understanding of the molecular and functional identities characteristic of atrial and ventricular iPSC-CMs and -EHM and supports their suitability in disease modeling and chamber-specific drug screening.
Lukas Cyganek, Malte Tiburcy, Karolina Sekeres, Kathleen Gerstenberg, Hanibal Bohnenberger, Christof Lenz, Sarah Henze, Michael Stauske, Gabriela Salinas, Wolfram-Hubertus Zimmermann, Gerd Hasenfuss, Kaomei Guan
Oncogenic Kras expression specifically in hematopoietic stem cells (HSCs) induces a rapidly fatal myeloproliferative neoplasm in mice, suggesting that Kras signaling plays a dominant role in normal hematopoiesis. However, such a conclusion is based on expression of an oncogenic version of Kras. Hence, we sought to determine the effect of simply increasing the amount of endogenous wild-type Kras on HSC fate. To this end, we utilized a codon-optimized version of the murine Kras gene (Krasex3op) that we developed, in which silent mutations in exon 3 render the encoded mRNA more efficiently translated, leading to increased protein expression without disruption to the normal gene architecture. We found that Kras protein levels were significantly increased in bone marrow (BM) HSCs in Krasex3op/ex3op mice, demonstrating that the translation of Kras in HSCs is normally constrained by rare codons. Krasex3op/ex3op mice displayed expansion of BM HSCs, progenitor cells, and B lymphocytes, but no evidence of myeloproliferative disease or leukemia in mice followed for 12 months. BM HSCs from Krasex3op/ex3op mice demonstrated increased multilineage repopulating capacity in primary competitive transplantation assays, but secondary competitive transplants revealed exhaustion of long-term HSCs. Following total body irradiation, Krasex3op/ex3op mice displayed accelerated hematologic recovery and increased survival. Mechanistically, HSCs from Krasex3op/ex3op mice demonstrated increased proliferation at baseline, with a corresponding increase in Erk1/2 phosphorylation and cyclin-dependent kinase 4 and 6 (Cdk4/6) activation. Furthermore, both the enhanced colony-forming capacity and in vivo repopulating capacity of HSCs from Krasex3op/ex3op mice were dependent on Cdk4/6 activation. Finally, BM transplantation studies revealed that augmented Kras expression produced expansion of HSCs, progenitor cells, and B cells in a hematopoietic cell–autonomous manner, independent from effects on the BM microenvironment. This study provides fundamental demonstration of codon usage in a mammal having a biological consequence, which may speak to the importance of codon usage in mammalian biology.
Joshua P. Sasine, Heather A. Himburg, Christina M. Termini, Martina Roos, Evelyn Tran, Liman Zhao, Jenny Kan, Michelle Li, Yurun Zhang, Stéphanie C. de Barros, Dinesh S. Rao, Christopher M. Counter, John P. Chute
Cancer stem cells (CSCs) — known to be resistant to genotoxic radiation and chemotherapy — are fundamental to therapy failure and cancer relapse. Here, we reveal that glioma CSCs are hypersensitive to radiation, but a temporal DNA repair mechanism converts the intrinsic sensitivity to genomic instability and treatment resistance. Transcriptome analysis identifies DNA-dependent protein kinase (DNA-PK) as a predominant DNA repair enzyme in CSCs. Notably, DNA-PK activity is suppressed after irradiation when ROS induce the dissociation of DNA-PKcs with Ku70/80, resulting in delayed DNA repair and radiosensitivity; subsequently, after ROS clearance, the accumulated DNA damage and robust activation of DNA-PK induce genomic instability, facilitated by Rad50-mediated cell-cycle arrest, leading to enhanced malignancy, CSC overgrowth, and radioresistance. Finally, we show a requisite in vivo role for DNA-PK in CSC-mediated radioresistance and glioma progression. These findings identify a time-sensitive mechanism controlling CSC resistance to DNA-damaging treatments and suggest DNA-PK/Rad50 as promising targets for CSC eradication.
Yanling Wang, Haineng Xu, Tianrun Liu, Menggui Huang, Param-Puneet Butter, Chunsheng Li, Lin Zhang, Gary D. Kao, Yanqing Gong, Amit Maity, Constantinos Koumenis, Yi Fan
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