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Rationale: Myocardial infarction causes spatial variation in collagen organization and phenotypic diversity in fibroblasts, which regulate the heart’s ECM (extracellular matrix). The relationship between collagen structure and fibroblast phenotype is poorly understood but could provide insights regarding the mechanistic basis for myofibroblast heterogeneity in the injured heart. Objective: To investigate the role of collagen organization in cardiac fibroblast fate determination. Methods and Results: Biomimetic topographies were nanofabricated to recapitulate differential collagen organization in the infarcted mouse heart. Here, adult cardiac fibroblasts were freshly isolated and cultured on ECM topographical mimetics for 72 hours. Aligned mimetics caused cardiac fibroblasts to elongate while randomly organized topographies induced circular morphology similar to the disparate myofibroblast morphologies measured in vivo. Alignment cues also induced myofibroblast differentiation, as >60% of fibroblasts formed αSMA (α-smooth muscle actin) stress fibers and expressed myofibroblast-specific ECM genes like Postn (periostin). By contrast, random organization caused 38% of cardiac fibroblasts to express αSMA albeit with downregulated myofibroblast-specific ECM genes. Coupling topographical cues with the profibrotic agonist, TGFβ (transforming growth factor beta), additively upregulated myofibroblast-specific ECM genes independent of topography, but only fibroblasts on flat and randomly oriented mimetics had increased percentages of fibroblasts with αSMA stress fibers. Increased tension sensation at focal adhesions induced myofibroblast differentiation on aligned mimetics. These signals were transduced by p38-YAP (yes-associated protein)-TEAD (transcriptional enhanced associate domain) interactions, in which both p38 and YAP-TEAD (yes-associated protein transcriptional enhanced associate domain) binding were required for myofibroblast differentiation. By contrast, randomly oriented mimetics did not change focal adhesion tension sensation or enrich for p38-YAP-TEAD interactions, which explains the topography-dependent diversity in fibroblast phenotypes observed here. Conclusions: Spatial variations in collagen organization regulate cardiac fibroblast phenotype through mechanical activation of p38-YAP-TEAD signaling, which likely contribute to myofibroblast heterogeneity in the infarcted myocardium.

Tissues- and organs-on-chips are microphysiological systems (MPSs) that model the architectural and functional complexity of human tissues and organs that is lacking in conventional cell monolayer cultures. While substantial progress has been made in a variety of tissues and organs, chips recapitulating immune responses have not advanced as rapidly. This review discusses recent progress in MPSs for the investigation of immune responses. To illustrate recent developments, we focus on two cases in point: immunocompetent tumor microenvironment-on-a-chip devices that incorporate stromal and immune cell components and pathomimetic modeling of human mucosal immunity and inflammatory crosstalk. More broadly, we discuss the development of systems immunology-on-a-chip devices that integrate microfluidic engineering approaches with high-throughput omics measurements and emerging immunological applications of MPSs.

Gene editing technologies hold great potential to enhance our ability to model inheritable neurodegenerative diseases. Specifically, engineering multiple amyotrophic lateral sclerosis (ALS) mutations into isogenic cell populations facilitates determination of whether different causal mutations cause pathology via shared mechanisms, and provides the capacity to separate these mechanisms from genotype-specific effects. As gene-edited, cell-based models of human disease become more commonplace, there is an urgent need to verify that these models constitute consistent and accurate representations of native biology. Here, commercially sourced, induced pluripotent stem cell-derived motor neurons from Cellular Dynamics International, edited to express the ALS-relevant mutations TDP-43M337V and TDP-43Q331K were compared with in-house derived lines engineered to express the TDP-43Q331K mutation within the WTC11 background. Our results highlight electrophysiological and mitochondrial deficits in these edited cells that correlate with patient-derived cells, suggesting a consistent cellular phenotype arising from TDP-43 mutation. However, significant differences in the transcriptomic profiles and splicing behavior of the edited cells underscores the need for careful comparison of multiple lines when attempting to use these cells as a means to better understand the onset and progression of ALS in humans.

Bio-sourced and biodegradable polymers for additive manufacturing could enable the rapid fabrication of parts for a broad spectrum of applications ranging from healthcare to aerospace. However, a limited number of these materials are suitable for vat photopolymerization processes. Herein, we report a two-step additive manufacturing process to fabricate robust protein-based constructs using a commercially available laser-based SLA printer. Methacrylated bovine serum albumin (MA-BSA) was synthesized and formulated into aqueous resins that were used to print complex 3D objects with a resolution comparable to a commercially available resin. The MA-BSA resins were characterized by rheometry to determine the viscosity and the cure rate, as both of these parameters can ultimately be used to predict the printability of the resin. In the first step of patterning these materials, the MA-BSA resin was 3D printed, and in the second step, the printed construct was thermally cured to denature the globular protein and increase the intermolecular noncovalent interactions. Thus, the final 3D printed part was comprised of both chemical and physical cross-links. Compression studies of hydrated and dehydrated constructs demonstrated a broad range of compressive strengths and Young’s moduli that could be further modulated by adjusting the type and amount of co-monomer. The printed hydrogel constructs demonstrated good cell viability (> 95%) after a 21-day culture period. These MA-BSA resins are expected to be compatible with other vat photopolymerization techniques including digital light projection (DLP) and continuous liquid interface production (CLIP).

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX. Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.

Mozzetta and Tedesco preview work from the Murry laboratory yielding insight into cardiac laminopathy pathogenesis mechanisms by analyzing chromatin compartment dynamics in a haploinsufficient model of the disease., Lamins A and C are intermediate filaments that provide structural support to the nuclear envelope and regulate gene expression. In this issue, Bertero et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201902117) report that although lamin A/C haploinsufficient cardiomyocytes show disease-associated phenotypes, those changes cannot be explained by alterations in chromatin compartmentalization.

Aims Heart failure invariably affects patients with various forms of muscular dystrophy (MD), but the onset and molecular sequelae of altered structure and function resulting from full-length dystrophin (Dp427) deficiency in MD heart tissue are poorly understood. To better understand the role of dystrophin in cardiomyocyte development and the earliest phase of Duchenne muscular dystrophy (DMD) cardiomyopathy, we studied human cardiomyocytes differentiated from induced pluripotent stem cells (hiPSC-CMs) obtained from the urine of a DMD patient. Methods and results The contractile properties of patient-specific hiPSC-CMs, with no detectable dystrophin (DMD-CMs with a deletion of exon 50), were compared to CMs containing a CRISPR-Cas9 mediated deletion of a single G base at position 263 of the dystrophin gene (c.263delG-CMs) isogenic to the parental line of hiPSC-CMs from a healthy individual. We hypothesized that the absence of a dystrophin-actin linkage would adversely affect myofibril and cardiomyocyte structure and function. Cardiomyocyte maturation was driven by culturing long-term (80–100 days) on a nanopatterned surface, which resulted in hiPSC-CMs with adult-like dimensions and aligned myofibrils. Conclusions Our data demonstrate that lack of Dp427 results in reduced myofibril contractile tension, slower relaxation kinetics, and to Ca2+ handling abnormalities, similar to DMD cells, suggesting either retarded or altered maturation of cardiomyocyte structures associated with these functions. This study offers new insights into the functional consequences of Dp427 deficiency at an early stage of cardiomyocyte development in both patient-derived and CRISPR-generated models of dystrophin deficiency.

Visual Abstract, Highlights • Many cardiomyopathy families have genetic variants whose significance is unknown. We studied a novel (E848G) mutation in MYH7, a sarcomeric protein. • Patient-specific induced pluripotent stem cell–derived cardiomyocytes and engineered heart tissues recapitulated the contractile dysfunction. • Overexpression of the E848G allele in MYH7-null induced pluripotent stem cell–derived cardiomyocytes confirms the causality of the E848G variant. • The E848G allele disrupts the protein–protein interaction between MYH7 and cardiac myosin binding protein C, presenting a potential mechanism of action. • Assessing the pathogenicity of new MYH7 variants by overexpressing them in a null background should accelerate their screening for disease causality., Summary A novel myosin heavy chain 7 mutation (E848G) identified in a familial cardiomyopathy was studied in patient-specific induced pluripotent stem cell–derived cardiomyocytes. The cardiomyopathic human induced pluripotent stem cell–derived cardiomyocytes exhibited reduced contractile function as single cells and engineered heart tissues, and genome-edited isogenic cells confirmed the pathogenic nature of the E848G mutation. Reduced contractility may result from impaired interaction between myosin heavy chain 7 and cardiac myosin binding protein C.

SUMMARYThe gut microbiota benefits the host by limiting enteric pathogen expansion (colonization resistance) partially via the production of inhibitory metabolites. Propionate, a short-chain fatty acid produced by microbiota members, is proposed to mediate colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm). Here, we show that S. Tm overcomes the inhibitory effects of propionate by using it as a carbon source for anaerobic respiration. We determined that propionate metabolism provides an inflammation-dependent colonization advantage to S. Tm during infection. Such benefit was abolished in the intestinal lumen of Salmonella-infected germ-free mice. Interestingly, S. Tm propionate-mediated intestinal expansion was restored when germ-free mice were monocolonized with Bacteroides thetaiotaomicron (B. theta), a prominent propionate producer in the gut, but not when mice were monocolonized with a propionate production-deficient B. theta strain. Taken together, our results reveal a novel strategy used by S. Tm to mitigate colonization resistance by metabolizing microbiota-derived propionate.

Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post-translational cancer hallmark and the consequences thereof remain elusive. Here we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In both cancer cell cultures and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes revealed that fucosylation of the antioxidant PON1 is a critical component of the therapy-induced secretome. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Non-specific and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.

Background: Human induced pluripotent stem cells (hiPSCs) are widely used in studies of developmental and regenerative biomedicine involving various cell types, including cardiomyocytes (CMs). Understanding the cellular and molecular processes during hiPSC-CM differentiation and maturation will be crucial to develop heart regeneration therapies. In addition, while bioengineered cellular cues have been shown to modulate cellular morphology and phenotypes, little is known regarding their effects on molecular and functional maturity of hiPSC-CMs. Aims: To determine the expression of cardiac and cell cycle genes and the electrophysiological properties of hiPSC-CMs during their differentiation and maturation on unpatterned (Flat) or isotropic/nanopatterned (Nano) cell culture surface. Methods and Results: Using small molecules, WTC-11 hiPSC were differentiated into CMs efficiently (92.5% TNNT2 + ; 95% NKX2-5 + ). The resultant hiPSC-CMs were re-plated in Flat or Nano surfaces and harvested at different time points. Cardiac genes Myh7, Tnnt2, Serca2a, Ryr2, Cacna1c , and Kcnj2 gradually and significantly increased during differentiation; this was accompanied by reduced expression of cell cycle genes. While myofilament genes expressions were similar between Nano and Flat cultured hiPSC-CMs, ion channel genes Scn5a, Cacna1c , and Kcnj2 were significantly higher in Nano group, suggesting that Nano cultured CMs were more matured. In addition, fewer hiPSC-CMs were proliferative (EdU + ) in 2-week Nano group compared to Flat group. This was associated with decreased expression of active cell cycle genes Ccne1, Cdk4, Cdk14, Ki67 and Plk1 in Nano 2-week CMs. Micro-electrode array (MEA) analysis demonstrated that Beat Period, Spike Amplitude, and Field Potential Duration were increased in the Nano group. Fluo-4 Ca 2+ imaging assay revealed improved Ca 2+ transition activities in isotropically cultured hiPSC-CMs. Conclusion: These results demonstrate a significant upregulation of cardiac genes along with a down-regulation of cell cycle genes during the differentiation and maturation of hiPSC-CM on Nano surfaces. Bioengineered nanotopographically patterned substrates promoted the maturation and electrophysiological functions of hiPSC-CMs.

International audience; The gastrointestinal (GI) tract is a complex system responsible for nutrient absorption, digestion, secretion, and elimination of waste products that also hosts immune surveillance, the intestinal microbiome, and interfaces with the nervous system. Traditional in vitro systems cannot harness the architectural and functional complexity of the GI tract. Recent advances in organoid engineering, microfluidic organs-on-a-chip technology, and microfabrication allows us to create better in vitro models of human organs/tissues. These micro-physiological systems could integrate the numerous cell types involved in GI development and physiology, including intestinal epithelium, endothelium (vascular), nerve cells, immune cells, and their interplay/cooperativity with the microbiome. In this review, we report recent progress in developing microphysiological models of the GI systems. We also discuss how these models could be used to study normal intestinal physiology such as nutrient absorption, digestion, and secretion as well as GI infection, inflammation, cancer, and metabolism.

The root cause of cancer mortality and morbidity is the metastatic spread of the primary tumor, but underlying mechanisms remain elusive. Here we investigate biomechanical interactions that may accompany invasive spread of melanoma cells. We find that metastatic cells can exert considerable traction forces and modify local collagen organization within a 3D matrix. When this re-organization is mimicked using a nano-fabricated model of aligned extracellular matrix fibers, metastatic cells, including less invasive melanoma cells, were in turn induced to align, elongate and migrate, guided by the local ridge orientations. Strikingly, we found that this aligned migration of melanoma cells was accompanied by long-range regulation of cytoskeletal remodeling that show anisotropic stiffening in the direction of fiber orientation suggestive of a positive feedback between ECM fiber alignment and forces exerted by cancer cells. Taken together, our findings suggest that the invasive spread of cancer cells can be defined by complex interplay with the surrounding matrix, during which they both modify the matrix and use the matrix alignment as a persistent migration cue, leading to more extensive and rapid invasive spread.

The cell reprogramming of somatic cells into pluripotent states and subsequent differentiation into certain phenotypes has helped progress regenerative medicine research and other medical applications. Recent research has used viral vectors to induce this reprogramming; however, limitations include low efficiency and safety concerns. In this review, we discuss how biomaterial methods offer potential avenues for either increasing viability and downstream applicability of viral methods, or providing a safer alternative. The use of non-viral delivery systems, such as electroporation, micro/nanoparticles, nucleic acids and the modulation of culture substrate topography and stiffness, has generated valuable insights regarding cell reprogramming.

Background: The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin ( TTN ) are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within TTN motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function. Methods: To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z −/− ) and A-band (TTN-A −/− ) regions of the TTN gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements. Results: After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z −/− -CMs had sarcomeres and visibly contracted, whereas TTN-A −/− -CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z −/− -CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z −/− -CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin. Conclusions: We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to human cardiac disease.

Pathogenic mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses (Hi-C) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared to gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape inactivation during cardiogenesis. These regions exhibit upregulation of multiple non-cardiac genes includingCACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. On the other hand, A/B compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. We conclude that global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency.SummaryBertero et al. observe that lamin A/C haploinsufficiency in human cardiomyocytes markedly alters electrophysiology, contractility, gene expression, and chromosomal topology. Contrary to expectations, however, changes in chromatin compartments involve just few regions, and most dysregulated genes lie outside these hotspots.Condensed titleGenomic effects of lamin A/C haploinsufficiency

Living cells and the extracellular matrix (ECM) can display complex interactions that define key developmental, physiological and pathological processes. Here, we report a new type of directed migration — which we term ‘topotaxis’ — by which cell movement is guided by the gradient of the nanoscale topographic features in the cells’ ECM environment. We show that the direction of topotaxis is reflective of the effective cell stiffness, and that it depends on the balance of the ECM-triggered signalling pathways PI3K-Akt and ROCK-MLCK. In melanoma cancer cells, this balance can be altered by different ECM inputs, pharmacological perturbations or genetic alterations, particularly a loss of PTEN in aggressive melanoma cells. We conclude that topotaxis is a product of the material properties of cells and the surrounding ECM, and propose that the invasive capacity of many cancers may depend broadly on topotactic responses, providing a potentially attractive mechanism for controlling invasive and metastatic behaviour.

Charcot-Marie-Tooth disease type 4H (CMT4H) is an autosomal recessive demyelinating subtype of peripheral enuropathies caused by mutations in the FGD4 gene. Most CMT4H patients are in consanguineous Mediterranean families characterized by early onset and slow progression. We identified two CMT4H patients from a Korean CMT cohort, and performed a detailed genetic and clinical analysis in both cases. Both patients from nonconsanguineous families showed characteristic clinical manifestations of CMT4H including early onset, scoliosis, areflexia, and slow disease progression. Exome sequencing revealed novel compound heterozygous mutations in FGD4 as the underlying cause in both families (p.Arg468Gln and c.1512-2A>C in FC73, p.Met345Thr and c.2043+1G>A (p.Trp663Trpfs*30) in FC646). The missense mutations were located in highly conserved RhoGEF and PH domains which were predicted to be pathogenic in nature by in silico modeling. The CMT4H occurrence frequency was calculated to 0.7% in the Korean demyelinating CMT patients. This study is the first report of CMT4H in Korea. FGD4 assay could be considered as a means of molecular diagnosis for sporadic cases of demyelinating CMT with slow progression.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer unprecedented opportunities to study inherited heart conditions in vitro, but are phenotypically immature, limiting their ability to effectively model adult-onset diseases. Cardiomyopathy is becoming the leading cause of death in patients with Duchenne muscular dystrophy (DMD), but the pathogenesis of this disease phenotype is not fully understood. Therefore, we aimed to test whether biomimetic nanotopography could further stratify the disease phenotype of DMD hiPSC-CMs to create more translationally relevant cardiomyocytes for disease modeling applications. We found that anisotropic nanotopography was necessary to distinguish structural differences between normal and DMD hiPSC-CMs, as these differences were masked on conventional flat substrates. DMD hiPSC-CMs exhibited a diminished structural and functional response to the underlying nanotopography compared to normal cardiomyocytes at both the macroscopic and subcellular levels. This blunted response may be due to a lower level of actin cytoskeleton turnover as measured by fluorescence recovery after photobleaching. Taken together these data suggest that DMD hiPSC-CMs are less adaptable to changes in their extracellular environment, and highlight the utility of nanotopographic substrates for effectively stratifying normal and structural cardiac disease phenotypes in vitro.

The cells of the vascular system are highly sensitive to biophysical cues from their local cellular microenvironment. To engineer improved materials for vascular devices and delivery of cell therapies, a key challenge is to understand the mechanisms that cells use to sense biophysical cues from their environment. Syndecans are heparan sulfate proteoglycans (HSPGs) that consist of a protein core modified with heparan sulfate glycosaminoglycan chains. Due to their presence on the cell surface and their interaction with cytoskeletal and focal adhesion associated molecules, cell surface proteoglycans are well poised to serve as mechanosensors of the cellular microenvironment. Nanotopological cues have become recognized as major regulators of cell growth, migration and phenotype. We hypothesized that syndecan-1 could serve as a mechanosensor for nanotopological cues and can mediate the responsiveness of vascular smooth muscle cells to nanoengineered materials. We created engineered substrates made of polyurethane acrylate with nanogrooves using ultraviolet-assisted capillary force lithography. We cultured vascular smooth muscle cells with knockout of syndecan-1 on engineered substrates with varying compliance and nanotopology. We found that knockout of syndecan-1 reduced alignment of vascular smooth muscle cells to the nanogrooves under inflammatory treatments. In addition, we found that loss of syndecan-1 increased nuclear localization of Yap/Taz and phospho-Smad2/3 in response to nanogrooves. Syndecan-1 knockout vascular smooth muscle cells also had elevated levels of Rho-associated protein kinase-1 (Rock1), leading to increased cell stiffness and an enhanced contractile state in the cells. Together, our findings support that syndecan-1 knockout leads to alterations in mechanosensing of nanotopographical cues through alterations of in rho-associated signaling pathways, cell mechanics and mediators of the Hippo and TGF-β signaling pathways.

In summary, the CRISPR-Cas9-treated mice do not exhibit more variants that are absent in the uncorrected control than vice versa. Furthermore, the observed variants are extremely unlikely a consequence of a mutagenic process in the investigated animal, but are much better explained by differences in the genetic background between the two gene-edited mice and the control mouse. We conclude that the data presented by Schaefer et al does not provide any evidence for off-target mutagenesis due to CRISPR-Cas9 treatment and see no reason to be doubtful about the previous findings that Cas9 does not cause SNVs and indels at non-homologous, PAM-free sites.

Periostin is a matricellular fascilin-like protein that was first described as a component of periodontal ligaments.1 Subsequently, periostin has been shown to have critical functions in development, pathologic fibrosis, and cancer biology (reviewed in2–5). In carcinomas, periostin, secreted by cancer and stromal cells, promotes malignancy by promoting invasion, cell survival, treatment resistance, and metastasis. Secreted periostin functions in part through integrin interactions (most commonly with αvβ3 and αvβ5), which in turn activates proinvasive and survival-signaling pathways including focal adhesion kinase (FAK) and PI3K/Akt.5 Periostin promotes epithelial-mesenchymal transition (EMT)4 and is accordingly implicated in metastatic cell colonization and survival.6,7 Unlike carcinomas, relatively little is known about the prognostic or functional significance of periostin in human gliomas, this despite the early recognition that the periostin gene (POSTN) is among the most upregulated genes in GBM compared with normal brain.8,9 The few studies of periostin in glioma have reported direct correlations between expression levels and increased tumor edema, tumor grade, and survival.10,11 Functionally, periostin is reported to promote glioma cell invasion in vitro.11 In addition to expression in glioma cells, periostin is also reported to be a tumor-specific marker of GBM-associated angiogenesis.12 These associations support the potential importance of periostin in glioma malignancy, but the mechanisms by which periostin may contribute to glioma malignancy require further investigation. This study broadly examined periostin expression and function to extend current understanding of its clinical relevance and therapeutic potential. Expression in clinical samples and public databases have confirmed robust prognostic significance across all glioma grades. Periostin gene signatures also predicted outcome and demonstrated relationships of periostin with biological hallmarks of malignancy. In functional analysis, periostin regulated glioma cell invasion, adhesion, migration, cell morphology, and glioma stem cell survival in vivo. Interactions with αvβ3 and αvβ5 integrins and alteration of FAK localization suggested putative mechanisms of action for secreted periostin. Together, these data strongly support the potential of periostin as a biomarker and therapeutic target in human gliomas and provide direction for future investigations critical to realizing this potential.

Vascular smooth muscle cells (vSMCs) retain the ability to undergo modulation in their phenotypic continuum, ranging from a mature contractile state to a proliferative, secretory state. vSMC differentiation is modulated by a complex array of microenvironmental cues, which include the biochemical milieu of the cells and the architecture and stiffness of the extracellular matrix. In this study, we demonstrate that by using UV-assisted capillary force lithography (CFL) to engineer a polyurethane substratum of defined nanotopography and stiffness, we can facilitate the differentiation of cultured vSMCs, reduce their inflammatory signature, and potentially promote the optimal functioning of the vSMC contractile and cytoskeletal machinery. Specifically, we found that the combination of medial tissue-like stiffness (11 MPa) and anisotropic nanotopography (ridge width_groove width_ridge height of 800_800_600 nm) resulted in significant upregulation of calponin, desmin, and smoothelin, in addition to the downregulation of intercellular adhesion molecule-1, tissue factor, interleukin-6, and monocyte chemoattractant protein-1. Further, our results allude to the mechanistic role of the RhoA/ROCK pathway and caveolin-1 in altered cellular mechanotransduction pathways via differential matrix nanotopography and stiffness. Notably, the nanopatterning of the stiffer substrata (1.1 GPa) resulted in the significant upregulation of RhoA, ROCK1, and ROCK2. This indicates that nanopatterning an 800_800_600 nm pattern on a stiff substratum may trigger the mechanical plasticity of vSMCs resulting in a hypercontractile vSMC phenotype, as observed in diabetes or hypertension. Given that matrix stiffness is an independent risk factor for cardiovascular disease and that CFL can create different matrix nanotopographic patterns with high pattern fidelity, we are poised to create a combinatorial library of arterial test beds, whether they are healthy, diseased, injured, or aged. Such high-throughput testing environments will pave the way for the evolution of the next generation of vascular scaffolds that can effectively crosstalk with the scaffold microenvironment and result in improved clinical outcomes.

Adult stem cells hold great promise as a source of diverse terminally differentiated cell types for tissue engineering applications. However, due to the complexity of chemical and mechanical cues specifying differentiation outcomes, development of arbitrarily complex geometric and structural arrangements of cells, adopting multiple fates from the same initial stem cell population, has been difficult. Here, we show that the topography of the cell adhesion substratum can be an instructive cue to adult stem cells and topographical variations can strongly bias the differentiation outcome of the cells towards adipocyte or osteocyte fates. Switches in cell fate decision from adipogenic to osteogenic lineages were accompanied by changes in cytoskeletal stiffness, spanning a considerable range in the cell softness/rigidity spectrum. Our findings suggest that human mesenchymal stem cells (hMSC) can respond to the varying density of nanotopographical cues by regulating their internal cytoskeletal network and use these mechanical changes to guide them toward making cell fate decisions. We used this finding to design a complex two-dimensional pattern of co-localized cells preferentially adopting two alternative fates, thus paving the road for designing and building more complex tissue constructs with diverse biomedical applications.

Nanoscale topography of artificial substrates can greatly influence the fate of stem cells including adhesion, proliferation, and differentiation. Thus the design and manipulation of nanoscale stem cell culture platforms or scaffolds are of great importance as a strategy in stem cell and tissue engineering applications. In this report, we propose that a graphene oxide (GO) film is an efficient platform for modulating structure and function of human adipose-derived stem cells (hASCs). Using a self-assembly method, we successfully coated GO on glass for fabricating GO films. The hASCs grown on the GO films showed increased adhesion, indicated by a large number of focal adhesions, and higher correlation between the orientations of actin filaments and vinculin bands compared to hASCs grown on the glass (uncoated GO substrate). It was also found that the GO films showed the stronger affinity for hASCs than the glass. In addition, the GO film proved to be a suitable environment for the time-dependent viability of hASCs. The enhanced differentiation of hASCs included osteogenesis, adipogenesis, and epithelial genesis, while chondrogenic differentiation of hASCs was decreased, compared to tissue culture polystyrene as a control substrate. The data obtained here collectively demonstrates that the GO film is an efficient substratum for the adhesion, proliferation, and differentiation of hASCs.

To investigate complex biophysical relationships driving directed cell migration, we developed a biomimetic platform that allows perturbation of microscale geometric constraints with concomitant nanoscale contact guidance architectures. This permits us to elucidate the influence, and parse out the relative contribution, of multiscale features, and define how these physical inputs are jointly processed with oncogenic signaling. We demonstrate that collective cell migration is profoundly enhanced by the addition of contract guidance cues when not otherwise constrained. However, while nanoscale cues promoted migration in all cases, microscale directed migration cues are dominant as the geometric constraint narrows, a behavior that is well explained by stochastic diffusion anisotropy modeling. Further, oncogene activation (i.e. mutant PIK3CA) resulted in profoundly increased migration where extracellular multiscale directed migration cues and intrinsic signaling synergistically conspire to greatly outperform normal cells or any extracellular guidance cues in isolation.

SummaryGlioblastoma multiforme is a heterogeneous and infiltrative cancer with dismal prognosis. Studying the migratory behavior of tumor-derived cell populations can be informative, but it places a high premium on the precision of in vitro methods and the relevance of in vivo conditions. In particular, the analysis of 2D cell migration may not reflect invasion into 3D extracellular matrices in vivo. Here, we describe a method that allows time-resolved studies of primary cell migration with single-cell resolution on a fibrillar surface that closely mimics in vivo 3D migration. We used this platform to screen 14 patient-derived glioblastoma samples. We observed that the migratory phenotype of a subset of cells in response to platelet-derived growth factor was highly predictive of tumor location and recurrence in the clinic. Therefore, migratory phenotypic classifiers analyzed at the single-cell level in a patient-specific way can provide high diagnostic and prognostic value for invasive cancers.

Fibrotic remodeling is a hallmark of most forms of cardiovascular disease and a strong prognostic indicator of the advancement towards heart failure. Myofibroblasts, which are a heterogeneous cell-type specialized for extracellular matrix (ECM) secretion and tissue contraction, are the primary effectors of the heart's fibrotic response. This review is focused on defining myofibroblast physiology, its progenitor cell populations, and the core signaling network that orchestrates myofibroblast differentiation as a way of understanding the basic determinants of fibrotic disease in the heart and other tissues.

Regenerative medicine holds great potential to address many shortcomings in current medical therapies. An emerging avenue of regenerative medicine is the use of self-assembling peptides (SAP) in conjunction with stem cells to improve the repair of damaged tissues. The specific peptide sequence, mechanical properties, and nanotopographical cues vary widely between different SAPs, many of which have been used for the regeneration of similar tissues. To evaluate the potential of SAPs to guide stem cell fate, we extensively reviewed the literature for reports of SAPs and stem cell differentiation. To portray the most accurate summary of these studies, we deliberately discuss both the successes and pitfalls, allowing us to make conclusions that span the breadth of this exciting field. We also expand on these conclusions by relating these findings to the fields of nanotopography, mechanotransduction, and the native composition of the extracellular matrix in specific tissues to identify potential directions for future research.

Engineered in vitro models using human cells, particularly patient-derived induced pluripotent stem cells (iPSCs), offer a potential solution to issues associated with the use of animals for studying disease pathology and drug efficacy. Given the prevalence of muscle diseases in human populations, an engineered tissue model of human skeletal muscle could provide a biologically accurate platform to study basic muscle physiology, disease progression, and drug efficacy and/or toxicity. Such platforms could be used as phenotypic drug screens to identify compounds capable of alleviating or reversing congenital myopathies, such as Duchene muscular dystrophy (DMD). Here, we review current skeletal muscle modeling technologies with a specific focus on efforts to generate biomimetic systems for investigating the pathophysiology of dystrophic muscle.

Cell polarization and directional cell migration can display random, persistent, and oscillatory dynamic patterns. However, it is not clear whether these polarity patterns can be explained by the same underlying regulatory mechanism. Here, we show that random, persistent, and oscillatory migration accompanied by polarization can simultaneously occur in populations of melanoma cells derived from tumors with different degrees of aggressiveness. We demonstrate that all of these patterns and the probabilities of their occurrence are quantitatively accounted for by a simple mechanism involving a spatially distributed, mechanochemical feedback coupling the dynamically changing extracellular matrix (ECM)-cell contacts to the activation of signaling downstream of the Rho-family small GTPases. This mechanism is supported by a predictive mathematical model and extensive experimental validation, and can explain previously reported results for diverse cell types. In melanoma, this mechanism also accounts for the effects of genetic and environmental perturbations, including mutations linked to invasive cell spread. The resulting mechanistic understanding of cell polarity quantitatively captures the relationship between population variability and phenotypic plasticity, with the potential to account for a wide variety of cell migration states in diverse pathological and physiological conditions.

Summary Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G). These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations., Highlights • The contractile properties of hiPSC-CM myofibrils have not been previously studied • hiPSC-CMs cultured on nanopatterned surfaces develop elongated, aligned myofibrils • hiPSC-CMs myofibrils have contractile properties similar to human fetal myofibrils • hiPSC-CMs can be used to study development of genetically based cardiac diseases, In this article, Pioner and colleagues reported contractile properties of isolated myofibrils from hiPSC-CMs with highly mature morphology. This approach permits quantitative assessment of maturation and contractile properties of hiPSC-CMs and can be used to study the development of contractile dysfunction in genetically based cardiac diseases. The authors present a patient-derived cell line carrying a novel familial cardiomyopathy MYH7 mutation (E848G).

The phenotypic expression and function of stem cells are regulated by their integrated response to variable microenvironmental cues, including growth factors and cytokines, matrix-mediated signals, and cell–cell interactions. Recently, growing evidence suggests that matrix-mediated signals include mechanical stimuli such as strain, shear stress, substrate rigidity and topography, and these stimuli have a more profound impact on stem cell phenotypes than had previously been recognized, e.g. self-renewal and differentiation through the control of gene transcription and signaling pathways. Using a variety of cell culture models enabled by micro and nanoscale technologies, we are beginning to systematically and quantitatively investigate the integrated response of cells to combinations of relevant mechanobiological stimuli. This paper reviews recent advances in engineering physical stimuli for stem cell mechanobiology and discusses how micro- and nanoscale engineered platforms can be used to control stem cell niche environments and regulate stem cell fate and function.

The architecture of the extracellular matrix (ECM) directs cell behavior by providing spatial and mechanical cues to which cells respond. In addition to soluble chemical factors, physical interactions between the cell and ECM regulate primary cell processes, including differentiation, migration, and proliferation. Advances in microtechnology and, more recently, nanotechnology provide a powerful means to study the influence of the ECM on cell behavior. By recapitulating local architectures that cells encounter in vivo, we can elucidate and dissect the fundamental signal transduction pathways that control cell behavior in critical developmental, physiological, and pathological processes.

Neurons in the central nervous system (CNS) fail to regenerate axons after injuries due to the diminished intrinsic axon growth capacity of mature neurons and the hostile extrinsic environment composed of a milieu of inhibitory factors. Recent studies revealed that targeting a particular group of extracellular inhibitory factors is insufficient to trigger long-distance axon regeneration. Instead of antagonizing the growing list of impediments, tackling a common target that mediates axon growth inhibition offers an alternative strategy to promote axon regeneration. Neuronal growth cone, the machinery that derives axon extension, is the final converging target of most, if not all, growth impediments in the CNS. In this study, we aim to promote axon growth by directly targeting the growth cone. Here we report that pharmacological inhibition or genetic silencing of nonmuscle myosin II (NMII) markedly accelerates axon growth over permissive and nonpermissive substrates, including major CNS inhibitors such as chondroitin sulfate proteoglycans and myelin-associated inhibitors. We find that NMII inhibition leads to the reorganization of both actin and microtubules (MTs) in the growth cone, resulting in MT reorganization that allows rapid axon extension over inhibitory substrates. In addition to enhancing axon extension, we show that local blockade of NMII activity in axons is sufficient to trigger axons to grow across the permissive–inhibitory border. Together, our study proposes NMII and growth cone cytoskeletal components as effective targets for promoting axon regeneration.

Duchenne's muscular dystrophy (DMD) is a neuromuscular disorder accompanied with muscle weakness and wasting. Since myostatin was reported to be a key regulator of muscle wasting, myostatin inhibitors have been investigated as therapeutic candidates for the treatment of muscular diseases. Here, we report an antigenic peptide of myostatin fragment (MstnF) conjugated to hyaluronate (HA) with a low molecular weight (MW, 17 kDa) for transdermal immunotherapy of DMD. Facilitating the transdermal delivery, the low MW HA showed a boosting effect on the immunization of MstnF possibly by engaging both toll-like receptors and cluster of differentiation 44 (CD44). In vivo two-photon microscopy clearly visualized the effective transdermal penetration of HA-MstnF conjugates into deep intact skin layers. The transdermal immunization of mdx mice significantly increased antibody titers against myostatin. Furthermore, the mdx mice immunized with HA-MstnF conjugates resulted in statistically significant improvement in the biochemical and pathological status of skeletal musculature as well as functional behaviors.

Endothelial physiology is regulated not only by humoral factors, but also by mechanical factors such as fluid shear stress and the underlying cellular matrix microenvironment. The purpose of the present study was to examine the effects of matrix topographical cues on the endothelial secretion of cytokines/chemokines in vitro. Human endothelial cells were cultured on nanopatterned polymeric substrates with different ratios of ridge to groove widths (1:1, 1:2, and 1:5) and with different stiffnesses (6.7 MPa and 2.5 GPa) in the presence and absence of 1.0 ng/mL TNF-α. The levels of cytokines/chemokines secreted into the conditioned media were analyzed with a multiplexed bead-based sandwich immunoassay. Of the nanopatterns tested, the 1:1 and 1:2 type patterns were found to induce the greatest degree of endothelial cell elongation and directional alignment. The 1:2 type nanopatterns lowered the secretion of inflammatory cytokines such as IL-1β, IL-3, and MCP-1, compared to unpatterned substrates. Additionally, of the two polymers tested, it was found that the stiffer substrate resulted in significant decreases in the secretion of IL-3 and MCP-1. These results suggest that substrates with specific extracellular nanotopographical cues or stiffnesses may provide anti-atherogenic effects like those seen with laminar shear stresses by suppressing the endothelial secretion of cytokines and chemokines involved in vascular inflammation and remodeling.

During early development, the chorion envelope of the zebrafish embryo undergoes a thinning process called "chorion softening," which has so far only been characterized chemically. In this study, a micromechanical force sensing system was used to characterize and quantitate mechanical modifications of the zebrafish embryo chorion during early development. Quantitative relationships between applied forces and chorion structural deformations were established at various embryonic stages. The measured penetration force into the chorion at the blastula stage was 1.3-fold greater than those at the prehatching stage. Furthermore, chorion elastic modulus values were determined by using a biomembrane elastic model. The elastic modulus of the chorion at the blastula stage was 1.66-fold greater than that at the prehatching stage, thus indicating that the chorion envelope become mechanically "softened" at the prehatching stage. The experimental results quantitatively describe "chorion softening," which is most likely due to proteolytic activities at the prehatching stage. Gradual chorion softening during embryonic development was also artificially achieved by treating blastula chorion with pronase, a proteolytic enzyme. The forces required to penetrate the pronase-treated chorion were similar to those at the prehatching stage. This similarity suggests that "chorion softening" may be induced by the release of protease from the embryos, and the chemical nature of the process involves proteolytic fragmentation of the ZP2 protein.

Most cells in the body secrete, or are in intimate contact with extracellular matrix (ECM), which provides structure to tissues and regulates various cellular phenotypes. Cells are well known to respond to biochemical signals from the ECM, but recent evidence has highlighted the mechanical properties of the matrix, including matrix elasticity and nanotopography, as fundamental instructive cues regulating signal transduction pathways and gene transcription. Recent observations also highlight the importance of matrix nanotopography as a regulator of cellular functions, but lack of facile experimental platforms has resulted in a continued negligence of this important microenvironmental cue in tissue culture experimentation. In this review, we present our opinion on the importance of nanotopography as a biological cue, contexts in which it plays a primary role influencing cell behavior, and detail advanced techniques to incorporate nanotopography into the design of experiments, or in cell culture environments. In addition, we highlight signal transduction pathways that are involved in conveying the extracellular matrix nanotopography information within the cells to influence cell behavior.

Based on recent results that 1,2-dibromopropane (1,2-DBP) causes hepatotoxicity and immunotoxicity in female BALB/c mice as well as a reduction of hepatic glutathione levels, the possible formation of glutathione conjugates and mercapturic acids of 1,2-DBP was investigated in vivo in the present studies. The following four metabolites were identified in the liver at 12 h after treatment with 1,2-DBP, by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS): M1, 2-hydroxypropylglutathione; M2, 2-oxopropylglutathione; M3, N-acetyl-S-(2-hydroxypropyl)-L-cysteine; and M4, N-acetyl-S-(2-oxopropyl)-L-cysteine. Ions of individual conjugates were observed at m/z 366, 364, 222 and 220, respectively. Characteristic product ions at m/z 237, 217, 204 and 202 for the identification of M1, M2, M3 and M4 were observed, respectively. In the sera isolated from the same animals, only mercapturic acids (M3 and M4) were observed by LC-ESI/MS. When female BALB/c mice were treated orally with 1,2-DBP at doses of 150, 300 and 600 mg kg(-1) once for 12 h, the production of glutathione conjugates and mercapturic acids in liver was apparently dose dependent, as were the concentrations of them in sera. When the production of metabolites from 1,2-DBP was investigated in liver following oral treatment with 600 mg kg(-1) 1,2-DBP for 6, 12, 24 and 48 h, metabolite concentrations were greatest at the first time point (6 h). The results explain the authors' previous studies that oral treatment with 1,2-DBP reduces the hepatic content of glutathione.

Most current drug screening assays used to identify new drug candidates are 2D cell-based systems, even though such in vitro assays do not adequately recreate the in vivo complexity of 3D tissues. Inadequate representation of the human tissue environment during a preclinical test can result in inaccurate predictions of compound effects on overall tissue functionality. Screening for compound efficacy by focusing on a single pathway or protein target, coupled with difficulties in maintaining long-term 2D monolayers, can serve to exacerbate these issues when utilizing such simplistic model systems for physiological drug screening applications. Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens. In this review, we discuss the development of microengineered 3D tissue models which accurately mimic the physiological properties of native tissue samples, and highlight the advantages of using such 3D micro-tissue models over conventional cell-based assays for future drug screening applications. We also discuss biomimetic 3D environments, based-on engineered tissues as potential preclinical models for the development of more predictive drug screening assays for specific disease models.

Eung-Sam Kim,1,2 Eun Hyun Ahn,3,4 Tal Dvir,5,6 Deok-Ho Kim1,4,71Department of Bioengineering, University of Washington, Seattle, WA, USA; 2Department of Biological Sciences, Chonnam National University, Gwangju, Korea; 3Department of Pathology, 4Institute of Stem Cell and Regenerative Medicine, School of Medicine, University of Washington, Seattle, WA, USA; 5Department of Molecular Microbiology and Biotechnology, 6Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, Israel; 7Center for Cardiovascular Biology, University of Washington, Seattle, WA, USAThe history of human kind suggests that there has been a correlation between global population growth and major events in science and technology over the last three centuries. Sharp increases in the world’s population have been triggered by the industrial revolution and scientific and technological breakthroughs including: the advent of the railways, discovery of penicillin and deoxyribonucleic acid (DNA), and the invention of the computer.1 Since the 20th century, interdisciplinary areas in the physical and biological sciences have accelerated the progress of biomedical applications. The recent integration of emerging nanotechnology into biology and biomedicine has resulted in a range of innovative nanoengineering efforts for the repair and regeneration of tissues and organs.2 Thus, it is expected that nanoengineering approaches to biomedical applications can contribute to addressing the present issue of personal and global health care and its economic burden for more than 7 billion people.Why are we paying attention to nanoengineering for biomedical applications? The size of most biomolecules ranges from 0.2 nm to 200 nm (Figure 1). Research has focused on control of the interaction and localization of biomolecules even at the single-molecule level using ever-evolving nanotechnology.3 The evidence indicates that cells can respond to nanoscale changes in the dynamic extracellular matrix and vice versa. Biomimetic nanopatterns alone can direct the differentiation of stem cells without involvement of exogenous soluble biochemical factors.4,5 This regulation of cellular behavior by nanotechnology is one of many examples demonstrating the significant applications of nanoengineering in biomedicine. This special issue includes four review papers and seven research articles that provide an insight into current nanoengineering approaches to the repair or regeneration of tissues and organs.

Microfluidic platforms provide several unique advantages for drug development. In the production of drug carriers, physical properties such as size and shape, and chemical properties such as drug composition and pharmacokinetic parameters, can be modified simply and effectively by tuning the flow rate and geometries. Large numbers of carriers can then be fabricated with minimal effort and with little to no batch-to-batch variation. Additionally, cell or tissue culture models in microfluidic systems can be used as in vitro drug screening tools. Compared to in vivo animal models, microfluidic drug screening platforms allow for high-throughput and reproducible screening at a significantly lower cost, and when combined with current advances in tissue engineering, are also capable of mimicking native tissues. In this review, various microfluidic platforms for drug and gene carrier fabrication are reviewed to provide guidelines for designing appropriate carriers. In vitro microfluidic drug screening platforms designed for high-throughput analysis and replication of in vivo conditions are also reviewed to highlight future directions for drug research and development.

Development of a small-diameter vascular graft (

Cell cycle arrest in response to hypoxia is a fundamental physiological mechanism to maintain a balance between O(2) supply and demand. Many of the cellular responses to reduced O(2) availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the minichromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. Although the interaction between Cdc6 and the MCM complex increased the association of the MCM proteins with chromatin, the binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. These findings establish a transcription-independent mechanism by which the stabilization of HIF-1α leads to cell cycle arrest in response to hypoxia.