Glioblastoma (GBM) is characterized by an aberrant yet druggable epigenetic landscape. One major family of epigenetic regulators, the histone deacetylases (HDACs), are considered promising therapeutic targets for GBM due to their repressive influences on transcription. Although HDACs share redundant functions and common substrates, the unique isoform-specific roles of different HDACs in GBM remain unclear. In neural stem cells, HDAC2 is the indispensable deacetylase to ensure normal brain development and survival in the absence of HDAC1. Surprisingly, we find that HDAC1 is the essential class I deacetylase in glioma stem cells, and its loss is not compensated for by HDAC2. Using cell-based and biochemical assays, transcriptomic analyses, and patient-derived xenograft models, we find that knockdown of HDAC1 alone has profound effects on the glioma stem cell phenotype in a p53-dependent manner. We demonstrate marked suppression in tumor growth upon targeting of HDAC1 and identify compensatory pathways that provide insights into combination therapies for GBM. Our study highlights the importance of HDAC1 in GBM and the need to develop isoform-specific drugs.
Costanza Lo Cascio, James B. McNamara, Ernesto L. Melendez, Erika M. Lewis, Matthew E. Dufault, Nader Sanai, Christopher L. Plaisier, Shwetal Mehta
Engineered heart tissue (EHT) strategies, by combining cells within a hydrogel matrix, may be a novel therapy for heart failure. EHTs restore cardiac function in rodent injury models, but more data are needed in clinically relevant settings. Accordingly, an upscaled EHT patch (2.5 cm × 1.5 cm × 1.5 mm) consisting of up to 20 million human induced pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) embedded in a fibrin-based hydrogel was developed. A rabbit myocardial infarction model was then established to test for feasibility and efficacy. Our data showed that hPSC-CMs in EHTs became more aligned over 28 days and had improved contraction kinetics and faster calcium transients. Blinded echocardiographic analysis revealed a significant improvement in function in infarcted hearts that received EHTs, along with reduction in infarct scar size by 35%. Vascularization from the host to the patch was observed at week 1 and stable to week 4, but electrical coupling between patch and host heart was not observed. In vivo telemetry recordings and ex vivo arrhythmia provocation protocols showed that the patch was not pro-arrhythmic. In summary, EHTs improved function and reduced scar size without causing arrhythmia, which may be due to the lack of electrical coupling between patch and host heart.
Richard J. Jabbour, Thomas J. Owen, Pragati Pandey, Marina Reinsch, Brian Wang, Oisín King, Liam Steven Couch, Dafni Pantou, David S. Pitcher, Rasheda A. Chowdhury, Fotios G. Pitoulis, Balvinder S. Handa, Worrapong Kit-Anan, Filippo Perbellini, Rachel C. Myles, Daniel J. Stuckey, Michael Dunne, Mayooran Shanmuganathan, Nicholas S. Peters, Fu Siong Ng, Florian Weinberger, Cesare M. Terracciano, Godfrey L. Smith, Thomas Eschenhagen, Sian E. Harding
Taspase1, a highly conserved threonine protease encoded by TASP1, cleaves nuclear histone modifying factors and basal transcription regulators to orchestrate diverse transcription programs. Hereditary loss-of-function mutation of TASP1 has recently been reported in human resulting in a novel anomaly complex syndrome manifested with hematological, facial, and skeletal abnormalities. Here, we demonstrate that Taspase1-mediated cleavage of TFIIAα-β, rather than of MLL1 or MLL2, in mouse embryos is required for proper fetal liver hematopoiesis and correct segmental identities of the axial skeleton. Homozygous genetic deletion of Taspase1 (Tasp1-/-) disrupted embryonic hematopoietic stem cell self-renewal and quiescence states, and axial skeleton fates. Strikingly, mice carrying knockin non-cleavable mutations of TFIIAα-β (Gtf2a1nc/nc), a well-characterized basal transcription factor, displayed more pronounced fetal liver and axial skeleton defects than those with non-cleavable MLL1 and MLL2 (Mll1nc/nc;2nc/nc), two trithorax group (Trx-G) histone H3 trimethyl transferases. Our study offers molecular insights concerning TASP1-loss human syndrome and discovers unexpected role of TFIIAα-β cleavage in embryonic cell fate decisions.
Hidetaka Niizuma, Adam C. Searleman, Shugaku Takeda, Scott A. Armstrong, Christopher Y. Park, Emily H. Cheng, James J. Hsieh
Abnormal action potential (AP) properties, as occurs in long or short QT syndromes (LQTS and SQTS, respectively), can cause life-threatening arrhythmias. Optogenetics strategies, utilizing light-sensitive proteins, have emerged as experimental platforms for cardiac pacing, resynchronization, and defibrillation. We tested the hypothesis that similar optogenetic tools can modulate the cardiomyocyte’s AP properties, as a potentially novel antiarrhythmic strategy. Healthy control and LQTS/SQTS patient–specific human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) were transduced to express the light-sensitive cationic channel channelrhodopsin-2 (ChR2) or the anionic-selective opsin, ACR2. Detailed patch-clamp, confocal-microscopy, and optical mapping studies evaluated the ability of spatiotemporally defined optogenetic protocols to modulate AP properties and prevent arrhythmogenesis in the hiPSC-CMs cell/tissue models. Depending on illumination timing, light-induced ChR2 activation induced robust prolongation or mild shortening of AP duration (APD), while ACR2 activation allowed effective APD shortening. Fine-tuning these approaches allowed for the normalization of pathological AP properties and suppression of arrhythmogenicity in the LQTS/SQTS hiPSC-CM cellular models. We next established a SQTS–hiPSC-CMs–based tissue model of reentrant-arrhythmias using optogenetic cross-field stimulation. An APD-modulating optogenetic protocol was then designed to dynamically prolong APD of the propagating wavefront, completely preventing arrhythmogenesis in this model. This work highlights the potential of optogenetics in studying repolarization abnormalities and in developing novel antiarrhythmic therapies.
Amit Gruber, Oded Edri, Irit Huber, Gil Arbel, Amira Gepstein, Assad Shiti, Naim Shaheen, Snizhana Chorna, Michal Landesberg, Lior Gepstein
BACKGROUND. Whether airspace biomarkers add value to plasma biomarkers in studying ARDS is not well understood. Mesenchymal stromal cells (MSCs) are an investigational therapy for ARDS, and airspace biomarkers may provide mechanistic evidence for MSCs' impact in patients with ARDS. METHODS. We carried out a nested cohort study within a phase 2a safety trial of treatment with allogeneic MSCs for moderate to severe ARDS. Non-bronchoscopic bronchoalveolar lavage and plasma samples were collected 48 hours after study drug infusion. Airspace and plasma biomarker concentrations were compared between the MSC (n = 17) and placebo (n = 10) treatment arms, and correlation between the two compartments was tested. Airspace biomarkers were also tested for associations with clinical and radiographic outcomes. RESULTS. Compared to placebo, MSC treatment significantly reduced airspace total protein, angiopoietin-2 (Ang-2), interleukin-6 (IL-6), and soluble tumor necrosis factor receptor-1 concentrations. Plasma biomarkers did not differ between groups. Each 10-fold increase in airspace Ang-2 was independently associated with 6.7 fewer days alive and free of mechanical ventilation (95% CI -12.3 to -1.0, p = 0.023), and each 10-fold increase in airspace receptor for advanced glycation end-products (RAGE) was independently associated with a 6.6 point increase in day 3 radiographic assessment of lung edema score (95% CI 2.4 to 10.7, p = 0.004). CONCLUSIONS. MSCs reduced biological evidence of lung injury in patients with ARDS. Biomarkers from the airspaces provide additional value for studying pathogenesis, treatment effects, and outcomes in ARDS. TRIAL REGISTRATION. NCT02097641 FUNDING. National Heart, Lung, and Blood Institute
Katherine D. Wick, Aleksandra Leligdowicz, Hanjing Zhuo, Lorraine B. Ware, Michael A. Matthay
Glioma stem cells (GSCs) drive propagation and therapeutic resistance of glioblastomas, the most aggressive diffuse brain tumors. However, the molecular mechanisms that maintain the stemness and promote therapy resistance remain poorly understood. Here we report CD109/STAT3 axis as crucial for the maintenance of stemness and tumorigenicity of GSCs and as a mediator of chemoresistance. Mechanistically, CD109 physically interacts with glycoprotein 130 to promote activation of the IL-6/STAT3 pathway in GSCs. Genetic depletion of CD109 abolished the stemness and self-renewal of GSCs and impaired tumorigenicity. Loss of stemness was accompanied with a phenotypic shift of GSCs to more differentiated astrocytic-like cells. Importantly, genetic or pharmacologic targeting of CD109/STAT3 axis sensitized the GSCs to chemotherapy, suggesting that targeting CD109/STAT3 axis has potential to overcome therapy resistance in glioblastoma.
Pauliina Filppu, Jayendrakishore Tanjore Ramanathan, Kirsi J. Granberg, Erika Gucciardo, Hannu Haapasalo, Kaisa Lehti, Matti Nykter, Vadim Le Joncour, Pirjo Laakkonen
Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early onset, severe and rapidly progressive form of muscular dystrophy affecting equally girls and boys. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and the heart. We isolated human muscle stem cells from two donors with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of two co-regulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with >90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base edited patient cells regenerated muscle and contributed to the Pax7 positive satellite cell compartment in vivo in mouse xenografts. We hereby provide the first evidence that autologous gene repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.
Helena Escobar, Anne Krause, Sandra Keiper, Janine Kieshauer, Stefanie Müthel, Manuel García de Paredes, Eric Metzler, Ralf Kühn, Florian Heyd, Simone Spuler
Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy–based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.
Tony Chour, Lei Tian, Edward Lau, Dilip Thomas, Ilanit Itzhaki, Olfat Malak, Joe Z. Zhang, Xulei Qin, Mirwais Wardak, Yonggang Liu, Mark Chandy, Katelyn E. Black, Maggie P.Y. Lam, Evgenios Neofytou, Joseph C. Wu
After 9/11, threat of nuclear attack on American urban centers prompted government agencies to develop medical radiation countermeasures to mitigate hematopoietic-acute radiation syndrome (H-ARS) and higher-dose gastrointestinal-ARS (GI-ARS) lethality. While re-purposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS in pre-clinical models, no mitigator potentially deliverable under mass casualty conditions preserves GI tract. Here we generate anti-ceramide 6B5 single-chain variable fragment (scFv) and show subcutaneous 6B5 scFv delivery at 24h after a 90% lethal GI-ARS dose of 15Gy mitigates mouse lethality, despite administration after DNA repair is complete. We define an alternate target to DNA repair, an evolving pattern of ceramide-mediated endothelial apoptosis post-radiation, which when disrupted by 6B5 scFv, initiates a durable program of tissue repair, permitting crypt, organ and mouse survival. We posit successful pre-clinical development will render anti-ceramide 6B5 scFv a candidate for inclusion in the Strategic National Stockpile for distribution after a radiation catastrophe.
Jimmy A. Rotolo, Chii Shyang Fong, Sahra Bodo, Prashanth K. B. Nagesh, John D. Fuller, Thivashnee Sharma, Alessandra Piersigilli, Zhigang Zhang, Zvi Fuks, Vijay K. Singh, Richard Kolesnick
Limitations in cell proliferation are important for normal function of differentiated tissues, and essential for the safty of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing S-phase entry, or S-phase completion and determined growth potential, differentiation and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S-phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.
Lina Sui, Yurong Xin, Qian Du, Daniela Georgieva, Giacomo Diedenhofen, Leena Haataja, Qi Su, Michael V. Zuccaro, Jinrang Kim, Jiayu Fu, Yuan Xing, Yi He, Danielle Baum, Robin S. Goland, Yong Wang, Jose Oberholzer, Fabrizio Barbetti, Peter Arvan, Sandra Kleiner, Dieter Egli
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