Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1α alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.
Alexander Waclawiczek, Ashley Hamilton, Kevin Rouault-Pierre, Ander Abarrategi, Manuel Garcia Albornoz, Farideh Miraki-Moud, Nourdine Bah, John Gribben, Jude Fitzgibbon, David Taussig, Dominique Bonnet
Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome–type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.
Stefanie Kreutmair, Miriam Erlacher, Geoffroy Andrieux, Rouzanna Istvanffy, Alina Mueller-Rudorf, Melissa Zwick, Tamina Rückert, Milena Pantic, Teresa Poggio, Khalid Shoumariyeh, Tony A. Mueller, Hiroyuki Kawaguchi, Marie Follo, Cathrin Klingeberg, Marcin Wlodarski, Irith Baumann, Dietmar Pfeifer, Michal Kulinski, Martina Rudelius, Simone Lemeer, Bernhard Kuster, Christine Dierks, Christian Peschel, Nina Cabezas-Wallscheid, Jesus Duque-Afonso, Robert Zeiser, Michael L. Cleary, Detlev Schindler, Annette Schmitt-Graeff, Melanie Boerries, Charlotte M. Niemeyer, Robert A.J. Oostendorp, Justus Duyster, Anna Lena Illert
Sensory nerve was recently identified as being involved in regulation of bone mass accrual. We previously discovered that PGE2 secreted by osteoblastic cells could activate sensory nerve EP4 receptor to promote bone formation by inhibiting sympathetic activity. However, the fundamental units of bone formation are active osteoblasts, which originate from skeletal stem cells. Here, we found that after sensory denervation, knockout of the EP4 receptor in sensory nerves, or knockout of cyclooxygenase-2 (COX2) in osteoblasts could significantly promote adipogenesis and inhibit osteogenesis in adult mice. Furthermore, injection of SW033291 (a small molecule that locally increases PGE2 level) or propranolol (a beta-blocker) significantly promoted osteogenesis and inhibited adipogenesis. This effect of SW033291, but not propranolol, was abolished in conditional EP4 knockout mice under normal conditions or in the bone repair process. We conclude that the PGE2-EP4 sensory nerve axis could regulate skeletal stem cell differentiation in bone marrow of adult mice.
Bo Hu, Xiao Lv, Hao Chen, Peng Xue, Bo Gao, Xiao Wang, Gehua Zhen, Janet L. Crane, Dayu Pan, Shen Liu, Shuangfei Ni, Panfeng Wu, Weiping Su, Xiaonan Liu, Zemin Ling, Mi Yang, Ruoxian Deng, Yusheng Li, Lei Wang, Ying Zhang, Mei Wan, Zengwu Shao, Huajiang Chen, Wen Yuan, Xu Cao
Glioblastoma (GBM) contains a subpopulation of cells, GBM stem cells (GSCs), that maintain the bulk tumor and represent a key therapeutic target. Norrin is a Wnt ligand that binds the Frizzled4 (FZD4) receptor to activate canonical Wnt signaling. While Norrin, encoded by NDP, has a well- described role in vascular development, its function in human tumorigenesis is largely unexplored. Here, we show that NDP expression is enriched in neurological cancers, including GBM, and its levels positively correlated with survival in a GBM subtype defined by low expression of ASCL1, a proneural factor. We investigated the function of Norrin and FZD4 in GSCs and found that it mediated opposing tumor-promoting and -suppressive effects on ASCL1lo and ASCL1hi GSCs. Consistent with a potential tumor suppressive effect of Norrin suggested by the tumour outcome data, we found that Norrin signaling through FZD4 inhibited growth in ASCL1lo GSCs. In contrast, in ASCL1hi GSCs Norrin promoted Notch signaling, independently of WNT, to promote tumor progression. Forced ASCL1 expression reversed the tumor suppressive effects of Norrin in ASCL1lo GSCs. Our results identify Norrin as a modulator of human brain cancer progression and reveal an unanticipated Notch mediated function of Norrin in regulating cancer stem cell biology.
Ahmed El-Sehemy, Hayden J. Selvadurai, Arturo Ortin-Martinez, Neno T. Pokrajac, Yasin Mamatjan, Nobuhiko Tachibana, Katherine J. Rowland, Lilian Lee, Nicole I. Park, Kenneth D. Aldape, Peter Dirks, Valerie A. Wallace
Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the Lamin A/C gene cause several diseases, belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of Lamin A-dependent dystrophies are still largely unknown. Polycomb group of proteins (PcG) are epigenetic repressors and Lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss Muscular Dystrophy (EDMD), we showed here that Lamin A loss deregulated PcG positioning in muscle satellite stem cells leading to de-repression of non-muscle specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional programme caused impairment in self-renewal, loss of cell identity and premature exhaustion of quiescent satellite cell pool. Genetic ablation of Cdkn2a locus restored muscle stem cell properties in Lamin A/C null dystrophic mice. Our findings established a direct link between Lamin A and PcG epigenetic silencing and indicated that Lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.
Andrea Bianchi, Chiara Mozzetta, Gloria Pegoli, Federica Lucini, Sara Valsoni, Valentina Rosti, Cristiano Petrini, Alice Cortesi, Francesco Gregoretti, Laura Antonelli, Gennaro Oliva, Marco De Bardi, Roberto Rizzi, Beatrice Bodega, Diego Pasini, Francesco Ferrari, Claudia Bearzi, Chiara Lanzuolo
Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon–mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies.
Andreas Marg, Helena Escobar, Sina Gloy, Markus Kufeld, Joseph Zacher, Andreas Spuler, Carmen Birchmeier, Zsuzsanna Izsvák, Simone Spuler
Cancer–related anemia is present in over 60% of newly diagnosed cancer patients and is associated with substantial morbidity and high medical costs. Drugs that enhance erythropoiesis are urgently required to decrease transfusion rates and improve quality of life. Clinical studies have observed an unexpected improvement in hemoglobin and red blood cell (RBC) transfusion-independence in AML patients treated with the isocitrate dehydrogenase 2 (IDH2) mutant-specific inhibitor, enasidenib, leading to improved quality of life without a reduction in AML disease burden. Here, we demonstrate that enasidenib enhanced human erythroid differentiation of hematopoietic progenitors. The phenomenon was not observed with other IDH1/2 inhibitors and occurred in IDH2-deficient CRIPSR-engineered progenitors independently of D-2-hydroxyglutarate. The effect of enasidenib on hematopoietic progenitors was mediated by protoporphyrin accumulation, driving heme production and erythroid differentiation in committed CD71+ progenitors rather than hematopoietic stem cells. Our results position enasidenib as a promising therapeutic agent for improvement of anemia and provide the basis for a clinical trial using enasidenib to decrease transfusion dependence in a wide array of clinical contexts.
Ritika Dutta, Tian Yi Zhang, Thomas Köhnke, Daniel Thomas, Miles Linde, Eric Gars, Melissa Stafford, Satinder Kaur, Yusuke Nakauchi, Raymond Yin, Armon Azizi, Anupama Narla, Ravindra Majeti
Parkinson's disease (PD) is a neurodegenerative disorder associated with loss of striatal dopamine, secondary to degeneration of midbrain dopamine (mDA) neurons in the substantia nigra, rendering cell transplantation a promising therapeutic strategy. To establish human induced pluripotent stem cell (hiPSC)-based autologous cell therapy, we report a platform of core techniques for the production of mDA progenitors as a safe and effective therapeutic product. First, by combining metabolism-regulating microRNAs with reprogramming factors, we developed a method to more efficiently generate clinical grade iPSCs, as evidenced by genomic integrity and unbiased pluripotent potential. Second, we established a “spotting”-based in vitro differentiation methodology to generate functional and healthy mDA cells in a scalable manner. Third, we developed a chemical method that safely eliminates undifferentiated cells from the final product. Dopaminergic cells thus produced express high levels of characteristic mDA markers, produce and secrete dopamine, and exhibit electrophysiological features typical of mDA cells. Transplantation of these cells into rodent models of PD robustly restores motor dysfunction and reinnervates host brain, while showing no evidence of tumor formation or redistribution of the implanted cells. We propose that this platform is suitable for the successful implementation of human personalized autologous cell therapy for PD.
Bin Song, Young Cha, Sanghyeok Ko, Jeha Jeon, Nayeon Lee, Hyemyung Seo, Kyung-joon Park, In-Hee Lee, Claudia Lopes, Melissa Feitosa, María José Luna, Jin Hyuk Jung, Jisun Kim, Dabin Hwang, Bruce Cohen, Martin Teicher, Pierre Leblanc, Bob Carter, Jeffrey H. Kordower, Vadim Y. Bolshakov, Sek Won Kong, Jeffrey S. Schweitzer, Kwang-Soo Kim
The proximal tubule has a remarkable capacity for repair after acute injury, but the cellular lineage and molecular mechanisms underlying this repair response are incompletely understood. Here, we developed a Kim1-GFPCreERt2 knockin mouse line (Kim1-GCE) in order to perform genetic lineage tracing of dedifferentiated cells while measuring the cellular transcriptome of proximal tubule during repair. Acutely injured genetically labeled clones coexpressed KIM1, VIMENTIN, SOX9, and KI67, indicating a dedifferentiated and proliferative state. Clonal analysis revealed clonal expansion of Kim1+ cells, indicating that acutely injured, dedifferentiated proximal tubule cells, rather than fixed tubular progenitor cells, account for repair. Translational profiling during injury and repair revealed signatures of both successful and unsuccessful maladaptive repair. The transcription factor Foxm1 was induced early in injury, was required for epithelial proliferation in vitro, and was dependent on epidermal growth factor receptor (EGFR) stimulation. In conclusion, dedifferentiated proximal tubule cells effect proximal tubule repair, and we reveal an EGFR/FOXM1-dependent signaling pathway that drives proliferative repair after injury.
Monica Chang-Panesso, Farid F. Kadyrov, Matthew Lalli, Haojia Wu, Shiyo Ikeda, Eirini Kefaloyianni, Mai M. Abdelmageed, Andreas Herrlich, Akio Kobayashi, Benjamin D. Humphreys
Tuberculosis (TB) remains a major infectious disease worldwide. TB treatment displays a bi-phasic bacterial clearance, in which the majority of bacteria clear within the first month of treatment, but residual bacteria remains non-responsive to treatment and eventually may become resistant. Here, we have shown that Mycobacterium tuberculosis (M.tb) is taken up by mesenchymal stem cells (MSCs), where it established dormancy and became highly non-responsive to isoniazid, a major constituent of Directly Observed Treatment Short-course (DOTS). Dormant M.tb induced quiescence in MSCs and promoted their long-term survival. Unlike macrophages, where M.tb resides in early-phagosomal compartments, in MSCs the majority of bacilli were found in the cytosol, where they promoted rapid lipid-synthesis, hiding within lipid-droplets. Inhibition of lipid-synthesis prevented dormancy and sensitized the organisms to isoniazid. Thus, we have established that M.tb gains dormancy in MSCs, which thus serve as a long-term natural-reservoir of dormant M.tb. Interestingly, in the murine-model of TB, induction of autophagy eliminated M.tb from MSCs and consequently, the addition of rapamycin to an isoniazid treatment regimen successfully attained sterile clearance and prevented disease reactivation.
Samreen Fatima, Shashank Shivaji Kamble, Ved Prakash Dwivedi, Debapriya Bhattacharya, Santosh Kumar, Anand Ranganathan, Luc Van Kaer, Sujata Mohanty, Gobardhan Das