ABSTRACT
Significance: Heart disease is the leading cause of death in the United States, yet research is limited by the inability to culture primary cardiac cells. Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) are a promising solution for drug screening and disease modeling. Aim: Induced pluripotent stem cell-derived CM (iPSC-CM) differentiation and maturation studies typically use heterogeneous substrates for growth and destructive verification methods. Reproducible, tunable substrates and touch-free monitoring are needed to identify ideal conditions to produce homogenous, functional CMs. Approach: We generated synthetic polyethylene glycol-based hydrogels for iPSC-CM differentiation and maturation. Peptide concentrations, combinations, and gel stiffness were tuned independently. Label-free optical redox imaging (ORI) was performed on a widefield microscope in a 96-well screen of gel formulations. We performed live-cell imaging throughout differentiation and early to late maturation to identify key metabolic shifts. Results: Label-free ORI confirmed the expected metabolic shifts toward oxidative phosphorylation throughout the differentiation and maturation processes of iPSC-CMs on synthetic hydrogels. Furthermore, ORI distinguished high and low differentiation efficiency cell batches in the cardiac progenitor stage. Conclusions: We established a workflow for medium throughput screening of synthetic hydrogel conditions with the ability to perform repeated live-cell measurements and confirm expected metabolic shifts. These methods have implications for reproducible iPSC-CM generation in biomanufacturing.
ABSTRACT
Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.
Subject(s)
Exosomes , Ligaments , Mesenchymal Stem Cells , Wound Healing , Mesenchymal Stem Cells/metabolism , Exosomes/metabolism , Humans , Animals , Rats , Wound Healing/drug effects , Ligaments/metabolism , Ligaments/injuries , Cellular Microenvironment , MicroRNAs/genetics , MicroRNAs/metabolism , Rats, Sprague-Dawley , MaleABSTRACT
The ability to locally deliver bioactive molecules to distinct regions of the skeleton may provide a novel means by which to improve fracture healing, treat neoplasms or infections, or modulate growth. In this study, we constructed single-sided mineral-coated poly-ε-caprolactone membranes capable of binding and releasing transforming growth factor beta 1 (TGF-ß1) and human growth hormone (hGH). After demonstrating biological activity in vitro and characterization of their release, these thin bioabsorbable membranes were surgically implanted using an immature rabbit model. Membranes were circumferentially wrapped under the periosteum, thus placed in direct contact with the proximal metaphysis to assess its bioactivity in vivo. The direct effects on the metaphyseal bone, bone marrow, and overlying periosteum were assessed using radiography and histology. Effects of membrane placement at the tibial growth plate were assessed via physeal heights, tibial growth rates (pulsed fluorochrome labeling), and tibial lengths. Subperiosteal placement of the mineralized membranes induced greater local chondrogenesis in the plain mineral and TGF-ß1 samples than the hGH. More exuberant and circumferential ossification was seen in the TGF-ß1 treated tibiae. The TGF-ß1 membranes also induced hypocellularity of the bone marrow with characteristics of gelatinous degeneration not seen in the other groups. While the proximal tibial growth plates were taller in the hGH treated than TGF-ß1, no differences in growth rates or overall tibial lengths were found. In conclusion, these data demonstrate the feasibility of using bioabsorbable mineral coated membranes to deliver biologically active compounds subperiosteally in a sustained fashion to affect cells at the insertion site, bone marrow, and even growth plate.
Subject(s)
Human Growth Hormone , Periosteum , Polyesters , Transforming Growth Factor beta1 , Animals , Human Growth Hormone/administration & dosage , Human Growth Hormone/pharmacology , Polyesters/chemistry , Humans , Rabbits , Transforming Growth Factor beta1/pharmacology , Periosteum/drug effects , Membranes, Artificial , Tibia/drug effectsABSTRACT
The tailorable properties of synthetic polyethylene glycol (PEG) hydrogels make them an attractive substrate for human organoid assembly. Here, we formed human neural organoids from iPSC-derived progenitor cells in two distinct formats: (i) cells seeded on a Matrigel surface; and (ii) cells seeded on a synthetic PEG hydrogel surface. Tissue assembly on synthetic PEG hydrogels resulted in three dimensional (3D) planar neural organoids with greater neuronal diversity, greater expression of neurovascular and neuroinflammatory genes, and reduced variability when compared with tissues assembled upon Matrigel. Further, our 3D human tissue assembly approach occurred in an open cell culture format and created a tissue that was sufficiently translucent to allow for continuous imaging. Planar neural organoids formed on PEG hydrogels also showed higher expression of neural, vascular, and neuroinflammatory genes when compared to traditional brain organoids grown in Matrigel suspensions. Further, planar neural organoids contained functional microglia that responded to pro-inflammatory stimuli, and were responsive to anti-inflammatory drugs. These results demonstrate that the PEG hydrogel neural organoids can be used as a physiologically relevant in vitro model of neuro-inflammation.
ABSTRACT
mRNA therapeutics can be readily designed, manufactured, and brought to scale, as demonstrated by widespread global vaccination against COVID-19. However, mRNA therapies require cold chain shipment and storage from manufacturing to administration, which may limit them to affluent communities. This problem could be addressed by mimicking the known ability of mineralized fossils to durably stabilize nucleic acids under extreme conditions. We synthesized and screened 40 calcium-phosphate minerals for their ability to store and maintain the activity of lyophilized mRNA complexes. The optimal mineral formulation incorporated mRNA complexes with high efficiency (77 %), and increased mRNA transfection efficiency by 5.6-fold. Lyophilized mRNA complexes stored with the optimized mineral formulation for 6 months at 25 °C were 3.2-fold more active than those stored with state-of-the-art excipients, but without a mineral. mRNA complexes stored with minerals at room temperature did not decline in transfection efficacy from 3 days to 6 months of storage, indicating that minerals can durably maintain activity of therapeutic mRNA complexes without cold chain storage. STATEMENT OF SIGNIFICANCE: Therapeutic mRNA, such as mRNA COVID-19 vaccines, require extensive cold chain storage that limits their general application. This work screened a library of minerals to maintain the activity of mRNA complexes with freeze-drying. The optimized mineral was able to maintain mRNA activity up to 6 months of storage at room temperature outperforming current methods of freeze-drying therapeutic mRNA complexes.
Subject(s)
Biomimetics , COVID-19 Vaccines , Humans , Drug Stability , Freeze Drying/methods , Minerals , TemperatureABSTRACT
Decellularized plant scaffolds have drawn attention as alternative tissue culture platforms due to their wide accessibility, biocompatibility, and diversity of innate microstructures. Particularly, in this work, monocot leaves with innate uniaxial micropatterned topography were utilized to promote cell alignment and elongation. The leaf scaffold was biofunctionalized with poly(PEGMEMA-r-VDM-r-GMA) copolymer that prevented non-specific protein adsorption and was modified with cell adhesive RGD peptide to enable cell adhesion and growth in serum-free media. The biofunctionalized leaf supported the adhesion, growth, and alignment of various human cells including embryonic stem cells (hESC) derived muscle cells. The hESC-derived myogenic progenitor cells cultured on the biofunctionalized leaf scaffold adopted a parallel orientation and were elongated along the leaf topography. These cells showed significant early myogenic differentiation and muscle-like bundled myotube formation. The aligned cells formed compact myotube assemblies and showed uniaxial muscle contraction under chemical stimulation, a critical requirement for developing functional skeletal muscle tissue. Polymer-functionalized plant leaf scaffolds offer a novel human cell culture platform and have potential in human tissue engineering applications that require parallel alignment of cells. STATEMENT OF SIGNIFICANCE: Plant scaffolds are plentiful sources in nature and present a prefabricated construct to present topographical cues to cells. Their feature width is ideal for human cell alignment and elongation, especially for muscle cells. However, plant scaffolds lack proteins that support mammalian cell culture. We have developed a polymer coated leaf scaffold that enables cell adhesion and growth in serum-free media. Human muscle cells cultured on the biofunctionalized leaf, aligned along the natural parallel micro-patterned leaf topography, and formed muscle-like bundled myotube assemblies. These assemblies showed uniaxial muscular contraction, a critical requirement for developing functional skeletal muscle tissue. The biodiversity of the plant materials offers a novel human cell culture platform with potential in human tissue engineering.
Subject(s)
Muscle, Skeletal , Tissue Scaffolds , Animals , Humans , Tissue Scaffolds/chemistry , Culture Media, Serum-Free/metabolism , Muscle, Skeletal/physiology , Muscle Fibers, Skeletal , Tissue Engineering , Cell Differentiation , Polymers/chemistry , MammalsABSTRACT
Treated herein are the 113 described species and two described subspecies in 25 genera of the family Sciomyzidae (snail-killing or marsh flies) known from the Americas south of the United States. Included are details on type specimens, references to generic transfers and synonymies, taxonomy, biology, gastropod hosts/prey, immature stages, chromosomes, biological and phenological groups, general distribution, and molecular data. Annotated keys are presented to adults of genera known from the Nearctic-Neotropical interface area and the Neotropics as well as the first key to all sciomyzid genera known from the Nearctic Region. Also presented is the first key to third-instar sciomyzid larvae in the Neotropical Region. Sepedonea isthmi (Steyskal) is placed as a junior synonym of S. annulata Macquart (new status), and Tetanocera plumifera Wulp is placed as a junior synonym of T. plumosa Loew (new status).
Subject(s)
Diptera , United States , Animals , Larva , South America , Snails , Animal DistributionABSTRACT
Spinal cord injury often results in devastating consequences for those afflicted, with very few therapeutic options. A central element of spinal cord injuries is astrogliosis, which forms a glial scar that inhibits neuronal regeneration post-injury. Chondroitinase ABC (ChABC) is an enzyme capable of degrading chondroitin sulfate proteoglycan (CSPG), the predominant extracellular matrix component of the glial scar. However, poor protein stability remains a challenge in its therapeutic use. Messenger RNA (mRNA) delivery is an emerging gene therapy technology for in vivo production of difficult-to-produce therapeutic proteins. Here, mineral-coated microparticles as an efficient, non-viral mRNA delivery vehicles to produce exogenous ChABC in situ within a spinal cord lesion are used. ChABC production reduces the deposition of CSPGs in an in vitro model of astrogliosis, and direct injection of these microparticles within a glial scar forces local overexpression of ChABC and improves recovery of motor function seven weeks post-injury.
Subject(s)
Chondroitin ABC Lyase , Spinal Cord Injuries , Animals , Chondroitin ABC Lyase/metabolism , Chondroitin ABC Lyase/pharmacology , Chondroitin ABC Lyase/therapeutic use , Chondroitin Sulfate Proteoglycans/metabolism , Chondroitin Sulfate Proteoglycans/therapeutic use , Gliosis/drug therapy , Hindlimb/pathology , Nerve Regeneration , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Spinal Cord/pathology , Spinal Cord Injuries/drug therapy , Spinal Cord Injuries/pathologyABSTRACT
A catalog of the Sciomyzidae of Chile is presented. Included are all valid names and synonyms for the 27 species and 11 genera known from Chile, including information about name, author, year of publication, page number, type species, type depository, type locality, and references. Tetanoceroides Malloch is the most species-rich genus in Chile, with seven species, followed by Pherbellia Robineau-Desvoidy, with five species; however, if undescribed species are included, Pherbellia is the most species-rich genus in Chile, with nine species. The geographic distribution of species was determined from examination of bibliographic data and label data on specimens in collections. A key is provided to the genera of Sciomyzidae in Chile.
Subject(s)
Diptera , Animal Distribution , Animals , ChileABSTRACT
Autologous platelet-rich plasma (PRP) has gained popularity as a less invasive treatment for various musculoskeletal tissue injuries and conditions due to its favorable safety profile, minimal manipulation and cost-effectiveness. Although PRP treatment has been clinically used for the treatment of osteoarthritis (OA) and damaged cartilage, evidence on therapeutic efficacy has been inconsistent, which calls for a methodology to achieve consistent and improved treatment outcomes. Given that PRP contains numerous proteins, we hypothesized that attenuation of a growth factor known to be detrimental to the healing tissue would enhance efficacy of PRP treatment. Considering that VEGF-mediated angiogenesis inhibits the repair of articular cartilage, we developed VEGF-attenuated PRP by sequestering VEGF in PRP using VEGF-binding microspheres. We demonstrated that VEGF attenuation in PRP did not inhibit the effect of PRP on chondrogenic differentiation of stem cells in vitro. In addition, healing of rat OA cartilage was significantly improved after treatment with VEGF-attenuated PRP when compared to the PRP treatment group or PBS control group. We expect that attenuation of unwanted biological activity using growth factor-binding microspheres could provide a new PRP customization method broadly applicable to various tissue repair processes.
Subject(s)
Cartilage, Articular , Osteoarthritis , Platelet-Rich Plasma , Animals , Cartilage, Articular/metabolism , Chondrogenesis , Intercellular Signaling Peptides and Proteins/metabolism , Osteoarthritis/metabolism , Osteoarthritis/therapy , Rats , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The separation and sorting of human cells is an important step in the bioprocessing of cell-based therapeutics. Heterogeneous mixtures of cells must be sorted to isolate the desired cell type and purify the final product. This process is often achieved by antibody-based sorting techniques. In this work, we demonstrate that magnetic microspheres may be functionalized with peptides that selectively bind to cells on the basis of their relative concentration of specific surface proteins. Five-micrometer-magnetic microspheres were coated with the synthetic copolymer PVG (poly(poly(ethylene glycol)methyl ether methacrylate-ran-vinyl dimethyl azlactone-ran-glycidyl methacrylate) and functionalized with the vascular endothelial growth factor receptor binding peptide (VRBP), which binds to the vascular endothelial growth factor receptor (VEGFR). These microspheres exhibited low cytotoxicity and bind to cells depending on their relative surface protein expression. Finally, coated, magnetic microspheres were used to separate heterogeneous populations of cells dependent on their VEGFR expression through magnetic-assisted cell sorting (MACS), demonstrating that peptide-based cell sorting mechanisms may be useful in the bioprocessing of human-cell-based products.
Subject(s)
Peptides , Vascular Endothelial Growth Factor A , Humans , Magnetic Phenomena , Microspheres , Polymers , Receptors, Growth FactorABSTRACT
The ongoing usefulness of Table 1 in the Zootaxa paper Comprehensive taxonomic, faunistic, biological, and geographic inventory and analysis of the Sciomyzidae (Diptera: Acalyptratae) of the Delmarva region and nearby states in eastern North America (Murphy et al. 2018) is compromised by impermanent literature-citation numbering. To secure Table 1 as a permanent resource for the study of Sciomyzidae, provided herein are bibliographic data for the 59 works cited in that paper by Bibliography of Sciomyzidae (ScioBiblio) number only. Details are provided regarding the history of the ScioBiblio and plans to reorganize and publish it.
Subject(s)
Diptera , AnimalsABSTRACT
Human mesenchymal stromal cells (MSCs) are promising candidates for cell therapy due to their ease of isolation and expansion and their ability to secrete antiapoptotic, pro-angiogenic, and immunomodulatory factors. Three-dimensional (3D) aggregation "self-activates" MSCs to augment their pro-angiogenic and immunomodulatory potential, but the microenvironmental features and culture parameters that promote optimal MSC immunomodulatory function in 3D aggregates are poorly understood. Here, we generated MSC aggregates via three distinct methods and compared them with regard to their (a) aggregate structure and (b) immunomodulatory phenotype under resting conditions and in response to inflammatory stimulus. Methods associated with fast aggregation kinetics formed aggregates with higher cell packing density and reduced extracellular matrix (ECM) synthesis compared to those with slow aggregation kinetics. While all three methods of 3D aggregation enhanced MSC expression of immunomodulatory factors compared to two-dimensional culture, different aggregation methods modulated cells' temporal expression of these factors. A Design of Experiments approach, in which aggregate size and aggregation kinetics were systematically covaried, identified a significant effect of both parameters on MSCs' ability to regulate immune cells. Compared to small aggregates formed with fast kinetics, large aggregates with slow assembly kinetics were more effective at T-cell suppression and macrophage polarization toward anti-inflammatory phenotypes. Thus, culture parameters including aggregation method, kinetics, and aggregate size influence both the structural properties of aggregates and their paracrine immunomodulatory function. These findings underscore the utility of engineering strategies to control properties of 3D MSC aggregates, which may identify new avenues for optimizing the immunomodulatory function of MSC-based cell therapies.
Subject(s)
Mesenchymal Stem Cells , Cell Aggregation , Cell Proliferation , Cells, Cultured , Extracellular Matrix , Immunomodulation , T-LymphocytesABSTRACT
The retinal pigment epithelium (RPE)-choriocapillaris (CC) complex in the eye is compromised in age-related macular degeneration (AMD) and related macular dystrophies (MDs), yet in vitro models of RPE-CC complex that enable investigation of AMD/MD pathophysiology are lacking. By incorporating iPSC-derived cells into a hydrogel-based extracellular matrix, we developed a 3D RPE-CC model that recapitulates key features of both healthy and AMD/MD eyes and provides modular control over RPE and CC layers. Using this 3D RPE-CC model, we demonstrated that both RPE- and mesenchyme-secreted factors are necessary for the formation of fenestrated CC-like vasculature. Our data show that choroidal neovascularization (CNV) and CC atrophy occur in the absence of endothelial cell dysfunction and are not necessarily secondary to drusen deposits underneath RPE cells, and CC atrophy and/or CNV can be initiated systemically by patient serum or locally by mutant RPE-secreted factors. Finally, we identify FGF2 and matrix metalloproteinases as potential therapeutic targets for AMD/MDs.
Subject(s)
Choroid Diseases , Induced Pluripotent Stem Cells , Macular Degeneration , Choroid , Humans , Retinal Pigment EpitheliumABSTRACT
Pericytes play a critical role in promoting, regulating, and maintaining numerous vascular functions. Their dysfunction is a major contributor to the progression of vascular and neurodegenerative diseases, making them an ideal candidate for large-scale production for disease modeling and regenerative cell therapy. This protocol describes the rapid and robust differentiation of pericytes from human induced pluripotent stem cells (hiPSCs) while simultaneously generating a population of hiPSC-derived endothelial progenitor cells. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2017).
Subject(s)
Cell Culture Techniques , Cell Differentiation , Induced Pluripotent Stem Cells/metabolism , Pericytes/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Pericytes/cytologyABSTRACT
Human mesenchymal stromal cells (hMSC), also called mesenchymal stem cells, are adult cells that have demonstrated their potential in therapeutic applications, highlighted by their ability to differentiate down different lineages, modulate the immune system, and produce biologics. There is a pressing need for scalable culture systems for hMSC due to the large number of cells needed for clinical applications. Most current methods for expanding hMSC fail to provide a reproducible cell product in clinically required cell numbers without the use of serum-containing media or harsh enzymes. In this work, we apply a tailorable, thin, synthetic polymer coating-poly(poly(ethylene glycol) methyl ether methacrylate-ran-vinyl dimethyl azlactone-ran-glycidyl methacrylate) (P(PEGMEMA-r-VDM-r-GMA), PVG)-to the surface of commercially available polystyrene (PS) microcarriers to create chemically defined three-dimensional (3D) surfaces for large-scale cell expansion. These chemically defined microcarriers provide a reproducible surface that does not rely on the adsorption of xenogeneic serum proteins to mediate cell adhesion, enabling their use in xeno-free culture systems. Specifically, this work demonstrates the improved adhesion of hMSC to coated microcarriers over PS microcarriers in xeno-free media and describes their use in a readily scalable, bioreactor-based culture system. Additionally, these surfaces resist the adsorption of media-borne and cell-produced proteins, which result in integrin-mediated cell adhesion throughout the culture period. This feature allows the cells to be efficiently passaged from the microcarrier using a chemical chelating agent (ethylenediaminetetraacetic acid (EDTA)) in the absence of cleavage enzymes, an improvement over other microcarrier products in the field. Bioreactor culture of hMSC on these microcarriers enabled the production of hMSC over 4 days from a scalable, xeno-free environment.
Subject(s)
Mesenchymal Stem Cells , Bioreactors , Cell Culture Techniques , Cell Proliferation , Culture Media , HumansABSTRACT
The gold standard for treating peripheral nerve injuries that have large nerve gaps where the nerves cannot be directly sutured back together because it creates tension on the nerve, is to incorporate an autologous nerve graft. However, even with the incorporation of a nerve graft, generally patients only regain a small portion of function in limbs affected by the injury. Although, there has been some promising results using growth factors to induce more axon growth through the nerve graft, many of these previous therapies are limited in their ability to release growth factors in a sustained manner and tailor them to a desired time frame. The ideal drug delivery platform would deliver growth factors at therapeutic levels for enough time to grow axons the entire length of the nerve graft. We hypothesized that mineral coated microparticles (MCMs) would bind, stabilize and release biologically active glial cell-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in a sustained manner. Therefore, the objective of this study was to test the ability of MCMs releasing growth factors at the distal end of a 10 mm sciatic nerve graft, to induce axon growth through the nerve graft and restore hind limb function. After sciatic nerve grafting in Lewis rats, the hind limb function was tested weekly by measuring the angle of the ankle at toe lift-off while walking down a track. Twelve weeks after grafting, the grafts were harvested and myelinated axons were analyzed proximal to the graft, in the center of the graft, and distal to the graft. Under physiological conditions in vitro, the MCMs delivered a burst release of NGF and GDNF for 3 days followed by a sustained release for at least 22 days. In vivo, MCMs releasing NGF and GDNF at the distal end of sciatic nerve grafts resulted in significantly more myelinated axons extending distal to the graft when compared to rats that received nerve grafts without growth factor treatment. The rats with nerve grafts incorporated with MCMs releasing NGF and GDNF also showed significant improvement in hind limb function starting at 7 weeks postoperatively and continuing through 12 weeks postoperatively when compared to rats that received nerve grafts without growth factor treatment. In conclusion, MCMs released biologically active NGF and GDNF in a sustained manner, which significantly enhanced axon growth resulting in a significant improvement of hind limb function in rats. The animal experiments were approved by University of Wisconsin-Madison Animal Care and Use Committee (ACUC, protocol# M5958) on January 3, 2018.
ABSTRACT
Prolonged and elevated transforming growth factor-ß1 (TGF-ß1) signaling can lead to undesired scar formation during tissue repair and fibrosis that is often a result of chronic inflammation in the lung, kidney, liver, heart, skin, and joints. We report new TGF-ß1 binding peptides that interfere with TGF-ß1 binding to its cognate receptors and thus attenuate its biological activity. We identified TGF-ß1 binding peptides from the TGF-ß1 binding domains of TGF-ß receptors and engineered their sequences to facilitate chemical conjugation to biomaterials using molecular docking simulations. The in vitro binding studies and cell-based assays showed that RIPΔ, which was derived from TGF-ß type I receptor, bound TGF-ß1 in a sequence-specific manner and reduced the biological activity of TGF-ß1 when the peptide was presented either in soluble form or conjugated to a commonly used synthetic biomaterial. This approach may have implications for clinical applications such as treatment of various fibrotic diseases and soft tissue repair and offer a design strategy for peptide antibodies based on the biomimicry of ligand-receptor interactions.
Subject(s)
Receptors, Transforming Growth Factor beta , Transforming Growth Factor beta1 , Molecular Docking Simulation , Peptides , Signal TransductionABSTRACT
All reared larvae of flies of the genus Colobaea Zetterstedt, 1837 (Diptera: Sciomyzidae), which comprises 15 valid species, kill and consume freshwater nonoperculate pulmonate snails. New data are presented on the geographic distribution, biology, morphology of immature stages, and classification of Colobaea. Life cycle information is provided from field data and laboratory rearings for four of the 11 Palearctic species-C. bifasciella (Fallén), C. deemingi Knutson Bratt n.sp., C. pectoralis (Zetterstedt), and C. punctata (Lundbeck)-and for one of the three Nearctic species, C. americana Steyskal. Colobaea bifasciella is shown to be one of the most highly specialized parasitoid Sciomyzini, laying eggs on shells of Galba truncatula (O.F. Müller) and Stagnicola palustris (O.F. Müller) in temporary, intermittent, or vernal semiterrestrial situations. Each larva feeds in only one host snail, which is not killed until shortly before the larva completes development. Puparia are strongly modified to fit tightly within the shell of the host. The other reared species are shown to be less specialized than C. bifasciella, with eggs being laid upon vegetation, the larvae behaving as parasitoids-predators-saprophages of exposed aquatic snails, and the puparia of all four species being adapted to a lesser degree than C. bifasciella to fitting within the shell of the host snail. In nature, C. americana attacks Gyraulus parvus (Say) and Physa Draparnaud sp.; C. pectoralis attacks Anisus vortex (L.) and Bathyomphalus contortus (L.); and C. punctata attacks Gyraulus albus O.F. Müller, Lymnaea "peregra," Planorbarius corneus (L.), and Planorbis planorbis (L.). In the laboratory, these species also attacked and consumed other freshwater nonoperculate snails; C. deemingi was reared on Gyraulus intermixtus (Mousson) and Radix gedrosiana (Say), and an adult fly of the Palearctic C. distincta (Meigen) emerged from a puparium found in the shell of Anisus spirorbis (L.) collected in nature. Described and figured are eggs, larvae of all three instars, and puparia of the five laboratory-reared species. To provide perspective on features of Colobaea, diagnostic features are summarized of the immature stages of the Sciomyzini and the suprageneric categories of Sciomyzidae. The biogeography of the tribe Sciomyzini is presented, along with details of known geographical distribution. The classification and phylogenetic position of Colobaea are discussed. Included are a checklist of all known taxa of Colobaea, maps of geographic distribution, and a key to adults of the 15 valid species.