Bone defects caused by trauma, tumour resection, infection and congenital deformities, together with articular cartilage defects and cartilage–subchondral bone complex defects caused by trauma and degenerative diseases, remain great challenges for clinicians. Novel strategies utilising cell sheet technology to enhance bone and cartilage regeneration are being developed. The cell sheet technology has shown great clinical potential in regenerative medicine due to its effective preservation of cell–cell connections and extracellular matrix and its scaffold-free nature. This review will first introduce several widely used cell sheet preparation systems, including traditional approaches and recent improvements, as well as their advantages and shortcomings. Recent advances in utilising cell sheet technology to regenerate bone or cartilage defects and bone–cartilage complex defects will be reviewed. The key challenges and future research directions for the application of cell sheet technology in bone and cartilage regeneration will also be discussed. With the injection of a cell suspension, locating the injected suspension and controlling the shape and size of the cell suspension after injection is difficult. The number of cells that can be delivered by one injection is quite limited, and the cells are easily lost after injection. Additionally, a uniform distribution of the injected suspension is difficult to achieve. Thus far, the cell injection technique cannot meet the requirements for regenerating tissue morphology and function. An ideal biodegradable scaffold material that can efficiently promote cell adhesion, proliferation and extracellular matrix (ECM) secretion with suitable mechanical properties is still being sought by researchers.8 Existing scaffold materials usually have several limitations, such as insufficient biological activity, unstable degradation rate and immunogenicity, resulting in immune responses and inflammation after transplantation. Cell–material interactions are usually uncontrollable and may result in high cell mortality.9 Cell–cell interactions and ECM formation contribute to maintaining tissue stability. Conventional tissue engineering techniques for harvesting cells by trypsin digestion damage cell–cell interactions, cell–ECM interactions and cell membrane proteins, resulting in decreased cell adhesion and proliferation.Bone defects caused by various aetiologies, such as trauma, tumours, infection and congenital deformities, together with articular cartilage defects and osteochondral complex defects caused by trauma and degenerative diseases, are common clinical diseases that significantly affect the patient’s quality of life. Repair and regenerating these defects in bone and cartilage is a considerable challenge for clinicians.11 The hydrophilic and hydrophobic properties of the temperature-sensitive material poly(N-isopropylacrylamide) (PIPAAm) could be altered by changing the temperature, resulting in control over cell attachment and detachment.12 Cell sheet technology can be used to harvest cells without utilising proteolytic enzymes, such as trypsin, or chelating agents, such as ethylenediaminetetraacetic acid. Thus the cell–cell junctions, ECM and cell sheet structure are effectively preserved, allowing the constructed tissue to have a high cell density and a uniform cell distribution and thus to mimic native tissue more closely. In addition, cell sheets are prepared by the formation of cell–cell junctions and the secretion of ECM and are free from the limitations of scaffold materials, such as the immune and inflammatory reactions caused by scaffold implantation, tissue collapse caused by a fast degradation rate and compromised tissue formation caused by a slow degradation rate.18 The application of this technology in bone and cartilage regeneration has been widely studied. On the one hand, cell sheets can be used without scaffolds for bone and cartilage regeneration; thus they more closely mimic native tissue and avoid the limitations and potential problems of scaffolds.20 On the other hand, cell sheets can also be used in combination with various scaffolds and may be a better choice than traditional scaffolds seeded with cell suspensions because cell sheets can effectively preserve cell–cell junctions and ECM.22To overcome the shortcomings of conventional tissue engineering technology, cell sheet technology, an alternative approach, has gradually attracted the attention of researchers in recent years. Cell sheet technology was developed based on a novel technique for culturing and harvesting cells using temperature-responsive culture dishes, which was first reported in 1990.Several widely used cell sheet preparation systems, including traditional methods, and recent improvements in these methods, as well as their advantages and shortcomings, will be reviewed. Recent advances in the application of cell sheet technology for the repair and regeneration of bone and cartilage defects will also be reviewed. Furthermore, the key limitations of cell sheet applications in bone and cartilage regeneration, along with directions for future research, will be discussed.23 With continuous advances in cell sheet technology in recent years, approaches to optimising the preparation of cell sheets have been proposed based on these systems  on temperature-responsive culture dishes accelerated cell sheet formation by promoting cell adhesion.28To accelerate the formation of cell sheets, biomolecules can be utilised to promote cell adhesion or proliferation. Immobilising insulin (INS) on temperature-responsive culture dishes shortened the period of cell sheet formation by promoting cell proliferation,29 Furthermore, the co-grafting of poly(ethylene glycol) (PEG) with PIPAAm onto the PM by EB irradiation to form a PIPAAm(PEG)-PM substrate was proposed and ultimately shortened the cell sheet detachment time to 19 min at 20 °C.30 2-Carboxyisopropylacrylamide (CIPAAm) was synthesised with a similar side chain structure as that of N-isopropylacrylamide (IPAAm) and a functional carboxylate group. A P(IPAAm-co-CIPAAm) substrate was developed to accelerate cell sheet detachment by reducing the exposure time to 35 min at 20 °C.31 A temperature and saccharide dual-responsive system consisting of both PIPAAm and the saccharide-sensitive material poly(3-acrylamidophenylboronic acid) was proposed for cell sheet preparation. Cell sheets could be harvested more efficiently by simultaneously reducing the temperature and increasing the saccharide concentration. The exposure time at 20 °C for cell sheet detachment was shortened to within 30 min in 10 g/L fructose.32 A small amount of 3-aminopropyltriethoxysilane (APTES) was utilised to develop a PIPAAm-APTES substrate, which remarkably shortened the detachment time of cell sheets to within 2.5 min in cold culture medium and improved cell adhesion and proliferation simultaneously.33Several improved systems have been developed to shorten the cell sheet detachment time, which could permit further complex manipulation of cell sheets and prevent potential damage to cell viability from long exposures to low, non-physiological temperatures. PIPAAm was grafted onto a porous membrane (PM) by electron beam (EB) irradiation to form a PIPAAm-PM substrate for cell sheet preparation. Compared with the traditional PIPAAm substrate, the PIPAAm-PM substrate showed a decrease in cell sheet detachment time from approximately 75 to 30 min at 20 °C.35 A patterned, bio-functional and temperature-responsive system was fabricated using EB patterning and appropriate metal masks, resulting in the formation of a surface pattern of carboxyl-functional thermo-responsive polymers; this process was followed by site-selective bio-functionalisation with RGDS-INS biomolecules. Cell sheet formation using this system was improved by the promotion of cell adhesion and proliferation, but the detachment time was prolonged to >3 h at 20 °C.36 A dual temperature-responsive substrate for the preparation of patterned cell sheets containing two different cell types was developed via the patterned polymerisation of n-butyl methacrylate into PIPAAm using EB irradiation.37 A temperature-responsive system for the preparation of micropatterned cell sheets was proposed by Isenberg et al. The polystyrene substrate for cell sheet preparation was micropatterned by hot embossing with polydimethylsiloxane moulds containing an array of parallel grooves and then grafted with PIPAAm.38 A similar micropatterned substrate was used to prepare monolayered cell sheets with micropatterns.40 Williams et al. developed a temperature-responsive system combined with a microcontact printing technique for the preparation of aligned cell sheets. Lanes were formed on the PIPAAm substrate by microcontact printing of fibronectin (FN) using a patterned stamp. Cells incubated in a serum-free environment preferentially adhered to the lanes containing FN, resulting in ordered alignment. With the addition of serum, the cells expanded into the regions without FN to form a cell sheet. A patterned monolayered cell sheet was then harvested by reducing the temperature.41 A micropatterned and co-cultured cell sheet preparation system was developed by microcontact printing of FN, followed by the seeding of one type of cell in serum-free conditions and the seeding of another type of cell in the presence of serum.42The incorporation of specific structures into cell sheets is of great importance for mimicking the complex architecture of native tissues and has drawn the attention of researchers. Cell patterning or micropatterning technology has been applied in cell sheet preparation systems.44For application in bone and cartilage regeneration, commercial temperature-responsive culture dishes were used to culture cells, such as mesenchymal stem cells (MSCs), chondrocytes and synovial cells. After the cells were cultured for several days or weeks to form cell sheets, the temperature-responsive culture dishes were exposed to 20 °C or room temperature for a period of time, and polyvinylidene fluoride (PVDF) membranes or forceps were used to harvest the cell sheets.45 Inaba et al. proposed another electro-responsive system for cell sheet preparation involving a gold surface modified with a self-assembled monolayer of alkanethiol and Arg-Gly-Asp (RGD) peptides. Cell sheets formed on the substrate and then could be harvested within 10 min by the application of a negative electrical potential.46 To alleviate the problem of potentially harmful chemicals used in the electro-responsive system remaining in the cell sheets, leading to an inflammatory response upon application, an oligopeptide containing a cell-adhesion domain, RGD, in the centre and cysteine residues at both terminals was utilised to modify the gold surface. The cell sheets were also detached by the application of a negative electrical potential.47 Similar electro-responsive systems for cell sheet preparation were developed using oligopeptides and a negative electrical potential.49 Cell micropatterns could be transferred to a hydrogel by means of cell-adhesive and cell-repulsive micropatterned oligopeptides.49 An electrochemically switchable system for the preparation of micropatterned heterotypic cell sheets was proposed; the system combined photolithographic processing and the local electrochemical dissolution of polyelectrolytes.50 A porous gold-coated membrane substrate that improved oxygen and nutrient supply was covered with an oligopeptide layer and utilised as an electro-responsive system for cell sheet preparation. A monolayered cell sheet could be detached in 7 min by the application of a negative electrical potential, and multilayered structures could be formed by subsequent stacking.51 Most studies have mainly focused on electro-responsive systems for cell sheet preparation for application in vascularised tissues, and the application of such systems in bone and cartilage regeneration has not been reported thus far.52 The specific culture substrates and devices needed in the electro-responsive system might be obstacles for widespread application.A new electro-responsive system for cell sheet preparation was proposed by Guillaume-Gentil et al. The cell sheet was formed by growing cells to confluence on a thin polyelectrolyte film. Then the cell sheet was detached from the polyelectrolyte substrate with electrochemical control, such as by applying a positive potential for electrochemical polarisation, or spontaneously without electrochemical control.2) nanodot-coated quartz substrate and formed a cell sheet. More than 90% of the cells could be detached from the substrate by the application of 365-nm ultraviolet illumination for 20 min.53 A polystyrene substrate covered with a composite polydopamine/TiO2 film was applied for cell sheet preparation. The application of 365-nm ultraviolet illumination for 20 min allowed the detachment of >90% of fibroblasts and >77% of osteoblasts from the substrate.54 A TiO2 nanodot film with surface-immobilised RGD was proposed for cell sheet preparation and was shown to promote cell sheet formation by improving cell adhesion, and 365-nm ultraviolet illumination for 30 min prompted cell sheet detachment.56 Immobilising human recombinant laminin-521 on a TiO2 nanodot film could promote cell sheet formation by improving cell adhesion and proliferation, and these cell sheets could be harvested by 365-nm ultraviolet illumination for 30 min.57 A photo-responsive system for the preparation of micropatterned multilayered cell sheets was developed using a TiO2 nanodot film and photo-cross-linkable gelatine methacrylate (GelMA). Photomask-assisted 254-nm ultraviolet illumination was used to micropattern a TiO2 nanodot film, and 365-nm ultraviolet illumination was used to harvest the cell sheet. The photo-cross-linkable GelMA was used to transfer and stack monolayer cell sheets into a multilayer structure.58 A visible light-responsive system for cell sheet preparation was proposed by Wang et al. The cell sheets could be detached from silicon wafer substrates with p/n junctions (Si(p/n)) after 10 min of illumination with low-energy visible light.59 Na et al. proposed a photo-responsive system for cell sheet preparation based on near infrared (NIR) light, which is highly transmissive through tissues and safer at an appropriate intensity than ultraviolet and visible light. Owing to the high photothermal efficiency of the poly (PEDOT) substrate and the photothermal pattern formed by the diffraction of NIR light through a micropatterned optical lens, cell sheets with various patterns could be harvested within 5 min  and anionic poly(styrene sulfonate) on conductive indium tin oxide electrodes. Then a monolayered cell sheet could be harvested by decreasing the local or global pH.64 When the cells formed a contiguous cell sheet, a cell scraper could be used to lift the cell sheet inward with gentle scraping from the periphery in phosphate-buffered saline.19 Forceps have also been used to mechanically peel off and detach cell sheets.62 A novel mechanical system for the preparation of osteogenic cell sheets involving the addition of gelatine to osteogenic medium was proposed. Compared to the conventional osteogenic cell sheet, the gelatine-induced osteogenic cell sheet had great cell proliferation and abundant ECM, resulting in a thick and strong cell sheet.63Mechanical systems may be easy to prepare a cell sheet without specific culture substrates or techniques, but the required manipulation can be challenging. While few studies have been conducted on simple mechanical techniques, these techniques have been widely used to prepare cell sheets for application. To form an osteogenic or chondrocytic cell sheet, MSCs or chondrocytes were cultured in osteogenic or chondrogenic medium for several days or weeks.65 A culture surface coated with RGD peptide-conjugated MCLs (RGD-MCLs) by magnetic attraction was proposed for cell sheet preparation. A contiguous cell sheet containing RGD-MCLs was formed by incubating cells on the RGD-MCL substrate, which facilitated cell adhesion, and was then detached by removing the magnet.66 Multilayered cell sheets containing heterotypic co-cultured cells labelled with MCLs were fabricated by the application of magnetic force.67A magnetic system for cell sheet preparation was first introduced by Ito et al. Magnetite cationic liposomes (MCLs) with a positive surface charge for improved adsorption were taken up by cells. Multilayered sheets of MCL-labelled cells were formed in an ultralow-attachment plate via magnetic attraction. The cell sheets were detached from the plates by removing the magnetic field and then harvested with a magnet.3O4 magnetic nanoparticles (MNPs) coated with nanoscale graphene oxide (nGO@Fe3O4). The nGO@Fe3O4 MNPs could be easily and rapidly taken up by dental pulp stem cells (DPSCs), MC3T3-E1 cells, bone marrow-derived mesenchymal stem cells (BMSCs), chondrocytes and human umbilical vein endothelial cells (HUVECs)  Fig. . MultilaCs) Fig. . In addiCs) Fig. . A monkeCs) Fig. . The sepCs) Fig. . In addiCs) Fig. . MoreoveCs) Fig. .68Fig. 276 In this review, we focus on recent progress in the application of cell sheet technology in bone and cartilage regeneration  scaffolds. Subcutaneous implantation of these cell sheet scaffold constructs into rabbits resulted in superior ectopic bone formation.83 β-TCP scaffolds containing BMSCs were wrapped in osteogenic matrix BMSC sheets to form BMSC/TCP/sheet constructs. The subcutaneous implantation of these constructs, as well as their implantation into femoral bone defects, showed very promising osteogenic potential.85 Porous β-TCP/COL-I composite scaffolds wrapped in BMSC sheets after osteogenic induction implanted in nude mice have also shown superior osteogenic ability.86 BMSC sheets from both young and old rats after osteogenic induction were combined with β-TCP scaffolds and transplanted subcutaneously into recipient rats; bone formation was promoted in both the young and old BMSC sheets groups, indicating that BMSC sheets from old donors were also capable of bone regeneration.87 In addition to the most widely used β-TCP ceramic scaffold, polymeric scaffolds, including poly(sebacoyl diglyceride) (PSeD) and poly(lactic-co-glycolic acid) (PLGA), combined with cell sheets have also been studied for bone regeneration. Xie et al. wrapped human ethmoid sinus mucosal membrane-derived MSC sheets around porous PSeD scaffolds pre-seeded with rat BMSCs and showed that implantation of these constructs into 8-mm-diameter critical-sized calvarial defects in rats promoted new bone regeneration.21 The utilisation of PLGA scaffolds wrapped in BMSC sheets after osteogenic induction also improved bone regeneration in canine mandibular bone defects.88 Furthermore, scaffolds derived from natural bone, including calcined bovine bones and allografts, have been studied. Liu et al. wrapped rat BMSCs after osteogenic induction around calcined bovine bones (CBBs) to form CBB-BMSC sheet constructs. Upon implantation of these constructs into 8-mm-diameter critical-sized calvarial bone defects in osteoporotic rats, the BMSC sheets survived in the scaffold and participated in new bone formation.89 Devitalised allografts wrapped in mouse BMSC sheets were transplanted into 4-mm critical-sized murine femoral segmental bone defects by Long et al., who showed that the BMSC sheets improved bone callus formation during allograft healing in bone defects and enhanced allograft osseointegration.90Intact cell sheet formation relies on cell–cell connections and the ECM secreted by cells. However, as cell sheets have poor mechanical properties, repairing bone defects with cell sheets alone remains a challenge. Thus, many studies have investigated the repair of bone defects by combining cell sheets prepared by mechanical or temperature-responsive systems with scaffolds, which could provide initial mechanical and spatial support. In a study by Zhou et al., multilayered porcine BMSC sheets were harvested after 1–2 weeks of osteogenic induction and then wrapped around pre-seeded polycaprolactone–calcium phosphate (PCL-CaP) scaffolds to form cell sheet scaffold constructs.91 Recently, growth factors have been utilised with cell sheets to promote bone regeneration. The platelet-rich plasma (PRP) gel contains several growth factors, including platelet-derived growth factor (PDGF), transforming growth factor , insulin-like growth factor  and vascular endothelial growth factor. Qi et al. combined rat BMSC sheets prepared by the mechanical system with the PRP gel and CaP particles and then transplanted the constructs into cortical bone defects in rat femurs. They discovered that incorporating PRP gel/CaP particles into BMSC sheets enhanced bone regeneration.92 Unlike the intramembranous ossification-based bone tissue engineering strategy, which mimics the embryological process and requires the immediate establishment of initial vascular networks for implant survival, the endochondral ossification-based bone tissue engineering strategy utilises engineered cartilage as a transient template that can survive in an avascular environment and is capable of inducing angiogenesis via hypertrophic chondrocytes to promote bone regeneration and fracture healing.93 Bioactive microparticles capable of not only the controlled delivery of TGF-β1 early to induce cartilage formation but also the sustained delivery of bone morphogenetic protein 2 (BMP-2) to promote bone remodelling were engineered by Dang et al. Human BMSC sheets combined with such microparticles containing growth factors were implanted into rat critical-sized calvarial bone defects and were shown to accelerate bone defect healing.94 Our group discovered that nGO@Fe3O4 MNPs are capable of binding and delivering proteins via the plentiful carboxyl groups provided by the nGO coating  and different concentrations of bovine serum albumin  cells, which were induced from human BMSCs. Subcutaneous implantation of the construct into nude mice revealed its excellent osteogenic and angiogenic potential.99 A double cell sheet complex was formed by combining an osteogenic cell sheet with a vascular endothelial cell sheet, with both sets of cells induced from rabbit adipose-derived MSCs. Subcutaneous implantation of this complex into nude mice showed that it had an excellent ability to promote osteogenesis and angiogenesis. In addition, the combination of the double cell sheet complex with a coral hydroxyapatite (HA) scaffold resulted in the synergistic promotion of both osteogenesis and blood vessel formation.100 A novel vascularised tissue-engineered bone scaffold was developed by wrapping osteogenic matrix cell sheets induced from rat BMSCs around a microporous β-TCP scaffold with a side groove. Subcutaneous implantation of the sheet scaffold constructs into rats with femoral vascular bundles passing through the groove promoted vascularisation and new bone formation.101 Another study used an arteriovenous loop (AVL) to circle a β-TCP scaffold by insertion into the lateral groove. Then the construct was wrapped in a rabbit BMSC sheet and transplanted into the thigh muscle pockets of rabbits. New bone formation and vascularisation were promoted by the combination of a BMSC sheet, β-TCP scaffold and AVL.102Vascularisation is essential for the repair of large bone defects. Recently, strategies consisting of co-culturing cell sheets with vascular endothelial cells, stacking vascular endothelial cell sheets with other types of cell sheets or even inserting vascular bundles in cell sheet scaffold constructs have been utilised to construct cell sheets capable of promoting vascularised bone regeneration. Mechanical or temperature-responsive systems were used to prepare the cell sheets. Asakawa et al. created three-dimensional (3D) stratified tissues by stacking cell sheets with HUVEC sheets and discovered that pre-vascular networks composed of HUVECs formed tubular structures, similar to those of native microvasculature in vitro.103 Multilayered cell sheets have been prepared using mechanical systems to promote vascularised bone regeneration by mimicking the native periosteum structure. Kang et al. seeded HUVECs on undifferentiated human MSC sheets to mimic the fibrous layer of native periosteum, while an osteogenic human MSC sheet was cultured to mimic the osteogenic layer of native periosteum. Combining these two cell sheets and wrapping them around a β-TCP scaffold resulted in a biomimetic, cell sheet-based engineered periosteum. Subcutaneous implantation of the cell sheet scaffold construct into nude mice enhanced angiogenesis, functional anastomosis with the host vasculature and osteogenesis , canines (3 mm in diameter and 1 mm in depth) and NOD/SCID mice (1 mm in diameter). The transplanted cell sheets promoted cartilage regeneration without causing chondrocyte hypertrophy.43Articular cartilage is a highly organised, resilient connective tissue that covers the surface of bone.111 Therefore, the repair of bone–cartilage complex defects has received much attention in the field of regenerative medicine, and many researchers have applied the cell sheet technology for the treatment of bone–cartilage complex defects. Multilayered chondrocyte sheets prepared by the temperature-responsive system were autologously transplanted into full-thickness femoral condylar cartilage defects 6 mm in diameter and 5 mm in depth in minipigs by Ebihara et al., who discovered that the multilayered chondrocyte sheets promoted cartilage repair and regeneration. However, in some cases, the effect on subchondral bone regeneration was unsatisfactory.20 Multilayered chondrocyte sheets combined with synovial cells using the temperature-responsive system were transplanted into osteochondral defects 5 mm in diameter and 3 mm in depth in the femoral patellar groove of rabbits by Ito et al., who reported increased repair and regeneration of both cartilage and subchondral bone in bone–cartilage complex defects.44 Wang et al. utilised minipig-derived chondrocyte sheets prepared by the mechanical system to cover PCL/HA scaffolds seeded with minipig-derived BMSCs. Subcutaneous implantation of the constructs into nude mice regenerated condyle-shaped osteochondral composites.64 In addition to immunodeficient animal models, minipigs have been used to show that subcutaneous and intramuscular construct transplantation results in the regeneration of biological condyles with an osteochondral construction similar to that of native condyles.22 Takaku et al. used the temperature-responsive system to prepare multilayered cell sheets consisting of both chondrocytes and synovial cells from firefly luciferase-expressing transgenic rats and transplanted them into wild-type rats. The transplanted cell sheets could remain and survive in osteochondral defects in the knee joint for up to 21 months, which confirmed the effects of the cell sheets on the repair and regeneration of bone–cartilage complex defects.112 The mechanism of cell sheet-mediated repair of bone–cartilage complex defects was discovered by Shimizu et al. via cell tracking experiments using green fluorescent protein (GFP) transgenic rats. Moreover, these researchers found that higher expression of TGF-β1 in chondrocyte sheets might contribute to cartilage repair.113In addition to the destruction of articular cartilage, the destruction of subchondral bone is usually involved in osteoarthritis.3O4 MNPs. Magnetically attracting nGO@Fe3O4 MNP-labelled DPSCs and nGO@Fe3O4 MNP-bound growth factors led to the creation of microtissues with a closely integrated double-layered structure mimicking the cartilage–bone interface (Fig. 68Our group constructed microtissues consisting of chondrogenic cell sheets and osteogenic cell sheets for the repair of bone–cartilage complex defects based on the magnetically controlled approach. The osteogenic growth factor BMP-2 and the chondrogenic growth factor TGF-β3 were bound to nGO@Feace Fig. , in whicace Fig. . One weeace Fig. .68Fig. 5In recent decades, much progress has been made in the preparation and application of cell sheets. With the efforts of many researchers, cell sheet technology is becoming a promising strategy for the repair and regeneration of bone and cartilage defects. However, some obstacles remain in the application of cell sheet technology. For example, constructing tissue or organ-like tissue with a complicated structure and morphology is difficult, and the constructed cell sheets usually lack mechanical properties. The cells in multilayered cell sheets are prone to necrosis. In addition, the components of cells and ECM in cell sheets are quite different from those in native cartilage.To construct 3D bone and cartilage, the cells or cell sheets must be stacked to form complex structures and shapes. Various systems are available for constructing cell sheets. Temperature-responsive and mechanical approaches are the most widely used to prepare cell sheets for bone and cartilage regeneration, likely because temperature-responsive systems are the most traditional and widely studied systems and because mechanical approaches are easy due to the lack of a need for specific culture substrates or techniques. The stacking technique is widely utilised for the preparation of multilayered cell sheets. With the application of magnetic technology, multilayered cell sheets can be prepared by magnetically attracting MNP-labelled cells layer by layer in a single step. Cell patterning and micropatterning technology has been introduced to modify the morphology of cell sheets. In a magnetic system, the shape of cell sheets can be precisely controlled using different numbers of magnets or magnet patterns. However, using existing cell sheet preparation techniques to prepare relatively more complex tissues and even organs is still challenging. Thus existing preparation systems need to be further improved, or novel effective preparation systems need to be proposed.The maintenance of a sufficient nutrient and oxygen supply and the efficient exchange of cellular metabolites contribute to the survival of cells in cell sheets. Cells deep in thick multilayered cell sheets are prone to necrosis during application for bone tissue regeneration due to the insufficient nutrient and oxygen supply and the poor exchange of cell waste. Vascularisation is of great importance for bone tissue regeneration. The rapid growth of blood vessels into transplanted cell sheets can supply nutrients and oxygen and exchange metabolites in a timely manner, thus effectively promoting the survival of transplanted cells and the growth of host tissues. The rapid vascularisation of cell sheets is an important research direction for bone regeneration.114 Constructing cell sheets with a composition similar to that of native cartilage is crucial; however, further research is needed for the application of such sheets in cartilage regeneration.Native cartilage is composed of a large amount of ECM components and a small number of chondrocytes. However, currently prepared cell sheets usually contain a large number of cells with a relatively small proportion of ECM and are quite different from native cartilage. Recently, a micropatterned collagen sheet was fabricated by the permeation of a piece of doughnut-shaped paper by a cell–collagen mixture, and the components of this sheet are more similar to native cartilage than the other cell sheets mentioned above.A permission of Advanced MaterialsA permission of Advanced Functional MaterialsA permission of ACS Applied Materials & Interfaces


Here, we show that the genetically tractable frog Xenopus tropicalis, paired with optical coherence tomography imaging, provides new insights into CSF flow dynamics and role of ciliary dysfunction in hydrocephalus pathogenesis. We can visualize CSF flow within the multi-chambered ventricular system and detect multiple distinct polarized CSF flow fields. Using CRISPR/Cas9 gene editing, we modeled human L1CAM and CRB2 mediated aqueductal stenosis. We propose that our high-throughput platform can prove invaluable for testing candidate human CH genes to understand CH pathophysiology.Cerebrospinal fluid (CSF) flow in the brain ventricles is critical for brain development. Altered CSF flow dynamics have been implicated in congenital hydrocephalus (CH) characterized by the potentially lethal expansion of cerebral ventricles if not treated. CH is the most common neurosurgical indication in children effecting 1 per 1000 infants. Current treatment modalities are limited to antiquated brain surgery techniques, mostly because of our poor understanding of the CH pathophysiology. We lack model systems where the interplay between ependymal cilia, embryonic CSF flow dynamics and brain development can be analyzed in depth. This is in part due to the poor accessibility of the vertebrate ventricular system to  After neural tube closure, CSF is secreted primarily by the choroid plexus in the lateral ventricles, flows sequentially through the third and fourth ventricles, and then is reabsorbed by the arachnoid granulations and the lymphatic system2. The cilia on ependymal cells that line the ventricular cavities are important for CSF production and CSF flow through the ventricles6. As the choroid plexus pulsation and the wall motions dominates CSF dynamics in the center regions of the ventricles, ependymal cilia dominates near-wall cerebrospinal fluid dynamics in the lateral ventricles7 and regulates neuroblast migration8. Based on human and mouse genetics, mutations in genes that alter the structure and/or function of cilia can cause congenital hydrocephalus (CH); however, the mechanism of these genes on disease pathogenesis, including physiological embryonic CSF flow patterns, are poorly understood14.Cerebrospinal fluid (CSF) provides protection for the brain in the rigid cranium, clears waste produced by brain cells, and circulates growth factors important for neurodevelopment8. Understanding interactions between CSF flow and brain development is important to decipher the pathogenesis of hydrocephalus. While recent studies in single brain ventricular explants have highlighted potential complexities of CSF flow fields15, a global understanding of these networks in an intact, multi-compartmental ventricular system remains rudimentary. This is in part due to technical limitations of accessing the brain ventricles in a live animal through the rigid cranial vault and multilayered cortex, which impedes real-time imaging. Importantly, studies in other organ systems with cilia-lined fluid cavities  have revealed the importance of the interaction between the cilia, chamber geometry, and fluid viscosity on the mechanical polarization of the fluid-flow. This is a severe limitation for explant experiments that are widely used to understand the polarized CSF circulation19. Therefore, studying explants of only one chamber of a post-mortem ventricle is suboptimal for analyzing the CSF flow network. Unfortunately, the lack of model systems enabling real time changes in CSF flow pattern and ventricular development has been a critical roadblock in understanding the pathogenesis of hydrocephalus.CSF flow is critical for proper neurogenesis as it regulates neuroblast migrationXenopus tropicalis has a particular advantage for studying brain ventricular development and associated CSF flow dynamics. At stage 45 (Nieuwkoop - Faber staging. ~ post fertilization day 4 at 25 °C), the Xenopus brain is transparent, enabling Optical Coherence Tomography (OCT), a cross-sectional imaging modality that dynamically detects in vivo morphology at micrometer resolution23. We recently demonstrated that OCT imaging effectively detects structural changes in Xenopus craniofacial and cardiac structures, a useful screening tool for human genetic cranio-cardiac malformation research24. Here, we expand this platform to analyze in vivo CSF dynamics during ventricular development and use this approach to better understand the pathogenesis of patients with congenital hydrocephalus.The frog Xenopus, coupled with OCT imaging, enables the simultaneous visualization of brain ventricle development, CSF flow dynamics, and ependymal cilia function. Fortuitously, we take advantage of endogenous, free-floating intraventricular particles for particle tracking revealing multiple, differentially polarized flow fields throughout the ventricular system. By adding F0 CRISPR, we can rapidly evaluate the impact of gene depletion on CSF flow and ventricular structure. By targeting human congenital hydrocephaly candidate genes, we show that we can recapitulate the aqueductal stenosis in frogs, as seen in non-communicating type of human hydrocephalus. Lastly, we gain insight into the pathogenesis of hydrocephalus by modeling the effects of the candidate CH disease genes. This work establishes a platform for the rapid testing of candidate human CH genes, expeditious given the recent large-scale sequencing efforts of this disease25.We show that Xenopus tadpoles in vivo by embedding stage 45 tadpoles (post fertilization day 4 raised at 25 °C) in an agarose gel (1%) with the dorsal CNS structures positioned towards the OCT beam . By combining multiple 2D images, the OCT system can also generate a 3D image of the underlying structure.We applied OCT imaging to eam Fig. . This isFor our purposes, the embryo is oriented such that the left-right axis is aligned along the x direction, the anteroposterior axis is aligned along the y direction, and the dorsal-ventral axis is aligned in the z direction Fig. . We thenCoronal plane  in the xy-plane as we make 2D sections through the z-axis. To measure ventricular diameter, the user can examine each optical slice through the Z axis for the largest diameter for quantification  Tem15, differential polarization between the ventricles was unexpected and requires precise organization of the ependymal cells that drive flow. In describing the different flow fields, we oriented the embryo such that anterior is to the left and posterior is to the right with dorsal to the top in all of the following sections. In analyzing these images, we also quantified the endogenous particle counts for each frame and didn’t detect any significant variation between frames (1000 frames/acquisition/15 sec) or different tadpoles at the same stage . We recorded the slowest flow within the dorsal aspect of the midbrain ventricle.FF4 - Anterior rhombencephalic flow: Interestingly, two discrete flow fields are observed within the rhombencephalon, each with an opposite polarization. Immediately posterior to the cerebellar region, the dorsal ventricular surface generates a clockwise flow field in which CSF is directed anteriorly to the midbrain ventricle . Based on these findings, we speculate that CSF secretion via the choroid plexus may be contributing to FF5 formation. Future work can explore the contribution of the choroid plexus to CSF circulation at these early stages of development.Ependymal cilia drive CSF flow but this is not the only mechanism. Another contributor to CSF flow is the choroid plexus; a network of blood vessels and modified ependymal cells that produce CSF. In tadpoles, FF5 is adjacent to where the choroid plexus forms. As these cells are also ciliated, it is difficult to differentiate to what degree FF5 is a function of the ciliated surface or CSF secretion by the plexus. Clearly in both morphants, FF5 velocities were diminished but not lost even in cases where the cilia were disrupted  and is the most common heritable form of hydrocephalus30. Several l1cam mouse knockouts exist with varying degrees of hydrocephalus, with or without aqueductal stenosis31. L1CAM is an integral membrane glycoprotein that mediates cell-to-cell adhesion at the cell surface but is not involved directly in cilia structure or function32. We depleted Xenopus l1cam using F0 CRISPR-Cas9. Briefly, we injected a guide RNA(sgRNA) and Cas9 protein into fertilized 1 cell embryos and raised F0 CRISPR mutant tadpoles to stage 45 (Day 4 at 25 °C) for OCT imaging. Injection of a sgRNA and Cas9 protein can readily lead to biallelic frameshift mutations in just a few hours after injection  mutations in ion Fig. 28.l1cam F0 CRISPR mutant tadpoles had normal external morphology with some narrowing of the cranium. To test for gene modification and depletion of gene product, we used a T7 Endonuclease I assay and detected modified alleles at the l1cam locus with F0 CRISPR and also saw a loss of anti-L1cam antibody signal in F0 CRISPR embryos . We identified compound heterozygous mutations: c.1062delA (p.Thr354fs) and c.2400C>G (p.Asn800Lys) in Crumbs homolog 2 (CRB2), a gene involved in ciliogenesis35 and previously implicated in a rare Mendelian syndrome of prenatal onset ventriculomegaly and steroid-resistant nephropathy (OMIM#609720)37. Sanger sequencing of PCR amplicons confirmed p.Thr354fs and p.Asn800Lys compound heterozygosity in the patient, and heterozygosity in each respective parent (primer sequences available on request).Prenatal and postnatal chromosome analysis, microarray, and a comprehensive genetic panel for craniofacial syndromes and Xenopus, we depleted crb2 using F0 CRISPR and examined the ventricular system with OCT. At stage 45, crb2 F0 CRISPR mutant tadpoles had smaller ventricles  and midbrain ventricles (FF3) was severely affected , the few tadpoles that survived to this stage developed signs of hydrocephalus and the cerebral aqueduct remained obstructed . Our work addresses this need in two ways: 1) we develop a vertebrate animal model where the architecture of the brain ventricles can be visualized in real-time to map out an intact CSF flow network and 2) we use this model system to functionally assay the effect of CH genes on brain ventricular morphology, ependymal cilia function, and multi-ventricular CSF flow patterns.Congenital hydrocephalus (CH) is a common (1 per 1000 infants) and potentially lethal expansion of the CSF filled cerebral ventricles. Unfortunately, current treatment modalities are limited to morbid and antiquated brain surgery techniquesXenopus. As our goal is to model human disease, choosing an animal model as close to human is ideal. In isolated third ventricular explants, exogenously administered fluorescent particles can detect cilia-dependent flow15. However, these explant preparations cannot reproduce the three-dimensional CSF flow patterns in intact ventricular systems. Alternatively, CSF flow has been documented in zebrafish by injecting contrast reagents into the ventricular system39. However, Xenopus has certain evolutionary advantages for studying CH including lateral ventricles which the teleost lack and a neurulation process which is more similar between humans and Xenopus compared to zebrafish. In addition, the speed and low cost of making Xenopus models of CH makes Xenopus ideal for functionally testing CH candidate genes especially compared to the high cost of mouse. Previous work in Xenopus has identified CSF flow in a portion of the brain41. Miskevich in his work showed that fluorescent microspheres introduced to the ventricles with microinjection can be tracked using high speed camera in Albino Xenopus leavis tadpoles. He observed different velocities in different compartments of the brain which aligns with our current findings, yet this work was limited to visible parts of the ventricular system and didn’t show a global circulatory pattern41. Work by Hagencloher et al. showed that cilia were absent in foxj1 morphants, causing impaired CSF flow and fourth ventricle hydrocephalus in tadpole-stage embryos. This study also tracked fluorescent microspheres in the fourth ventricle using high speed imaging40. In our work, we comprehensively describe CSF flow through the entire ventricular system with its distinct polarity. We also reveal that the nature of ciliary gene affected can lead to distinct phenotypes and demonstrate its utility as a hydrocephalus model. When paired with OCT imaging, Xenopus provides a genetically tractable system where brain development, ventricular patterning, ependymal cilia function, and CSF microcirculatory dynamics can be simultaneously investigated and genetically perturbed in a non-destructive, label-free fashion. The candidate genes which show disease causality in our model, can be further studied using previously established methods to get at the underlying mechanism for gene function.CSF dynamics have been analyzed in various genetically tractable animal models including zebrafish, mice and Xenopus-OCT platform, we can detect the global CSF flow network mapped to CNS structures. We detect five discrete flow fields nested within three ventricles. Interestingly, unlike other ciliated surfaces that are polarized along a single axis, the CSF flow fields are location- and context-specific, suggesting patterning and polarization of the underlying ependymal cilia. For example, the telencephalic lateral ventricle has two discrete ciliary surfaces polarized in opposite directions generating a flow that unites in the center and forms a flow vector oriented towards the ventrally located optical tectum. Future work will define the cilia polarity and beating characteristics that are location specific to create these unique flow fields as well as the analysis of the 3D flow. Analyzing the subcellular architecture required for cilia polarization will prove fruitful for understanding how these flow fields are generated and how they change in disease states.Using our In analyzing flow fields with particle tracking, the presence of endogenous particles was fortuitous. By tracking particles, we can compute velocities in real time since the number and speed of these particles can be resolved with OCT imaging. Based on this data, we calculated average velocity maps to compare regional changes in velocities among the control and perturbed embryos. The average velocity maps can quantify mean velocity changes across multiple samples and highlight the dramatic changes in CSF flow across conditions Fig. .2 also remained constant over time 28. If the candidate genes recapitulate the hydrocephalus phenotype in our Xenopus model, this data bolsters the evidence for disease relevance and is also crucial to provide mechanistic insight about hydrocephalus pathophysiology. Our results with L1CAM and CRB2 indicate that Xenopus is efficient and robust for CH modeling. As WES identifies novel CH candidate genes, our Xenopus model may serve as a robust system to validate implicated genes, as well as to use for drug screening to develop of targeted therapeutics for specific CH subtypes  in accordance with NIH guidelines. Female and male mature Xenopus tropicalis were obtained from National Xenopus Resource.All study procedures and protocols comply with Yale University’s Human Investigation Committee and Human Research Protection Program. The study has been approved by Yale Human Investigation Committee (HIC protocol #1602017144) and by Yale IRB board. Written informed consent for genetic studies was obtained from all participants. Our patient and her participating family members provided buccal swab samples , medical records, radiological imaging studies, operative reports, and congenital hydrocephalus phenotype data.Xenopus embryo in vitro fertilization and microinjections were performed as per standard protocols24. Embryos were injected with CRISPR sgRNA and mRNA at the one cell stage. For microinjections, borosilicate glass needles were calibrated to inject 1.6 ng Cas9 protein with 200 pg of the CRISPR sgRNA and a fluorescent tracer Alexa488 (Invitrogen). The target sequence for the l1cam CRISPRs are GGGGACTGCTCAGTAATCAC and GGTGGCTGTGCCCAGATCAT and for crb2 CRISPRs are GGCAGCCCTGTCAGAATGGA and GGTCTCCATCATAGCCCGCT. To make F0 CRISPR mutants, we generated sgRNAs using the EnGen sgRNA synthesis kit (NEB) following the manufacturer’s instructions. Full length human WT CRB2 RNA construct was a kind gift from Dr. Nishimura, Shiga University of Medical Science. It was sub-cloned in the standard Xenopus vector, pCS2 and capped mRNA was synthesized in vitro using the mMessage machine kit (Ambion) as per the manufacturer’s instructions. We injected 12.5 and 25 pg wild type human CRB2 RNA for rescue of crb2 F0 CRISPR mutants. The following morpholino oligonucleotide was injected at 4-cell stage: foxj1 translation blocking (8 ng/embryo 5′-ATGCTGCTGTTGGGCATCTTCCTCC-3′) and the c21orf59 translation blocking (8 ng/embryo 5′-CCTTCTTAACGTGTAAGCGCACCAT-3′). Post-injections, embryos were incubated in 1/9X MR supplemented with 50 μg/ml of gentamycin at 25 °C. Injections were confirmed by fluorescent lineage tracing with a Zeiss Lumar fluorescence stereomicroscope at stage 28 and tadpoles further raised at until stage 45.Genomic DNA was extracted from each embryo using a DNeasy Blood & Tissue Kit (Qiagen). The target regions were amplified by PCR with 35 cycles to facilitate the heteroduplex formation using Phusion® High-Fidelity DNA Polymerase (NEB). PCR products were then annealed and digested with T7 Endonuclease I (NEB) as per the manufacturer’s instructions and run on a 2% agarose gel to determine genome targeting efficiency.We fixed control and mutant tadpoles at stage 45. We then dissected the brains and performed immunofluorescence of L1CAM (Abcam – ab24345) at 1:500. Alexa 488 (1:500) was used as a secondary antibody. Alexa 647 phalloidin (1:40) was used for actin staining. Embryos were mounted in Pro-Long Gold (Invitrogen) before imaging. Images were captured using a Zeiss 710 confocal microscope. Images were processed in Fiji, Image J, or Adobe Photoshop. Final figures were made in Adobe illustrator.X. tropicalis embryos were collected at stages 28 and stage 45. in-situ hybridization was performed according to standard protocols43. Additional probe information is available upon request.Stage 45 (post-fertilization Day 4) tadpoles were immobilized in 2.5 ml of 1% low melt agar in a 35 mm diameter −10 mm height polystyrene petri dish. The tadpoles were aligned such that the dorsal side of the body was exposed en face to the OCT imaging field. We used a Thorlabs Ganymede 900 nm spectral domain-OCT Imaging System, which allows for 1.4 mm imaging depth in air + water −2.2 µm Axial Resolution in water. We obtained 2D cross sectional and 3D images by scanning at 36 kHz (36000 A-scans per second) with maximal 101 dB sensitivity. Measurements obtained from Thorlabs software ThorImageOCT version 4.4.6, analyzed using Prism7 statistical software. The data was analyzed to check if it fits normal distribution and an independent-samples t-test was conducted to compare different ventricular measurements for control and mutant embryos.Flow velocimetry was performed in four steps: (1) anisotropy removal, (2) particle tracking, (3) region of interest selection, (4) particle displacement averaging to yield flow velocities. Further, we analyzed spatial particle distribution and present it as heatmaps (5). Next, (6) flow maps and heatmaps across multiple animals were created. The ventricle volume and 3D shape were also examined (7).All image and data processing was performed using Matlab  and ImageJ .Images from OCT are usually not stored as isotropic images . Various settings and factors during acquisition dictate the aspect ratio of a pixel (or voxel respectively), such as refractive index and beam movement. Using this information, width and height of each pixel was adjusted to have square aspect ratio.44 plugin build in ImageJ45. The plugin allows two-dimensional tracking for automated detection and analysis of particle trajectories. It is self-initializing, discriminates spurious detections, can handle temporary occlusion as well as particle appearance and disappearance from the image region to some degree.Particle tracking was performed using Mosaic particle trackerIn order to exclude any particles tracked outside the ventricle, the region of interest (ROI) was segmented. This was done manually using the MATLAB tool CROIEditor. All particle tracking data outside of the ROI was removed before any analysis.Particle trajectories were averaged across all frames of each sample to yield mean flow at each position. For visualization purposes, the number of vectors in the flow velocity plots was reduced to 10-by-10 pixel non-overlapping windows. Within each window, the average flow was calculated. After averaging, magnitudes above the 97th percentile were considered outliers caused by incorrect particle tracking and therefore removed.foxj1 morphants where particle counts were significantly high), we shifted to a single-image-based approach consisting of H-dome transform46 and subsequent particle detection using boundary detection47.The number of particles in a given sample was calculated as the mean number of particles in each frame (extracted from particle tracking information) and was normalized using the sample’s ROI area. Where particle tracking yielded a high rate of false negatives  in order to transform them such that they match another set of input points (another ventricle outline/ROI) as closely as possible (based on minimizing the sum of squared errors). This resulting transformation information was applied to corresponding flow vectors/heatmaps. Finally, all transformed flow vectors/heatmaps of a group were averaged.Averaged flow maps and particle distribution heatmaps of animals of a group were created by first matching the ROIs to each other using Procrustes analysis49 we extracted 3D representations of the ventricle and its volume. Seed points for the region growing algorithm were chosen manually. High particle density caused unsatisfactory performance of this approach. In these cases, manual segmentation of several slices was performed and the full volume was interpolated. Validation of this method was conducted by comparing volume measurements using the above described automatic approach with the manual approach on 12 different samples. Volume measurements of the automatic and manual approach showed a mean error of 0.005602574 mm3 and a standard deviation of the error of 0.006480865 mm3. This is based on 11–12 slices of the z-stack with manual ventricle segmentation.Employing a region growing algorithm50. Sequence reads were mapped and aligned to the GRCh38/hg19 human reference genome using Burrows-Wheeler Aligner-MEM51 and further processed using Genome Analysis Toolkit (GATK) base quality score recalibration52, indel realignment, duplication marking and removal, and base quality score recalibration in accordance with GATK Best Practices recommendations54. Single nucleotide variants and small indels were called using GATK Haplotype Caller and annotated using ANNOVAR55, DbSNP56, 1000 Genomes57, NHLBI exome variant server58, and gnomAD and ExAC databases59.DNA was obtained from buccal swab samples in accordance with manufacturer protocol. Whole-exome sequencing was performed using the Nimblegen SeqxCap EZ MedExome Target Enrichment Kit  followed by 99 base paired-end sequencing on the Illumina HiSeq 2000 instrument at the Yale Center for Genome Analysis as previously described60.Pedigree information and participant relationships were confirmed utilizing pairwise PLINK identity-by-descent calculationde novo mutations in the parent-offspring trio, consisting of our patient and her biological parents61. De novo candidates were filtered based on the following criteria of: (1) possessing a minor allele frequency (MAF)  ≤ 5 × 10−3 in ExAC, (2) having a GATK variant quality score recalibration (VQSR) of ‘pass’, (3) meeting a minimum sequencing depth of 8 reads in the proband and 12 reads in each parent, (4) having a genotype quality (GQ) score ≥20 and alternate allele ratio ≥40%, (5) earning a TrioDeNovo data quality (DQ) score ≥7, and (6) being an exonic or canonical splice-site variant.The Bayesian framework TrioDeNovo was used to call −5 in ExAC, (2) GQ ≥ 20 and alternate allele ratio ≥40%, (3) GATK VQSR of ‘pass’, and (4) minimum sequencing depth of 8 reads in each participant. Recessive variants were filtered for rare (MAF ≤ 10−3 in ExAC) homozygous and compound heterozygous mutations that met read quality criteria as above .Transmitted variants were filtered similarly, with dominant variants meeting the following criteria of rareness and quality: (1) MAF ≤ 2 × 1062, identifying mutations with rank scores greater than 0.83357 as deleterious (‘D-mis’). Only D-Mis and loss-of function mutations  were considered potentially damaging to protein function.We predicted the impact of nonsynonymous single nucleotide variants on protein function using the MetaSVM algorithmde novo and transmitted calls were verified by in silico visualization of aligned reads using the Integrative Genomics Viewer (IGV)63 and BLAT search64. Resultant de novo and compound heterozygous calls were then verified in the patient and both parents by direct Sanger sequencing of PCR amplicons containing the mutation. A compound heterozygous mutations: c.1062delA (p.Thr354fs) and c.2400C>G (p.Asn800Lys) was identified in Crumbs homolog 2 (CRB2), The maternally-inherited p.Thr354fs mutation is a novel, frameshift deletion not reported in large, reference databases . The second, paternally inherited p.Asn800Lys mutation occurs in a highly conserved residue and is extremely rare in the general population (MAF of 1.4 × 10−4 in the ExAC database). Sparse case reports demonstrate pathogenicity of this substitution in compound heterozygous state with other CRB2 mutations; interestingly, all previously reported patients harboring the p.Asn800Lys mutation were of Ashkenazi Jewish descent36. Analysis from 128 genomes of Ashkenazi Jewish ancestry identified two CRB2-p.Asn800Lys heterozygotes, demonstrating a 1:64 carrier frequency in the population34.All de novo variants were prioritized. Variants were filtered for predicted deleteriousness using a series of in silico prediction algorithms, and excluded if above standard rare minor allele frequency thresholds in ExAC and other established databases.The average depth of coverage of the whole-exome sequencing data was 41.2×, with greater than 8x coverage in 96.40% of the target region for exome capture. The pedigree suggested a sporadic or autosomal recessive mode of inheritance, so compound heterozygous, homozygous, and The data was analyzed to check if it fits normal distribution. Measurements via OCT Thorlabs software represented by the mean value ± SEM for each group. Two-tailed Student’s t test analysis or a chi-square test of independence was used to examine all significant differences between groups as indicated in the figure legends. Significance was determined when the p value is lower than 0.05.Supplementary DataMovie 1Movie 2Movie 3Movie 4Movie 5Movie 6Movie 7


Mercury (Hg) is a contaminant of major concern in Arctic marine ecosystems. Decades of Hg observations in marine biota from across the Canadian Arctic show generally higher concentrations in the west than in the east. Various hypotheses have attributed this longitudinal biotic Hg gradient to regional differences in atmospheric or terrestrial inputs of inorganic Hg, but it is methylmercury (MeHg) that accumulates and biomagnifies in marine biota. Here, we present high-resolution vertical profiles of total Hg and MeHg in seawater along a transect from the Canada Basin, across the Canadian Arctic Archipelago (CAA) and Baffin Bay, and into the Labrador Sea. Total Hg concentrations are lower in the western Arctic, opposing the biotic Hg distributions. In contrast, MeHg exhibits a distinctive subsurface maximum at shallow depths of 100–300 m, with its peak concentration decreasing eastwards. As this subsurface MeHg maximum lies within the habitat of zooplankton and other lower trophic-level biota, biological uptake of subsurface MeHg and subsequent biomagnification readily explains the biotic Hg concentration gradient. Understanding the risk of MeHg to the Arctic marine ecosystem and Indigenous Peoples will thus require an elucidation of the processes that generate and maintain this subsurface MeHg maximum. This has raised major concerns over the health of marine mammals and Indigenous Peoples whose traditional diets include marine mammal tissues. Mercury concentrations in marine biota are generally higher in the Beaufort Sea and western Canadian Arctic Archipelago (CAA) than in the eastern CAA and Baffin Bay3. This longitudinal gradient is not limited to apex predators5, but extends to organisms at lower trophic levels such as zooplankton 6  to be highly elevated, frequently exceeding toxicity thresholds.)6 Fig. . Whereas3, which is enhanced locally by atmospheric mercury depletion events (AMDEs) during polar sunrise7; (2) riverine Hg input from the Mackenzie River9, which may be enhanced by tundra uptake of atmospheric elemental Hg10 [and permafrost thawing11; and (3) a naturally high geological background of Hg12. These inorganic Hg-based hypotheses do not account for the fact that it is methylmercury (MeHg), not inorganic Hg, that accumulates and biomagnifies in marine biota2. The discovery of a subsurface MeHg enrichment in global oceans16 suggests that seawater MeHg may play a more important role in determining marine biotic Hg concentrations18, especially in regions such as the Beaufort Sea15 and the central Arctic Ocean16 where the maximum MeHg concentration was observed at shallow depths.Extensive efforts have been made to identify factors that control the spatial trends in marine biota and to develop appropriate mitigation strategies to reduce biotic Hg concentrations. Most hypotheses attribute higher marine biotic Hg concentrations in the western Canadian Arctic to elevated inputs of inorganic Hg to these regions. These inputs include (1) atmospheric deposition of anthropogenic Hg from Asian sourcesCCGS Amundsen, we measured high-resolution vertical profiles of total mercury (HgT) , slightly higher than those  measured in June 2014 in a similar area21.During the 2015 Canadian Arctic GEOTRACES cruises  aboard the Canadian Research Icebreaker gT) Fig.  in unfilgT) Fig. , S1. TotgT) Fig. , Table 1T concentrations in the Canada Basin, to the west, contrast with expectations based on the hypothesized elevated atmospheric and riverine inputs of Hg into this region. Whereas AMDEs do result in high springtime deposition, the low Canada Basin HgT concentrations are consistent with findings that most AMDEs occur in coastal regions and most of the deposited Hg is re-emitted to the atmosphere before snow melts22, thus limiting its transfer into the ocean. Likewise, Hg transported by rivers, possibly enhanced by tundra uptake, permafrost thawing, or geological enrichment, is likely deposited with sediment in coastal areas23 or escapes rapidly from the river plume to the atmosphere9. Furthermore, because MeHg accounts for <1% of the HgT in the Mackenzie River water23, and the atmospheric input of Hg is predominantly inorganic25, the Hg delivered to Canada Basin waters requires transformation to MeHg, the biomagnifying Hg species, to account for observed higher biotic Hg concentrations. Therefore, elevated input of inorganic Hg in the western relative to the eastern Arctic does not provide, by itself, a plausible mechanism to explain higher biotic Hg concentrations in the western Canadian Arctic , measured during the 2015 Canadian Arctic GEOTRACES cruises Fig. , are comumn Fig. . The impumn Fig. , Table 118, but high-resolution  water-column MeHg concentration data were not available at that time to support this hypothesis. The distribution of the subsurface MeHg peak along our transect directly links the spatial distribution of aqueous MeHg concentrations to biotic uptake , in agreement with recent reports from the Beaufort Sea15 and the central Arctic Ocean16. This MeHg maximum occurs at shallow depths that are just below the surface productive layer  except during winter, whereas C. glacialis spend all life stages in the top 300 m31. The amphipod consumers of these Calanus species, Themisto spp., also inhabit shallow waters32, as does Arctic cod , key species in the Arctic marine food web33. Given that the MeHg-enriched waters lie within the main habitat of low trophic level marine biota in these waters, spatial variations in MeHg concentrations within the subsurface zone can readily explain the higher biotic Hg concentrations in the western compared to the eastern regions of the section.Enrichment of MeHg in the subsurface water column (300–1000 m) is a common feature of many ocean basinssee Fig. , which min situ MeHg production associated with organic matter remineralization16. In the central Arctic Ocean, Heimbürger et al.16 suggested that sinking particles are slowed down at the shallow pycnocline where they undergo remineralization and stimulate in situ MeHg production. It remains unclear what microbial or abiotic processes are responsible for Hg methylation at such shallow depths where dissolved oxygen is well above 75% of the saturation value  ultraclean acid  and stored at 4 °C until analysis. The acidification breaks down dimethylmercury (DMHg) to monomethylmercury (MMHg)38 and, thus, the MeHg reported herein represents the sum of MMHg and DMHg.Seawater sampling and analyses were carried out following ultraclean techniques recommended for the GEOTRACES programT was analyzed in the Portable In-situ Laboratory for Mercury Speciation (PILMS) onboard the icebreaker . The analysis was carried out on a Tekran 2600 Hg analyzer following U.S EPA Method 1631, which involves BrCl oxidation, SnCl2 reduction, gold trap pre-concentration and measurement by cold vapor atomic fluorescence spectrometry (CVAFS). Water samples were analyzed for MeHg at the PILMS or at the Ultra-Clean Trace Elements Laboratory (UCTEL) at the University of Manitoba. Concentrations of MeHg were measured on an automated MeHg analyzer  following an adapted ascorbic acid-assisted direct ethylation method39, which involves ethylation, Tenax trap pre-concentration, gas chromatographic separation and CVAFS quantification. The original method39 was modified for use with ~40-mL sample volumes using acetate buffers to adjust pH. Daily calibration curves were prepared by adding standards solutions to filtered seawater to improve recovery from the seawater matrix. The detection limit (DL) was estimated at 0.25 pM and 0.014 pM for HgT and MeHg, respectively, as three times the standard deviation of seven laboratory blank replicates. Whenever seawater was sampled, Milli-Q water was collected in pre-cleaned 250-mL amber glass bottles to serve as field blanks, the concentrations of which were always lower than the DL for both HgT and MeHg. Certified reference seawater BCR579  was analyzed for HgT and the recovery was 93–116%. Since no certified reference seawater is available for MeHg, a 0.01 pmol MMHg spike was used during sample analysis and its recovery was 87–114%.Within 48 hr of sampling, HgSeawater fluorescence was measured in real-time with a chlorophyll fluorometer (Seapoint) installed on the rosette. To calibrate the fluorometer output, discrete seawater samples were measured for chlorophyll-α fluorescence concentrations.Supplementary Information


To find new potentially therapeutic drugs against clear cell Renal Cell Carcinoma (ccRCC), within drugs currently prescribed for other diseases (drug repositioning), we previously searched for drugs which are expected to bring the gene expression of 500 + ccRCC samples from The Cancer Genome Atlas closer to that of healthy kidney tissue samples. An inherent limitation of this bulk RNA-seq data is that tumour samples consist of a varying mixture of cancerous and non-cancerous cells, which influences differential gene expression analyses. Here, we investigate whether the drug repositioning candidates are expected to target the genes dysregulated in ccRCC cells by studying the association with tumour purity. When all ccRCC samples are analysed together, the drug repositioning potential of identified drugs start decreasing above 80% estimated tumour purity. Because ccRCC is a highly vascular tumour, attributed to frequent loss of VHL function and subsequent activation of Hypoxia-Inducible Factor (HIF), we stratified the samples by observed activation of the HIF-pathway. After stratification, the association between estimated tumour purity and drug repositioning potential disappears for HIF-activated samples. This result suggests that the identified drug repositioning candidates specifically target the genes expressed by HIF-activated ccRCC tumour cells, instead of genes expressed by other cell types part of the tumour micro-environment. To discover new potentially therapeutic drugs against ccRCC within drugs already prescribed for diseases (drug repositioning), we previously developed an individualised drug repositioning approach based on the gene expression profiles of over 500 ccRCC tumours generated using bulk RNA-Seq by The Cancer Genome Atlas (TCGA)2.Tumours of metastatic clear cell renal carcinoma (ccRCC) patients typically become resistant to available treatments within 1.5 years3. The presence of non-cancerous cells might be an especially big concern for ccRCC samples, as ccRCC estimated tumour purity was ranked the third most impure tumour type out of the total 21 solid tumours analysed, despite the lower mutational burden typical for other highly impure cancers4. The relatively low content of cancerous cells in ccRCC tumours is likely due to the high degree of vascularity often observed in ccRCC tumours6. This hypervascularity is attributed to the frequent inactivation of the Von Hippel-Lindau gene, which leads to activation of the Hypoxia Inducible Factor (HIF) pathway and the subsequent release of vascularizing growth factors: Vascular Endothelial Growth Factor (VEGF), platelet-derived growth factor beta (PDGFβ), and transforming growth factor alpha (TGFα)7.With bulk RNA-seq the gene expression of all cell types present in the sample is measured simultaneouslyet al. reported in their systematic pan-cancer analysis of TCGA tumour sample purity that variation in estimated tumour purity can significantly influence the results of differential gene expression analyses4. After adjusting for estimated tumour purity, on average 14% of differentially expressed genes lost statistical significance and 11% of expressed genes were now shown to be statistically differentially expressed when they were not before adjustment4. Depending on whether tumour purity is seen as a factor that needs to be corrected, such as in the case when only tumour cells are of interest, the differential gene expression profile can therefore change drastically.Aran Computational drug repositioning methods which rely on transcriptomic data commonly use this data type without accounting for the potential influence of tumour sample composition. The drug repositioning method we used, gene expression signature reversal, functions by searching for drugs which can normalize the genes which are differentially expressed in the tumour tissue . Specifically, drugs which can get tumour gene expression closer to that to normal tissue are considered potentially therapeutic drugs for this tumour. However, if genes are incorrectly classified as differentially expressed due to the confounding presence of non-cancerous cells present in the sample, it naturally follows that this can reduce the predictive validity of the procedure if the intention is to target the tumour cells with the drug. Excluding these drugs early on would therefore save vital time and money spent on laboratory validation experiments to determine whether the drug is likely to be safe and effective at clinically tolerated dosing regimens. Furthermore, while we considered all differentially expressed genes to be of interest in our initial drug repositioning analysis, the same issue comes up when only genes of a specific biological pathway are of interest if these genes are sufficiently expressed in the surrounding non-cancerous cells.The goal of the current study is to investigate whether the previously identified drug repositioning candidates are indeed expected to target the dysregulated genes expressed by ccRCC tumour cells and are not systematically influenced by variation in tumour purity between the ccRCC samples.8.Tumour purity estimations and connectivity scores  were available for 529 tumour sample gene expression profiles, of which 521 also passed the independent Toil quality control−13 for all 8 drugs, Supplementary Figs The distribution of tumour purity estimations of all ccRCC samples (with estimated tumour purity and gene expression results available) has a mode of 70% and an interquartile range of 57–75% Fig. . This find line treatments (the mTOR inhibitor temsirolimus). Therefore, we investigated whether the same increase in connectivity scores occurred for inhibitors of the HIF-pathway, as activation of the HIF-pathway within the cancerous cells is a frequently observed hallmark of ccRCC7 .It is striking that all eight drugs Fig.  show exaCC7 Fig. . Indeed,9), and the 3 most frequent chromosomal alterations observed in ccRCC: loss of chromosome 3p , gain of chromosome 5q  and loss of chromosome 14q . Furthermore, the total number of chromosomal alterations (deletions of negative regulators and gains of positive regulators) in the pathway of genes that regulate p53 and the G1/S cell cycle transition were tested as potential predictors, as genes in this pathway are frequently chromosomally altered in ccRCC12. Batch ID, plate ID, ship date and tissue source site were included as random variables as these were considered potential sources of batch effects. The distribution of all included variables is listed in Table To determine which variables predict HIF-activation and/or the variation in estimated tumour purity, two separate linear regression analyses combined with backward elimination were performed. The input for these analyses were: VHL mutation/methylation status, tumour stage, mRNA subtype  and missing VHL mutation/methylation data (N = 196) occurred frequently, missingness was used as a separate category. Otherwise, a complete case analysis was performed . The variable selection and estimation therefore took place on 510 out of the total 521 remaining ccRCC samples.−9) and tumour stage is associated with estimated tumour purity . As 4 out of the 7 variables are univariately associated with both outcomes, we performed stepwise linear regression using backward elimination, starting with all 7 variables including all possible sources of batch effects in both models . Gain of chromosome 5q further decreases the mean connectivity score by an average of 0.05 points (P = 3 × 10−4). Samples with a missing mRNA subtype had on average a 0.1 points higher connectivity score (P = 4 × 10−6). In addition, each chromosomal alteration in p53 associated genes increases the mean connectivity score by an average of 0.013 points per alteration (P = 1.8 × 10−9). This is likely caused by the fact that the number of chromosomal alterations in p53 associated genes is strongly positively associated with the number of genes in the tumour sample signature . The 2nd strongest effect size with regard to predicting estimated tumour purity was loss of chromosome 3p, estimated to decrease the estimated tumour purity by an average of 4.2% (P = 0.02). Loss of chromosome 14q  and loss of chromosome 14q  further contribute to decreases in estimated tumour purity.With respect to tumour purity, the by far the strongest predictor of estimated tumour purity was mRNA subtype, with samples belonging to the m3 subtype being estimated to have an average 12.5% decrease compared to the m1 subtype  are associated with both HIF activation (as evidenced by on average more negative connectivity scores with HIF inhibitors) and decreased estimated tumour purity regardless of analysis method .et al.13 reported that the methylome of 25 ccRCC samples in the TCGA dataset clustered closer to papillary and chromophobe RCC samples than typical ccRCC samples. 24 of these samples  and far more samples  show no loss of chromosome 3p compared to samples with a typical ccRCC methylome , which is highly statistically significant . Lastly, the 12 samples which upon histopathological re-evaluation were still considered ccRCC or unconventional ccRCC had lower median connectivity scores with PX-12 and CAY-10585 than the 12 samples deemed non-ccRCC .In an earlier paper published in 2016, Winter et al.4, which automatically eliminates genes which are more strongly differentially expressed at lower estimated tumour purity, we would have incorrectly excluded genes representing a real biological signal.We found that HIF-activation likely acts as a confounder between estimated tumour purity and estimated drug repositioning potential of the previously reported top 8 drugs for ccRCC Fig. . If inst5014.To classify samples by probability of HIF-activation, we used the same assumptions underlying the computational drug repositioning method we used in the original analysis, namely that of gene expression signature reversal. This method predicts that drugs which downregulate genes upregulated by HIF-activation, and vice versa, are expected to produce negative connectivity scores with HIF-activated samples. This worked well for 2 out of 3 HIF-inhibitors (PX-12 and CAY-10585): the connectivity scores of tumour samples with these drugs produced concordant negative connectivity scores for the majority (58%) of samples. The third HIF-inhibitor (YC-1) did not correlate well with the other two or even produced more negative connectivity scores than expected by chance. This discrepancy is as of yet unexplained: YC-1 was tested in 13/14 of the same cell lines as CAY-10585 and in concentrations above the ICBy studying predictors of the probability of HIF-activation in samples (with the mean connectivity score of PX-12 and CAY-10585 as the dependent variable), we learned that it can be attributed to two main underlying causes:7.Firstly, observing HIF-activation depends on inactivation of the VHL gene due to loss of chromosome 3p Table  or, in s15. When genes which are not associated with HIF expression are added to the tumour sample signature, on average the genes rank closer to the middle when they are compared to the genes ordered by changes in expression after administration of HIF-inhibitors. Statistical procedures which only look at the overlap of genes differentially expressed between the tumour sample signature and drug signature may be less sensitive to this effect16. Missing mRNA subtype was also found to be an important negative predictor of HIF-activation, for which we have not found a good explanation yet.Secondly, observing HIF-activation can be interfered with by other factors, the most biologically plausible factor being deletions of negative regulators and gains of positive regulators in the genes that regulate p53 and the G1/S cell cycle transition. These copy number alterations result in many more genes differentially expressed in the tumour samples Fig. , which s9) produced the largest contribution. Compared to the m1 subtype, samples belonging to the m3 subtype have on average a 12.5% lower estimated tumour purity .When we explored possible predictors of estimated tumour purity, we found that mRNA subtype  estimated tumour purity of the scale. However, as the 12 samples which were independently histopathologically reclassified as conventional ccRCC (N = 2) or unconventional ccRCC (N = 10) had lower median connectivity scores with the HIF-inhibitors than the 12 samples reclassified as chromophobe or papillary RCC, observed HIF-activation (as a correlate of ccRCC-likeness) does seem to correlate better with independent histopathological review than methylation clustering alone.When we compared our gene expression-based results to the methylome clustering results produced by Winter 9.For both models presented in Tables It is biologically plausible that the cancer cells behave differently (and thus gene expression within tumour cells changes) if the tumour microenvironment becomes purer or less pure (e.g. as a reaction to the invasion of immune cells or infiltration of blood vessels). However, the bulk RNA-seq data from TCGA do not allow us to distinguish directly which mRNA is produced by cancer cells and which is produced by non-cancerous cells. We therefore heavily relied on previously established knowledge to interpret our findings . To conclusively answer this question and explore new hypotheses, it would require a large single cell RNA-seq dataset of the ccRCC tumour micro-environment from many different patients, so that the impact of variation in cancer cell genetic identity  can be distinguished from variation in cell type composition of the micro-environment. However, because of the expense and newness of single cell RNA-seq no such dataset exists yet (to our knowledge), and only the TCGA dataset contains enough samples  to really quantify the impact of inter-sample variation in gene expression and relate this to other genomic determinants.In conclusion, we demonstrated using the example of HIF-activation that confounding between tumour purity and gene expression can occur. Therefore, we recommend correcting for tumour purity in bulk RNA-seq differential expression analyses only after any other possible biological causes are included in the model.All data was processed with R version 3.4.0 and R/Bioconductor packages. All tests produced two-sided P-values and were considered statistically significant at P < 0.05.2. Briefly, the tumour sample signatures were generated by comparing 538 individual tumour gene expression profiles to 72 normal kidney tissue gene expression profiles, both generated by TCGA9. After filtering out genes with low expression (counts per million <0.5) and applying the Benjamini-Hochberg procedure to calculate the False Discovery Rate (FDR), genes with an FDR <1% were included in the tumour sample signatures. As an additional quality control filter, only samples which passed the independent quality control check of the Toil recompute effort  were included8. Drug signatures, based on data generated by The Library of Integrated Network-Based Cellular Signatures (LINCS) Program17, were calculated with the goal of approximating the effect in the average tumour cell line. This was accomplished through the use of a linear model with drug concentration as a linear parameter, and cell line, exposure duration, and batch as categorical variables.The tumour sample and drug signatures were calculated as described previously4. In summary, CPE is based on 3 different data types and algorithms: copy number data (ABSOLUTE), methylation data (LUMP) and transcriptomic data (ESTIMATE). For simplicity and clarity’s sake, CPE is hereafter simply referred to as “Estimated tumour purity”. TCGA batch information  were downloaded using the TCGA Batch Effects Tool18 (version 2.0).To estimate tumour sample purity, we used the previously published consensus measurements of tumour purity estimations (CPE)16 (version 1.8.3). A negative connectivity score implies that the drug is expected to  reverse the tumour signature. The correlation between the connectivity scores and estimated tumour purity was tested non-parametrically using the Spearman rank correlation test. A positive association (i.e. more positive connectivity scores at higher tumour purity %) suggests that the gene expression reversal occurs mainly in non-cancerous cells. Conversely, a negative association implies the opposite, namely that as the % of tumour material in a sample increases so does the drug repositioning potential, which suggests that the drug is targeting the tumour cells.Connectivity scores (the method used to compare the drug and tumour signatures) were calculated using the PharmacoGx packageGenes were classified as more differentially expressed with increasing tumour purity if upregulated and downregulated genes showed a statistically significant (P < 0.05) positive, respectively negative Spearman rank correlation with increasing tumour purity. If it showed the opposite relationship, the gene was classified as becoming less differentially expressed with increasing tumour purity. Lastly, if the Spearman rank correlation with estimated tumour purity was statistically insignificant (P > 0.05) regardless of direction, the gene was classified as insensitive to increasing tumour purity.19. Only mutations and methylation of the VHL gene were considered because this gene is considered the key trigger in starting the angiogenesis cascade. VHL was considered methylated if the beta value for probe cg15267345 exceeded 0.2 . The copy number data processed by GISTIC  was downloaded using the RTCGAToolbox package (version 2.8.0)23. For tumour samples analysed in duplicate, the average value of the variable was used in the analysis.Mutation and methylation data of tumour samples were downloaded using the TCGAbiolinks package (version 2.6.12)The following variables were investigated for their association with observed HIF activation (for which we used negative connectivity scores with the HIF inhibitors PX-12 and CAY-10585 as a proxy) and estimated tumour purity: Estimated tumour purity, VHL mutation/methylation status, tumour stage, mRNA subtype, loss of chromosome 3p, gain of chromosome 5q, loss of chromosome 14q and number of chromosomal alterations in the p53 and the G1/S cell cycle transition pathway. Selected variables were univariately associated with HIF-activation and estimated tumour purity using non-parametric statistical tests: the Wilcoxon Rank-sum test for categorical variables with 2 groups (e.g. no 3p loss versus 3p loss), the Kruskal-Wallis test for variables with more than 2 groups (e.g. mRNA subtype) and Spearman’s rank correlation coefficient for continuous variables . To explain inter-sample variability in HIF-activation and estimated tumour purity, linear mixed models with all variables included were fitted. Batch ID, plate ID, tissue source site and ship date were parameterized as random effects. Estimated tumour purity, plate ID and ship date were only included as a variable to predict inter-sample variability in HIF-activation because estimated tumour purity can’t be used to predict itself and plate ID + ship date are specific only to the RNA-seq experiments. Variables were sequentially dropped from the model until only variables with P values lower than 0.05 were left. This process was repeated on 1,000 bootstrapped datasets (sampled with replacement). Variables which remained in the model after backward selection at least 50% of the time were kept in the final 2 models presented in the main article. The bootstrapped P values of the final models were determined by sampling the original dataset with replacement 10,000 times. Lastly, for each final model, repeated  10-fold cross-validation was performed to detect possible overfitting.et al. analysed 319 TCGA samples for which Illumina HumanMethylation 450 BeadChip results were available and concluded that based on the methylation patterns, 15 of the ccRCC samples showed closer similarity to chromophobe RCC and 10 to papillary RCC13. Additionally, they asked 2 independent pathologists with expertise in renal tumour pathology to re-evaluate the ccRCC samples based on publicly available TCGA diagnostic and histological slide images. The discordant samples and re-evaluation results were extracted from Supplementary Table In a previously published methylation analysis, Winter Supplementary material


Thermomicrobium roseum (class Chloroflexia) extensively remodels its respiratory chain upon entry into stationary phase due to nutrient limitation. Whereas primary dehydrogenases associated with heterotrophic respiration were downregulated, putative operons encoding enzymes involved in molecular hydrogen (H2), carbon monoxide (CO), and sulfur compound oxidation were significantly upregulated. Gas chromatography and microsensor experiments showed that T. roseum aerobically respires H2 and CO at a range of environmentally relevant concentrations to sub-atmospheric levels. Phylogenetic analysis suggests that the hydrogenases and carbon monoxide dehydrogenases mediating these processes are widely distributed in Chloroflexi genomes and have probably been horizontally acquired on more than one occasion. Consistently, we confirmed that the sporulating isolate Thermogemmatispora sp. T81 (class Ktedonobacteria) also oxidises atmospheric H2 and CO during persistence, though further studies are required to determine if these findings extend to mesophilic strains. This study provides axenic culture evidence that atmospheric CO supports bacterial persistence and reports the third phylum, following Actinobacteria and Acidobacteria, to be experimentally shown to mediate the biogeochemically and ecologically important process of atmospheric H2 oxidation. This adds to the growing body of evidence that atmospheric trace gases are dependable energy sources for bacterial persistence.Most aerobic bacteria exist in dormant states within natural environments. In these states, they endure adverse environmental conditions such as nutrient starvation by decreasing metabolic expenditure and using alternative energy sources. In this study, we investigated the energy sources that support persistence of two aerobic thermophilic strains of the environmentally widespread but understudied phylum Chloroflexi. A transcriptome study revealed that  Bacteria from the phylum Chloroflexi are widespread and abundant in free-living microbial communities –4. One r2) of both geothermal and atmospheric origin are available in such environments and may be particularly important energy sources for sustaining growth and persistence -hydrogenase -hydrogenases (HhyL and homologues) and type I carbon monoxide dehydrogenases (CoxL) were retrieved from Chloroflexi genomes and metagenome-assembled genomes (MAGs) in the NCBI RefSeq database via protein BLAST  in OctobT. roseum cultures under nutrient-rich  and nutrient-limited (stationary phase) conditions. A total of 401 genes were significantly upregulated and 539 genes were significantly downregulated by at least two-fold (p < 10−6) in response to nutrient limitation . Three major trends were observed with respect to energy acquisition and utilisation. Firstly, genes associated with energetically expensive processes were downregulated, including those encoding ribosomal proteins, cytochrome c and menaquinone biosynthesis enzymes, and the megaplasmid-encoded chemotactic and flagellar apparatus (Table ). Secondly, there was evidence of mobilisation of internal carbon stores, including an acetoin dehydrogenase complex and an electron transfer flavoprotein complex (ETF). Thirdly, the expression profiles indicate there is extensive remodelling of the respiratory chain. Two primary respiratory dehydrogenases involved in heterotrophic growth (type I and II NADH dehydrogenases) were downregulated, whereas complexes involved in lithotrophic energy generation and a succinate dehydrogenase were upregulated . In both conditions, the terminal oxidases that mediate aerobic respiration were highly expressed and there was no evidence of the use of other electron acceptors; the cytochrome aa3 oxidase was expressed in both phases and the alternative cytochrome bo3 oxidase was upregulated during stationary phase. In contrast, the F1Fo-ATPase (ATP synthase) was downregulated, a finding consistent with an expected decrease in the availability of respiratory electron donors during nutrient limitation (Table ).We compared the transcriptomes of triplicate ion Fig. . Three mus Table . Secondlted Fig. . In bothon Table .Fig. 1DiThermomicrobium roseum upregulates genes associated with H2 and CO metabolism under nutrient-limiting conditions. The genes encoding the structural subunits of a group 1h [NiFe]-hydrogenase  -hydrogenase and type I carbon monoxide dehydrogenase suggested that els Fig. . This coT. roseum efficiently oxidises H2 and CO across a wide range of concentrations through hydrogenase and carbon monoxide dehydrogenase activity. In cultures, the enzymes display a moderate apparent velocity (Vmax app of 376 nmol H2 and 149 nmol CO g−1 of protein min−1) and moderate apparent affinity (Km app of 569 nM H2 and 285 nM CO) for these substrates . With respect to carbon monoxide dehydrogenase, these observations are consistent with the organism being able to utilise CO at elevated concentrations for growth  and slightly below the expected molecular weight of a hydrogenase dimer . This is compatible with biochemical studies in other organisms that have shown type I carbon monoxide dehydrogenases and group 1h [NiFe]-hydrogenases form homodimers -hydrogenase and type I carbon monoxide dehydrogenase directly use electrons derived from atmospheric H2 and CO to support aerobic respiration , terminal oxidases , and ATP synthase (atp). The genome also encodes putative operons for the structural subunits of a group 1h [NiFe]-hydrogenase, the maturation factors of this hydrogenase, and structural subunits of a type I carbon monoxide dehydrogenase . However, homologues of the putative heterodisulfide reductase and ETF complexes encoded by T. roseum are absent from the Thermogemmatispora sp. T81 genome.Having demonstrated that  Zealand , 40. Anae Figure . HoweverThermogemmatispora sp. T81 actively consume H2 and CO. The organism slowly oxidised available H2 and CO in the headspace to sub-atmospheric levels  over ~320 h . Although this strain has previously been shown to oxidise carbon monoxide -hydrogenases known to facilitate dehalorespiration -hydrogenases and type I carbon monoxide dehydrogenases were encoded in three of the four reference genomes within the Thermomicrobiales (class Chloroflexia) and four of the five reference genomes within the Ktedonobacteriales (class Ktedonobacteria) Fig. . The latacemifer  and the llandica . In addi -hydrogenases from Chloroflexia and Ktedonobacteria are divergent and fall into two distinct, robustly supported branches . It is therefore more likely that Chloroflexia and Ktedonobacteria independently acquired these enzymes, for example as a result of horizontal gene transfer events from other Terrabacteria, rather than vertically inheriting them from a common ancestor. Phylogenetic analysis also suggests that the type I carbon monoxide dehydrogenase may have also been acquired on two or three occasions in this phylum . In line with their probable independent acquisition, the putative operons encoding the hydrogenase and carbon monoxide dehydrogenase in T. roseum, but are separated into a structural operon (coxGSLM) and accessory operon (including coxG and coxE) in T. roseum. These findings agree with previous inferences of horizontal dissemination of hhyL and coxL genes -hydrogenases and carbon monoxide dehydrogenases within these organisms fall into different clades and are relatively divergent. Hence, it is probable that these organisms have horizontally acquired the capacity to oxidise atmospheric H2 and CO via separate events, though other explanations are possible. The apparent convergence in persistence strategies is notable given the distinct evolutionary histories, persistence morphologies , and ecological niches of these bacteria. Resource generalism is therefore likely to be a common ecological strategy for the survival of Chloroflexi in environments where organic carbon and other nutrients may be periodically scarce.Aerobic heterotrophic bacteria from the phylum Chloroflexi are more metabolically versatile than previously thought. The transcriptome analyses clearly show that 2, atmospheric CO is likely to be a dependable energy source for microbial survival given its ubiquity, diffusibility, and energy density. Integrating these findings with the wider literature, it is probable that atmospheric CO oxidation is a general strategy supporting long-term survival of aerobic heterotrophic bacteria. Indeed, various heterotrophic bacteria have previously been inferred to be capable of oxidising atmospheric CO, including Proteobacteria -hydrogenases. Given at least four other cultured phyla  and two candidate phyla [2 serves as a general energy source for aerobic heterotrophic bacteria. This observation is also potentially biogeochemically significant, given aerobic soil bacteria are known to be the main sink in the global hydrogen cycle [Finally, this study establishes Chloroflexi as the third phylum experimentally shown to scavenge atmospheric Hbacteria , 37, 57 bacteria , 38. Themegmatis , 60 and atogenes , both ofyla Fig.  and two te phyla  also encen cycle . FurtherSupplemental materialTable S1