ABSTRACT
Undifferentiated neural stem and progenitor cells (NSPCs) encounter extracellular signals that bind plasma membrane proteins and influence differentiation. Membrane proteins are regulated by N-linked glycosylation, making it possible that glycosylation plays a critical role in cell differentiation. We assessed enzymes that control N-glycosylation in NSPCs and found that loss of the enzyme responsible for generating ß1,6-branched N-glycans, N-acetylglucosaminyltransferase V (MGAT5), led to specific changes in NSPC differentiation in vitro and in vivo. Mgat5 homozygous null NSPCs in culture formed more neurons and fewer astrocytes compared with wild-type controls. In the brain cerebral cortex, loss of MGAT5 caused accelerated neuronal differentiation. Rapid neuronal differentiation led to depletion of cells in the NSPC niche, resulting in a shift in cortical neuron layers in Mgat5 null mice. Glycosylation enzyme MGAT5 plays a critical and previously unrecognized role in cell differentiation and early brain development.
Subject(s)
Brain , Membrane Proteins , Neurogenesis , Animals , Mice , Brain/growth & development , Glycosylation , Mice, KnockoutABSTRACT
PIEZO1 channels play a critical role in numerous physiological processes by transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of endogenous PIEZO1 activity and localization in regulating mechanotransduction. To enable physiologically and clinically relevant human-based studies, we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with super-resolution imaging, our chemogenetic approach allows precise visualization of PIEZO1 in various cell types. Further, the PIEZO1-HaloTag hiPSC technology allows non-invasive monitoring of channel activity via Ca2+-sensitive HaloTag ligands, with temporal resolution approaching that of patch clamp electrophysiology. Using lightsheet imaging of hiPSC-derived neural organoids, we also achieve molecular scale PIEZO1 imaging in three-dimensional tissue samples. Our advances offer a novel platform for studying PIEZO1 mechanotransduction in human cells and tissues, with potential for elucidating disease mechanisms and development of targeted therapeutics.
ABSTRACT
Mechanical forces and tissue mechanics influence the morphology of the developing brain, but the underlying molecular mechanisms have been elusive. Here, we examine the role of mechanotransduction in brain development by focusing on Piezo1, a mechanically activated ion channel. We find that Piezo1 deletion results in a thinner neuroepithelial layer, disrupts pseudostratification, and reduces neurogenesis in E10.5 mouse embryos. Proliferation and differentiation of Piezo1 knockout (KO) mouse neural stem cells (NSCs) isolated from E10.5 embryos are reduced in vitro compared to littermate WT NSCs. Transcriptome analysis of E10.5 Piezo1 KO brains reveals downregulation of the cholesterol biosynthesis superpathway, in which 16 genes, including Hmgcr, the gene encoding the rate-limiting enzyme of the cholesterol biosynthesis pathway, are downregulated by 1.5-fold or more. Consistent with this finding, membrane lipid composition is altered, and the cholesterol levels are reduced in Piezo1 KO NSCs. Cholesterol supplementation of Piezo1 KO NSCs partially rescues the phenotype in vitro. These findings demonstrate a role for Piezo1 in the neurodevelopmental process that modulates the quantity, quality, and organization of cells by influencing cellular cholesterol metabolism. Our study establishes a direct link in NSCs between PIEZO1, intracellular cholesterol levels, and neural development.
Subject(s)
Ion Channels/metabolism , Mechanotransduction, Cellular , Neural Stem Cells , Animals , Brain/metabolism , Cholesterol , Mechanotransduction, Cellular/physiology , Mice , Mice, Knockout , Neural Stem Cells/metabolismABSTRACT
T lymphocytes encounter complex mechanical cues during an immune response. The mechanosensitive ion channel, Piezo1, drives inflammatory responses to bacterial infections, wound healing, and cancer; however, its role in helper T cell function remains unclear. In an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we found that mice with genetic deletion of Piezo1 in T cells showed diminished disease severity. Unexpectedly, Piezo1 was not essential for lymph node homing, interstitial motility, Ca2+ signaling, T cell proliferation, or differentiation into proinflammatory T helper 1 (TH1) and TH17 subsets. However, Piezo1 deletion in T cells resulted in enhanced transforming growth factor-ß (TGFß) signaling and an expanded pool of regulatory T (Treg) cells. Moreover, mice with deletion of Piezo1 specifically in Treg cells showed significant attenuation of EAE. Our results indicate that Piezo1 selectively restrains Treg cells, without influencing activation events or effector T cell functions.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Multiple Sclerosis , Animals , Cell Differentiation , Encephalomyelitis, Autoimmune, Experimental/pathology , Ion Channels/genetics , Lymphocyte Activation , Mice , Mice, Inbred C57BL , T-Lymphocytes, Regulatory , Th1 CellsABSTRACT
Piezo channels transduce mechanical stimuli into electrical and chemical signals to powerfully influence development, tissue homeostasis, and regeneration. Studies on Piezo1 have largely focused on transduction of "outside-in" mechanical forces, and its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that cellular traction forces generate spatially-restricted Piezo1-mediated Ca2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched near force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca2+ flickers allow spatial segregation of mechanotransduction events, and that mobility allows Piezo1 channels to explore a large number of mechanical microdomains and thus respond to a greater diversity of mechanical cues.
Subject(s)
Calcium Signaling , Calcium/metabolism , Fibroblasts/metabolism , Ion Channels/metabolism , Mechanotransduction, Cellular , Myosin Type II/metabolism , Neural Stem Cells/metabolism , Animals , Cells, Cultured , Humans , Ion Channels/deficiency , Ion Channels/genetics , Male , Mice, Knockout , Time FactorsABSTRACT
Adipocytes function as an energy buffer and undergo significant size and volume changes in response to nutritional cues. This adipocyte plasticity is important for systemic lipid metabolism and insulin sensitivity. Accompanying the adipocyte size and volume changes, the mechanical pressure against cell membrane also changes. However, the role that mechanical pressure plays in lipid metabolism and insulin sensitivity remains to be elucidated. Here we show that Piezo1, a mechanically-activated cation channel stimulated by membrane tension and stretch, was highly expressed in adipocytes. Adipose Piezo1 expression was increased in obese mice. Adipose-specific piezo1 knockout mice (adipose-Piezo1-/-) developed insulin resistance, especially when challenged with a high-fat diet (HFD). Perigonadal white adipose tissue (pgWAT) weight was reduced while pro-inflammatory and lipolysis genes were increased in the pgWAT of HFD-fed adipose-Piezo1-/- mice. The adipose-Piezo1-/- mice also developed hepatic steatosis with elevated expression of fatty acid synthesis genes. In cultured adipocytes, Piezo1 activation decreased, while Piezo1 inhibition elevated pro-inflammatory gene expression. TLR4 antagonist TAK-242 abolished adipocyte inflammation induced by Piezo1 inhibition. Thus, adipose Piezo1 may serve as an adaptive mechanism for adipocyte plasticity restraining pro-inflammatory response in obesity.
ABSTRACT
Nature's wisdom resides in achieving a joint enhancement of strength and toughness by constructing intelligent, hierarchical architectures from extremely limited resources. A representative example is nacre, in which a brick-and-mortar structure enables a confluence of toughening mechanisms on multiple length scales. The result is an outstanding combination of strength and toughness which is hardly achieved by engineering materials. Here, a bioinspired Ni/Ni3C composite with nacre-like, brick-and-mortar structure was constructed from Ni powders and graphene sheets. This composite achieved a 73% increase in strength with only a 28% compromise on ductility, leading to a notable improvement in toughness. The graphene-derived Ni-Ti-Al/Ni3C composite retained high hardness up to 1000°C. The present study unveiled a method to smartly use 2D materials to fabricate high-performance metal matrix composites with brick-and-mortar structure through interfacial reactions and, furthermore, created an opportunity of developing advanced Ni-C-based alloys for high-temperature environments.
ABSTRACT
Understanding the cellular properties controlling neural stem and progenitor cell (NSPC) fate choice will improve their therapeutic potential. The electrophysiological measure whole-cell membrane capacitance reflects fate bias in the neural lineage but the cellular properties underlying membrane capacitance are poorly understood. We tested the hypothesis that cell surface carbohydrates contribute to NSPC membrane capacitance and fate. We found NSPCs differing in fate potential express distinct patterns of glycosylation enzymes. Screening several glycosylation pathways revealed that the one forming highly branched N-glycans differs between neurogenic and astrogenic populations of cells in vitro and in vivo. Enhancing highly branched N-glycans on NSPCs significantly increases membrane capacitance and leads to the generation of more astrocytes at the expense of neurons with no effect on cell size, viability, or proliferation. These data identify the N-glycan branching pathway as a significant regulator of membrane capacitance and fate choice in the neural lineage.
Subject(s)
Cell Lineage , Cell Membrane/metabolism , Electrophysiological Phenomena , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Polysaccharides/metabolism , Acetylglucosamine/metabolism , Animals , Astrocytes/cytology , Brain/cytology , Cell Differentiation , Cell Proliferation , Cell Size , Cell Survival , Fucose/metabolism , Gene Expression Regulation , Glycosylation , Mice , N-Acetylneuraminic Acid/metabolism , Neurogenesis , Stem Cell NicheABSTRACT
Transforming growth factor beta (TGF-ß) signaling has pleiotropic functions regulating cancer initiation, development, and metastasis, and also plays important roles in the interaction between stromal and cancer cells, making the pathway a potential therapeutic target. LY2157299 monohydrate (LY), an inhibitor of TGF-ß receptor I (TGFBRI), was examined for its ability to inhibit ovarian cancer (OC) growth both in high-grade serous ovarian cancer (HGSOC) cell lines and xenograft models. Immunohistochemistry, qRT-PCR, and Western blot were performed to study the effect of LY treatment on expression of cancer- and fibroblast-derived genes. Results showed that exposure to TGF-ß1 induced phosphorylation of SMAD2 and SMAD3 in all tested OC cell lines, but this induction was suppressed by pretreatment with LY. LY alone inhibited the proliferation, migration, and invasion of HGSOC cells in vitro. TGF-ß1-induced fibroblast activation was blocked by LY. LY also delayed tumor growth and suppressed ascites formation in vivo. In addition, independent of tumor inhibition, LY reduces ascites formation in vivo. Using OVCAR8 xenograft specimens we confirmed the inhibitory effect of LY on TGF-ß signaling and tumor stromal expression of collagen type XI chain 1 (COL11A1) and versican (VCAN). These observations suggest a role for anti-TGF-ß signaling-directed therapy in ovarian cancer.
ABSTRACT
Cells constantly encounter mechanical stimuli in their environment, such as dynamic forces and mechanical features of the extracellular matrix. These mechanical cues are transduced into biochemical signals, and integrated with genetic and chemical signals to modulate diverse physiological processes. Cells also actively generate forces to internally transport cargo, to explore the physical properties of their environment and to spatially position themselves and other cells during development. Mechanical forces are therefore central to development, homeostasis, and repair. Several molecular and biophysical strategies are utilized by cells for detecting and generating mechanical forces. Here we discuss an important class of molecules involved in sensing and transducing mechanical forces - mechanically-activated ion channels. We focus primarily on the Piezo1 ion channel, and examine its relationship with the cellular cytoskeleton.
Subject(s)
Cytoskeleton/metabolism , Ion Channels/metabolism , Stress, Physiological , Animals , Extracellular Matrix/metabolism , Humans , Ion Channel GatingABSTRACT
Neural stem and progenitor cell (NSPC) fate is strongly influenced by mechanotransduction as modulation of substrate stiffness affects lineage choice. Other types of mechanical stimuli, such as stretch (tensile strain), occur during CNS development and trauma, but their consequences for NSPC differentiation have not been reported. We delivered a 10% static equibiaxial stretch to NSPCs and examined effects on differentiation. We found static stretch specifically impacts NSPC differentiation into oligodendrocytes, but not neurons or astrocytes, and this effect is dependent on particular extracellular matrix (ECM)-integrin linkages. Generation of oligodendrocytes from NSPCs was reduced on laminin, an outcome likely mediated by the α6 laminin-binding integrin, whereas similar effects were not observed for NSPCs on fibronectin. Our data demonstrate a direct role for tensile strain in dictating the lineage choice of NSPCs and indicate the dependence of this phenomenon on specific substrate materials, which should be taken into account for the design of biomaterials for NSPC transplantation.
Subject(s)
Cell Differentiation , Extracellular Matrix , Neural Stem Cells/cytology , Stress, Mechanical , Animals , Cells, Cultured , Integrins/metabolism , Laminin/metabolism , Mice , Oligodendroglia/cytology , Protein BindingABSTRACT
Neural stem cells are multipotent cells with the ability to differentiate into neurons, astrocytes, and oligodendrocytes. Lineage specification is strongly sensitive to the mechanical properties of the cellular environment. However, molecular pathways transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification remain unclear. We found that the mechanically gated ion channel Piezo1 is expressed by brain-derived human neural stem/progenitor cells and is responsible for a mechanically induced ionic current. Piezo1 activity triggered by traction forces elicited influx of Ca(2+), a known modulator of differentiation, in a substrate-stiffness-dependent manner. Inhibition of channel activity by the pharmacological inhibitor GsMTx-4 or by siRNA-mediated Piezo1 knockdown suppressed neurogenesis and enhanced astrogenesis. Piezo1 knockdown also reduced the nuclear localization of the mechanoreactive transcriptional coactivator Yes-associated protein. We propose that the mechanically gated ion channel Piezo1 is an important determinant of mechanosensitive lineage choice in neural stem cells and may play similar roles in other multipotent stem cells.
Subject(s)
Calcium Signaling/physiology , Ion Channel Gating/physiology , Ion Channels/metabolism , Mechanotransduction, Cellular/physiology , Multipotent Stem Cells/metabolism , Neural Stem Cells/metabolism , Neurogenesis/physiology , Cell Differentiation/physiology , Cells, Cultured , Female , Gene Knockdown Techniques , Humans , Ion Channels/genetics , Male , Multipotent Stem Cells/cytology , Neural Stem Cells/cytologyABSTRACT
We describe a method based on fluorescence-lifetime imaging microscopy (FLIM) to assess the fluidity of various membranes in neuronal cells at different stages of development [day 12 (E12) and day 16 (E16) of gestation]. For the FLIM measurements, we use the Laurdan probe which is commonly used to assess membrane water penetration in model and in biological membranes using spectral information. Using the FLIM approach, we build a fluidity scale based on calibration with model systems of different lipid compositions. In neuronal cells, we found a marked difference in fluidity between the internal membranes and the plasma membrane, being the plasma membrane the less fluid. However, we found no significant differences between the two cell groups, E12 and E16. Comparison with NIH3T3 cells shows that the plasma membranes of E12 and E16 cells are significantly more fluid than the plasma membrane of the cancer cells.
Subject(s)
2-Naphthylamine/analogs & derivatives , Cell Membrane/metabolism , Embryonic Development , Fluorescent Dyes/chemistry , Laurates/chemistry , Lipid Metabolism , Microscopy, Fluorescence/methods , Neurons/cytology , 2-Naphthylamine/chemistry , Animals , Cholesterol/metabolism , Female , Membrane Fluidity , Mice , NIH 3T3 Cells , Pregnancy , Time FactorsABSTRACT
Dielectrophoresis (DEP) has proven an invaluable tool for the enrichment of populations of stem and progenitor cells owing to its ability to sort cells in a label-free manner and its biological safety. However, DEP separation devices have suffered from a low throughput preventing researchers from undertaking studies requiring large numbers of cells, such as needed for cell transplantation. We developed a microfluidic device designed for the enrichment of stem and progenitor cell populations that sorts cells at a rate of 150,000 cells/h, corresponding to an improvement in the throughput achieved with our previous device designs by over an order of magnitude. This advancement, coupled with data showing the DEP-sorted cells retain their enrichment and differentiation capacity when expanded in culture for periods of up to 2 weeks, provides sufficient throughput and cell numbers to enable a wider variety of experiments with enriched stem and progenitor cell populations. Furthermore, the sorting devices presented here provide ease of setup and operation, a simple fabrication process, and a low associated cost to use that makes them more amenable for use in common biological research laboratories. To our knowledge, this work represents the first to enrich stem cells and expand them in culture to generate transplantation-scale numbers of differentiation-competent cells using DEP.
ABSTRACT
In the stem cell field there is a lack of non invasive and fast methods to identify stem cell's metabolic state, differentiation state and cell-lineage commitment. Here we describe a label-free method that uses NADH as an intrinsic biomarker and the Phasor approach to Fluorescence Lifetime microscopy to measure the metabolic fingerprint of cells. We show that different metabolic states are related to different cell differentiation stages and to stem cell bias to neuronal and glial fate, prior the expression of lineage markers. Our data demonstrate that the NADH FLIM signature distinguishes non-invasively neurons from undifferentiated neural progenitor and stem cells (NPSCs) at two different developmental stages (E12 and E16). NPSCs follow a metabolic trajectory from a glycolytic phenotype to an oxidative phosphorylation phenotype through different stages of differentiation. NSPCs are characterized by high free/bound NADH ratio, while differentiated neurons are characterized by low free/bound NADH ratio. We demonstrate that the metabolic signature of NPSCs correlates with their differentiation potential, showing that neuronal progenitors and glial progenitors have a different free/bound NADH ratio. Reducing conditions in NPSCs correlates with their neurogenic potential, while oxidative conditions correlate with glial potential. For the first time we show that FLIM NADH metabolic fingerprint provides a novel, and quantitative measure of stem cell potential and a label-free and non-invasive means to identify neuron- or glial- biased progenitors.
Subject(s)
Cell Differentiation , NAD/metabolism , Neural Stem Cells/physiology , Animals , Biomarkers/metabolism , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Female , Glycolysis , Mice , Microscopy, Fluorescence , Neural Stem Cells/metabolism , Neuroglia/metabolism , Oxidation-Reduction , Oxidative Phosphorylation , Phenotype , Primary Cell Culture , Protein Binding , Spheroids, Cellular/metabolism , Spheroids, Cellular/physiologyABSTRACT
The integration of microscale engineering, microfluidics, and AC electrokinetics such as dielectrophoresis has generated novel microsystems that enable quantitative analysis of cellular phenotype, function, and physiology. These systems are increasingly being used to assess diverse cell types, such as stem cells, so it becomes critical to thoroughly evaluate whether the systems themselves impact cell function. For example, engineered microsystems have been utilized to investigate neural stem/progenitor cells (NSPCs), which are of interest due to their potential to treat CNS disease and injury. Analysis by dielectrophoresis (DEP) microsystems determined that unlabeled NSPCs with distinct fate potential have previously unrecognized distinguishing electrophysiological characteristics, suggesting that NSPCs could be isolated by DEP microsystems without the use of cell type specific labels. To gauge the potential impact of DEP sorting on NSPCs, we investigated whether electric field exposure of varying times affected survival, proliferation, or fate potential of NSPCs in suspension. We found short-term DEP exposure (1 min or less) had no effect on NSPC survival, proliferation, or fate potential revealed by differentiation. Moreover, NSPC proliferation (measured by DNA synthesis and cell cycle kinetics) and fate potential were not altered by any length of DEP exposure (up to 30 min). However, lengthy exposure (>5 min) to frequencies near the crossover frequency (50-100 kHz) led to decreased survival of NSPCs (maximum â¼30% cell loss after 30 min). Based on experimental observations and mathematical simulations of cells in suspension, we find that frequencies near the crossover frequency generate an induced transmembrane potential that results in cell swelling and rupture. This is in contrast to the case for adherent cells since negative DEP frequencies lower than the crossover frequency generate the highest induced transmembrane potential and damage for these cells. We clarify contrasting effects of DEP on adherent and suspended cells, which are related to the cell position within the electric field and the strength of the electric field at specific distances from the electrodes. Modeling of electrode configurations predicts optimal designs to induce cell movement by DEP while limiting the induced transmembrane potential. We find DEP electric fields are not harmful to stem cells in suspension at short exposure times, thus providing a basis for developing DEP-based applications for stem cells.
Subject(s)
Electrophoresis/methods , Neural Stem Cells/cytology , Animals , Astrocytes/cytology , Cell Line , Cell Lineage , Cell Proliferation , Cell Survival , Cells, Cultured , Cerebral Cortex/cytology , DNA/metabolism , Electrophysiology/methods , Equipment Design , Gestational Age , Humans , Kinetics , Membrane Potentials , Mice , Microtechnology , Models, Statistical , Stem Cells/cytology , Time FactorsABSTRACT
We present an automated dielectrophoretic assisted cell sorting (DACS) device for dielectric characterization and isolation of neural cells. Dielectrophoretic (DEP) principles are often used to develop cell sorting techniques. Here we report the first statistically significant neuronal sorting using DACS to enrich neurons from a heterogeneous population of mouse derived neural stem/progenitor cells (NSPCs) and neurons. We also study the dielectric dispersions within a heterogeneous cell population using a Monte-Carlo (MC) simulation. This simulation model explains the trapping behavior of populations as a function of frequency and predicts sorting efficiencies. The platform consists of a DEP electrode array with three multiplexed trapping regions that can be independently activated at different frequencies. A novel microfluidic manifold enables cell sorting by trapping and collecting cells at discrete frequency bands rather than single frequencies. The device is used to first determine the percentage of cells trapped at these frequency bands. With this characterization and the MC simulation we choose the optimal parameters for neuronal sorting. Cell sorting experiments presented achieve a 1.4-fold neuronal enrichment as predicted by our model.
Subject(s)
Cell Separation/methods , Neurons/cytology , Animals , Dimethylpolysiloxanes/chemistry , Mice , Monte Carlo Method , Stem Cells/cytologyABSTRACT
BACKGROUND: Cisplatin-based chemotherapy followed by surgical extirpation of residual benign disease represents the usual sequence of curative therapy for metastatic nonseminomatous germ cell cancer of testicular origin. Occasionally, residual disease is malignant in the form of either a persistent nonseminomatous germ cell cancer tumor or degeneration into non-germ cell cancer. We reviewed our institution's experience with patients undergoing salvage operations to remove malignant intrathoracic metastases. METHODS: From 1981 through 2001, 438 patients with nonseminomatous germ cell cancer had operations to remove residual intrathoracic disease after cisplatin-based chemotherapy at Indiana University Hospital. A subset of 134 patients who underwent 186 surgical procedures to remove malignant metastases is the basis of this review. Fifty-nine patients had removal of pulmonary metastases, 49 had removal of mediastinal metastases, and 26 had removal of both pulmonary and mediastinal metastases. Surgical pathology demonstrated 84 patients with persistent nonseminomatous germ cell cancer tumors, 38 with degeneration into non-germ cell cancer, and 12 with both malignant pathologic categories. RESULTS: There were 4 (3.7%) operative deaths. The overall median survival was 5.6 years, with 55 (42.3%) patients alive and well after a mean follow-up of 5.1 years. Seventeen variables were analyzed by using Cox regression. Of these, older age, pulmonary metastases (vs mediastinal metastases), and 4 or more (vs 1) total intrathoracic metastases were significantly (P < or = .01) predictive of inferior long-term survival. CONCLUSIONS: Salvage thoracic surgery to remove malignant metastases from nonseminomatous germ cell cancer tumors of testicular origin can result in long-term survival in select patients. We identified variables that influence survival in this subset.
Subject(s)
Lung Neoplasms/therapy , Mediastinal Neoplasms/therapy , Neoplasms, Germ Cell and Embryonal/therapy , Testicular Neoplasms/therapy , Thoracic Surgical Procedures/methods , Adolescent , Adult , Antineoplastic Agents/therapeutic use , Cisplatin/therapeutic use , Combined Modality Therapy , Follow-Up Studies , Humans , Lung Neoplasms/secondary , Male , Mediastinal Neoplasms/secondary , Middle Aged , Neoplasm Staging , Neoplasms, Germ Cell and Embryonal/secondary , Survival Analysis , Testicular Neoplasms/pathology , Treatment OutcomeABSTRACT
BACKGROUND: Treatment of nonseminomatous germ cell tumors frequently requires bleomycin-combination chemotherapy followed by resection of residual disease. Bleomycin administration however raises concerns with respect to postoperative respiratory complications, particularly for patients undergoing large pulmonary resections. We undertook an institutional review to determine the outcome of large pulmonary resections after bleomycin-combination chemotherapy. METHODS: Between 1981 and 2001, 530 patients presented to our institution for resection of residual intrathoracic disease for either metastatic testicular or primary mediastinal nonseminomatous germ cell tumors. We subsequently reviewed 32 of these patients who required pneumonectomy (n = 19; RIGHT = 9, LEFT = 10) or bilobectomy (n = 13) after bleomycin-combination chemotherapy. RESULTS: There were four operative deaths (13%). All postoperative deaths occurred in patients undergoing right-sided resections (pneumonectomy, n = 2; bilobectomy, n = 2) as a consequence of pulmonary complications. Operative survivors had a pulmonary morbidity of 18%. Fourteen of 20 long-term survivors were found to have a satisfactory performance status at follow-up. CONCLUSIONS: Otherwise young and healthy male nonseminomatous germ cell tumors patients requiring large pulmonary resections after bleomycin-combination chemotherapy appear to be at higher than anticipated risk for pulmonary-related morbidity and mortality. However long-term survivors report an acceptable functional status.