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Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3–6 weeks in order to understand genetic determinants of disease and precision medicine.

Summary For those people with cystic fibrosis carrying rare CFTR mutations not responding to currently available therapies, there is an unmet need for relevant tissue models for therapy development. Here, we describe a new testing platform that employs patient-specific induced pluripotent stem cells (iPSCs) differentiated to lung progenitor cells that can be studied using a dynamic, high-throughput fluorescence-based assay of CFTR channel activity. Our proof-of-concept studies support the potential use of this platform, together with a Canadian bioresource that contains iPSC lines and matched nasal cultures from people with rare mutations, to advance patient-oriented therapy development. Interventions identified in the high-throughput, stem cell-based model and validated in primary nasal cultures from the same person have the potential to be advanced as therapies., Highlights • A Canadian resource (CFIT) has CF donor-matched iPSCs and nasal epithelial cells • Lung progenitor cells (LPCs) differentiated from iPSCs express CFTR • LPCs from people with rare CFTR mutations enable high-throughput therapy testing • Matching nasal cultures can validate patient-specific drug responses in LPCs, Bear and colleagues show that lung progenitor cells (LPCs) differentiated from cystic fibrosis (CF) iPSCs recapitulate the primary defects conferred by different types of CF mutations. LPCs enable high-throughput testing of interventions, and pilot studies show that responses in LPCs can be validated in patient-matched primary nasal epithelial cultures, confirming the potential utility of LPCs in precision CF therapy development.

Human embryonic stem cells (ES) and induced pluripotent stem cells (iPSC) are powerful tools that have the potential to generate in vitro human lung epithelial cells. However, challenges in efficiency and reproducibility remain in utilizing the cells for therapy discovery platforms. Here, we optimize our previously published protocols to efficiently generate three developmental stages of the lung model (fetal lung epithelial progenitors, fLEP; immature airway epithelial spheroid, AES; air-liquid interface culture, ALI), and demonstrate its potential for cystic fibrosis (CF) drug discovery platforms. The stepwise approach directs differentiation from hPSC to definitive endoderm, anterior ventral foregut endoderm, and fetal lung progenitor cells. The article also describes the generation of immature airway epithelial spheroids in Matrigel with epithelial cells sorted by a magnetic-activated cell sorting system, and the generation of adult-like airway epithelia through air-liquid interface conditions. We demonstrate that this optimized procedure generates remarkably higher cystic fibrosis transmembrane conductance regulator (CFTR) expression and function than our previous method, and thus is uniquely suitable for CF research applications. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hESC/hiPSC differentiation to fetal lung progenitors Basic Protocol 2: Formation of airway epithelial spheroids Alternate Protocol 1: Cryopreservation of airway epithelial spheroids Basic Protocol 3: Differentiation and maturation in air-liquid interface culture Alternate Protocol 2: Differentiation and maturation of epithelial progenitors from airway epithelial spheroids in ALI culture.

Human PSC (hPSC) differentiations can capture developmental phenotypes and processes and are useful for studying fundamental biological mechanisms driving tissue morphogenesis and cell lineage development. Here, we show for the first time the temporal development of lung cell lineages using hPSC that model developmental milestones observed in primary tissue, the generation of renewable fetal lung epithelial spheroids, and the functional utility of the lung models at different differentiation stages for Cystic fibrosis disease modeling. We first show the presence of hPSC-derived lung progenitor cells reminiscent of early trimester lung development and containing basal stem cells that generate renewable airway spheroids. Maturation and polarization in air liquid interface (ALI) generates additional epithelial cell lineages found in adult airways including pulmonary neuroendocrine, brush, mature basal, ciliated and secretory cell types. Finally, pseudotime and RNA velocity analyses of the integrated datasets from the fetal and ALI stages reveal previously identified and new cell lineage relationships. Overall, hPSC differentiation can capture aspects of human lung development and potentially provide important insight into congenital causes of diseases.

Human pluripotent stem cells (hPSC) differentiations can capture developmental phenotypes and processes. They are useful for studying fundamental biological mechanisms driving tissue morphogenesis and cell lineage development. Here, we show temporal development of lung cell lineages using hPSC that recapitulate developmental milestones observed in primary tissue, the generation of renewable fetal lung epithelial spheroids, and the functional utility of the lung models at different differentiation stages for cystic fibrosis disease modeling. We first show the presence of hPSC-derived lung progenitor cells reminiscent of early trimester lung development and containing basal stem cells that generate renewable airway spheroids. Maturation and polarization in air liquid interface (ALI) generates additional epithelial cell lineages found in adult airways, including pulmonary neuroendocrine, brush, mature basal, ciliated and secretory cell types. Finally, pseudotime and RNA velocity analyses of the integrated datasets from fetal and ALI stages reveal both previously identified and new cell lineage relationships. Overall, hPSC differentiation can capture aspects of human lung development and potentially provide important insight into congenital causes of diseases.

Induced pluripotent, stem cell (iPSC)-derived models of airway tissue have successfully modeled the primary defect in regulated chloride conductance caused by the major Cystic Fibrosis causing mutation, F508del. However, it remains unclear if iPSC-derived airway cultures can be used in high-throughput therapy development for F508del and rarer mutations. There is an urgent need for airway tissue models that reflect the variability of patient-specific responses and are scalable for therapy development. In the current work, we describe a robust, high-throughput fluorescence assay of mutant CFTR function in iPSCs differentiated to immature airway epithelium. This assay measures reproducible functional responses to modulators targeting either the major CF mutant F508del or the nonsense mutant: W1282X-CFTR. We show that the ranking of patient-specific responses to interventions in this stem-cell based model recapitulates the ranking observed in primary nasal epithelial cultures obtained from the same individuals. In summary, these proof-of-concept studies show that this novel platform has the potential to support therapy development and precision medicine for Cystic Fibrosis patients. One Sentence Summary We describe a fluorescence-based platform that enables high-throughput Cystic Fibrosis therapy testing using iPSCs differentiated to immature lung.

Background Therapies targeting certain CFTR mutants have been approved, yet variations in clinical response highlight the need for in-vitro and genetic tools that predict patient-specific clinical outcomes. Toward this goal, the CF Canada-Sick Kids Program in Individual CF Therapy (CFIT) is generating a "first of its kind", comprehensive resource containing patient-specific cell cultures and data from 100 CF individuals that will enable modeling of therapeutic responses. Methods The CFIT program is generating: 1) nasal cells from drug naive patients suitable for culture and the study of drug responses in vitro , 2) matched gene expression data obtained by sequencing the RNA from the primary nasal tissue, 3) whole genome sequencing of blood derived DNA from each of the 100 participants, 4) induced pluripotent stem cells (iPSCs) generated from each participant's blood sample, 5) CRISPR-edited isogenic control iPSC lines and 6) prospective clinical data from patients treated with CF modulators. Results To date, we have recruited 57 of 100 individuals to CFIT, most of whom are homozygous for F508del (to assess in-vitro: in-vivo correlations with respect to ORKAMBI response) or heterozygous for F508del and a minimal function mutation. In addition, several donors are homozygous for rare nonsense and missense mutations. Nasal epithelial cell cultures and matched iPSC lines are available for many of these donors. Conclusions This accessible resource will enable development of tools that predict individual outcomes to current and emerging modulators targeting F508del-CFTR and facilitate therapy discovery for rare CF causing mutations.

Fetal lung underdevelopment, also known as pulmonary hypoplasia, is characterized by decreased lung growth and maturation. The most common birth defect found in babies with pulmonary hypoplasia is congenital diaphragmatic hernia (CDH). Despite research and clinical advances, babies with CDH still have high morbidity and mortality rates, which are directly related to the severity of lung underdevelopment. To date, there is no effective treatment that promotes fetal lung growth and maturation. Here, we describe a stem cell-based approach in rodents that enhances fetal lung development via the administration of extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs). Using fetal rodent models of pulmonary hypoplasia (primary epithelial cells, organoids, explants, and in vivo), we demonstrated that AFSC-EV administration promoted branching morphogenesis and alveolarization, rescued tissue homeostasis, and stimulated epithelial cell and fibroblast differentiation. We confirmed this regenerative ability in in vitro models of lung injury using human material, where human AFSC-EVs obtained following good manufacturing practices restored pulmonary epithelial homeostasis. Investigating EV mechanism of action, we found that AFSC-EV beneficial effects were exerted via the release of RNA cargo. MicroRNAs regulating the expression of genes involved in lung development, such as the miR17-92 cluster and its paralogs, were highly enriched in AFSC-EVs and were increased in AFSC-EV-treated primary lung epithelial cells compared to untreated cells. Our findings suggest that AFSC-EVs hold regenerative ability for underdeveloped fetal lungs, demonstrating potential for therapeutic application in patients with pulmonary hypoplasia.

Incomplete lung development, also known as pulmonary hypoplasia, is a recognized cause of neonatal death and poor outcome for survivors. To date, there is no effective treatment that promotes fetal lung growth and maturation. Herein, we describe a novel stem cell-based approach that enhances fetal lung development via the administration of extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs). In experimental models of pulmonary hypoplasia, administration of AFSC-EVs promoted lung branching morphogenesis and alveolarization, and stimulated pulmonary epithelial cell and fibroblast differentiation. This regenerative ability was confirmed in two models of injured human lung cells, where human AFSC-EVs obtained following good manufacturing practices restored pulmonary epithelial homeostasis. AFSC-EV beneficial effects were exerted via the release of RNA cargo, primarily miRNAs, that regulate the expression of genes involved in fetal lung development. Our findings suggest that AFSC-EVs hold regenerative ability for underdeveloped fetal lungs, demonstrating potential for therapeutic application.One Sentence SummaryFetal lung regeneration via administration of extracellular vesicles derived from amniotic fluid stem cells

Pulmonary disease is the major cause of morbidity and mortality in patients with cystic fibrosis, a disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Heterogeneity in CFTR genotype–phenotype relationships in affected individuals plus the escalation of drug discovery targeting specific mutations highlights the need to develop robust in vitro platforms with which to stratify therapeutic options using relevant tissue. Toward this goal, we adapted a fluorescence plate reader assay of apical CFTR-mediated chloride conductance to enable profiling of a panel of modulators on primary nasal epithelial cultures derived from patients bearing different CFTR mutations. This platform faithfully recapitulated patient-specific responses previously observed in the “gold-standard” but relatively low-throughput Ussing chamber. Moreover, using this approach, we identified a novel strategy with which to augment the response to an approved drug in specific patients. In proof of concept studies, we also validated the use of this platform in measuring drug responses in lung cultures differentiated from cystic fibrosis iPS cells. Taken together, we show that this medium throughput assay of CFTR activity has the potential to stratify cystic fibrosis patient-specific responses to approved drugs and investigational compounds in vitro in primary and iPS cell-derived airway cultures., Cystic fibrosis: toward personalized therapies A new method for evaluating drug responses in patient-derived respiratory tissue promises to help determine the best treatment for each patient with cystic fibrosis (CF). CF patients are highly susceptible to lung infections due to the build-up of thick mucus in the airways. Over 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified in patients with CF, which partly explains their varied response to treatment. Saumel Ahmadi, Christine E. Bear, and colleagues at the Hospital for Sick Children in Toronto developed a fluorescence-based method for measuring improvements in mutant CFTR function in patient-derived nasal and induced pluripotent stem cell-derived lung tissue. This method enables comparison of approved and investigational drugs on airway cells from each individual patient and in the longer term will accelerate the development of personalized therapeutic strategies.

Cystic fibrosis (CF) is a monogenic recessive disorder, affecting 70,000 people worldwide. CF is due to mutations in the CFTR gene, resulting in a defective protein that leads to symptoms in numerous organs, especially lungs. Despite recent advances, the lack of CF material contributes to the unmet need to find effective treatments for 55% of CF patients, and to restore the function of all affected tissues. A potential strategy would be to use gene-editing technologies (TALENs or CRISPR/Cas9) to introduce into or correct specific mutations in pluripotent stem cells (hPSCs) that can be turned into any other cell type. But current protocols for gene-editing hPSCs are often complicated, lengthy (6-8 months) and costly, thus, being restricted to a few specialised labs. This study describes an optimised protocol to correct the W1282X mutation in 3 CF hPSC lines derived from an adult and children with CF (iPSCs), by transfecting a CRISPR/Cas9 plasmid, a selection plasmid and a donor template. Corrected iPSCs were produced in 3-6 weeks. This protocol was previously used to generate the most common CF genotype, ∆F508/∆F508, from healthy hPSCs using TALENs. This work demonstrated that optimising each step of the gene-editing process, including an efficient transfection of hPSCs, and rapid isolation and screening for corrected cells, can produce in vitro models for CF in less than one month with minimum costs. These cells generated from paediatric or adult CF patients can be used to generate mutation-customised tissue-specific cultures holding great promise to advance drug testing and personalised medicine for CF. Supported by CF Trust-SRC003, CFIT (SickKids, CF-Canada Foundation), Emily’s Entourage.

Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.

Terrestrial insects are susceptible to desiccation and conserve internal water stores by preventing the loss of water due to transpiration across the cuticle. The epicuticle, a thin waxy layer on the outer surface of the insect cuticle is comprised primarily of a complex blend of cuticular hydrocarbons (CHCs) and is integral to preventing cuticular water loss. How the composition of epicuticular lipids (quantity and quality of the specific hydrocarbons) relates to desiccation resistance, however, has been difficult to determine. Here, we establish a model system to test the capacity of CHCs to protect against desiccation in the vinegar fly, Drosophila melanogaster. Using this system, we demonstrate that the oenocytes and CHCs produced by these cells are critically important for desiccation resistance, as measured by survival under desiccative conditions. Additionally, we show that both mating status and developmental temperature influence desiccation resistance. Prior mating increased desiccation survival through the direct transfer of CHCs between sexual partners, as well as through a female-specific response to a male-derived factor transferred during copulation. Together, our results demonstrate that desiccation resistance is an adaptive life-history trait dependent upon CHCs and influenced by prior social interactions and environmental conditions.

The contribution of bone marrow cells (BMC) in lung repair is controversial. We previously reported a subpopulation of BMC that express Clara cell secretory protein (CCSP). To determine the contribution of endogenous CCSP(+) BMC to airway regeneration, we performed bone marrow transplantation studies using the CCtk mouse, which expresses a thymidine kinase suicide gene under regulation of the CCSP promoter. Mice were transplanted with wild-type or CCtk BMC and treated with ganciclovir to eliminate CCSP(+) cells. After airway injury using naphthalene, mice depleted of CCSP(+) BMC had more inflammatory cells in lung and decreased levels of oxygen in arterial blood. They also had reduced expression of airway epithelial genes and less Clara cells compared to control mice that had intact CCSP(+) BMC and bone marrow derived CCSP(+) cells in the airways. After naphthalene injury, administration of CCSP reproduced the beneficial effect of CCSP(+) BMC by improving recovery of airway epithelium, reducing lung inflammation and increasing oxygen in arterial blood from mice depleted of CCSP(+) BMC. Our data demonstrate that ablation of CCSP(+) BMC delays airway recovery and suggests the beneficial effect of CCSP(+) BMC in lung recovery is in part due to production of CCSP itself.

Airway epithelial cells are of great interest for research on lung development, regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation, and full maturation of the cells in air-liquid interface cultures occurs in

Low-dose metronomic chemotherapy is an emerging form of chemotherapy with distinct mechanisms of action from conventional chemotherapy (e.g., antiangiogenesis). Although developed to overcome resistance to conventional chemotherapy, metronomic chemotherapy is subject to resistance on its own. However, there is a paucity of information on mechanisms of resistance, on cross-resistance between metronomic regimens using different cytotoxic drugs, and on cross-resistance between metronomic versus conventional chemotherapy, or versus targeted antiangiogenic therapy. Herein we show that PC-3 human prostate cancer xenografts were sensitive to both metronomic cyclophosphamide and metronomic docetaxel, but resistant to metronomic topotecan. Conventional docetaxel was only moderately active in parental PC-3 and in metronomic cyclophosphamide resistant PC-3 tumors. However, in metronomic cyclophosphamide resistant PC-3 tumors combining conventional docetaxel or bolus cyclophosphamide therapy with continued metronomic cyclophosphamide was superior to each treatment alone. Furthermore, bevacizumab had single-agent activity against metronomic cyclophosphamide resistant PC-3 tumors. Microarray analyses identified altered regulation of protein translation as a potential mechanism of resistance to metronomic cyclophosphamide. Our results suggest that sensitivity to metronomic chemotherapy regimens using different cytotoxic drugs not only depends on shared mechanisms of action such as antiangiogenesis, but also on as yet unknown additional antitumor effects that appear to be drug-specific. As clinically observed with targeted antiangiogenic agents, the continued use of metronomic chemotherapy beyond progression may amplify the effects of added second-line therapies or vice versa. However, metronomic chemotherapy is no different from other systemic therapies in that predictive biomarkers will be essential to fully exploit this novel use of conventional chemotherapeutics.

We have previously reported a subpopulation of bone marrow cells (BMC) that express Clara cell secretory protein (CCSP), generally felt to be specific to lung Clara cells. Ablation of lung Clara cells has been reported using a transgenic mouse that expresses thymidine kinase under control of the CCSP promoter. Treatment with ganciclovir results in permanent elimination of CCSP(+) cells, failure of airway regeneration, and death. To determine if transtracheal delivery of wild-type bone marrow CCSP(+) cells is beneficial after ablation of lung CCSP(+) cells, transgenic mice were treated with ganciclovir followed by transtracheal administration of CCSP(+) or CCSP(-) BMC. Compared with mice administered CCSP(-) cells, mice treated with CCSP(+) cells had more donor cells lining the airway epithelium, where they expressed epithelial markers including CCSP. Although donor CCSP(+) cells did not substantially repopulate the airway, their administration resulted in increased host ciliated cells, better preservation of airway epithelium, reduction of inflammatory cells, and an increase in animal survival time. Administration of CCSP(+) BMC is beneficial after permanent ablation of lung Clara cells by increasing bronchial epithelial repair. Therefore, CCSP(+) BMC could be important for treatment of lung diseases where airways re-epithelialization is compromised.

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene that regulates chloride and water transport across all epithelia and affects multiple organs including the lungs. Here we report an in vitro directed differentiation protocol for generating functional CFTR-expressing airway epithelia from human embryonic stem cells. Carefully timed treatment by exogenous growth factors that mimic endoderm developmental pathways in vivo followed by air-liquid interface culture results in maturation of patches of tight junction-coupled differentiated airway epithelial cells that demonstrate active CFTR transport function. As a proof-of-concept, treatment of CF patient induced pluripotent stem cells (iPSC)-derived epithelial cells with a novel small molecule compound to correct for the common CF-processing mutation resulted in enhanced plasma membrane localization of mature CFTR protein. Our study provides a method for generating patient-specific airway epithelial cells for disease modeling and in vitro drug testing.

Background Studies suggest that inflammation is involved in the neurodegenerative cascade leading to Alzheimer's disease (AD) pathology and symptoms. This study sought to quantitatively summarize the clinical cytokine data. Methods Original English language peer-reviewed studies measuring cytokine concentrations in AD and healthy control subjects were included. Mean (±standard deviation) cytokine concentrations for AD and control subjects were extracted. Results Forty studies measuring peripheral blood cytokine concentrations and 14 measuring cerebrospinal fluid (CSF) cytokine concentrations were included. In peripheral blood, there were significantly higher concentrations (weighted mean difference [95% confidence interval]) of interleukin (IL)-6 (2.86 [1.68, 4.04] pg/mL, p N [AD/control subjects] = 985/680, 14 studies), tumor necrosis factor (TNF)-α (3.25 [.76, 5.74] pg/mL, p = .01, N = 680/447, 14 studies), IL-1β (.55 [.32, .78] pg/mL, p N = 574/370, 10 studies), transforming growth factor (TGF)-β (67.23 [28.62, 105.83] pg/mL, p = .0006, N = 190/158, 5 studies), IL-12 (7.60 [5.58, 9.62] pg/mL, p N = 148/106, 5 studies), and IL-18 (15.82 [1.98, 29.66] pg/mL, p = .03, N = 131/94, 4 studies) but not of IL-4, IL-8, IL-10, interferon-γ, or C-reactive protein in AD subjects compared with control subjects. There were significantly higher concentrations of TGF-β (7.81 [2.27, 13.35] pg/mL, p =.006, N = 113/114, 5 studies) but not IL-6, TNF-α, and IL-1β in the CSF of AD subjects compared with control subjects. Conclusions These results strengthen the clinical evidence that AD is accompanied by an inflammatory response, particularly higher peripheral concentrations of IL-6, TNF-α, IL-1β, TGF-β, IL-12 and IL-18 and higher CSF concentrations of TGF-β.

Inadequate invasion of the uterus by cytotrophoblasts is speculated to result in pregnancy-induced disorders such as preeclampsia. However, the molecular mechanisms that govern appropriate invasion of cytotrophoblasts are unknown. Here, we demonstrate that under low-oxygen conditions (2.5% oxygen), 2-methoxyestradiol (2-ME), which is a metabolite of estradiol and is generated by catechol-o-methyltransferase (COMT), induces invasion of cytotrophoblasts into a naturally-derived, extracellular matrix. Neither low-oxygen conditions nor 2-ME alone induces the invasion of cytotrophoblasts in this system; however, low-oxygen conditions combined with 2-ME result in the appropriate invasion of cytotrophoblasts into the extracellular matrix. Cytotrophoblast invasion under these conditions is also associated with a decrease in the expression of hypoxia-inducible factor-1α (HIF-1α), transforming growth factor-β3 (TGF-β3), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Pregnant COMT-deficient mice with hypoxic placentas and preeclampsia-like features demonstrate an up-regulation of HIF-1α, TGF-β3, and TIMP-2 when compared with wild-type mice; normal levels are restored on administration of 2-ME, which also results in the resolution of preeclampsia-like features in these mice. Indeed, placentas from patients with preeclampsia reveal lower levels of COMT and higher levels of HIF-1α, TGF-β3, and TIMP-2 when compared with those from normal pregnant women. We demonstrate that low-oxygen conditions of the placenta are a critical co-stimulator along with 2-ME for the proper invasion of cytotrophoblasts to facilitate appropriate vascular development and oxygenation during pregnancy.

Clara cell secretory protein (CCSP) is one of the most abundant proteins in the airway surface fluid, and has many putative functions. Recent advances in the field of stem cells and lung regeneration have identified potentially new roles of CCSP and CCSP-expressing cell populations in airway maintenance, repair and regeneration. This review focuses on the airway regenerative potential of CCSP and the cells that express this protein. The use of this protein or CCSP-expressing cells as an indication of biologic processes that contribute to lung injury or repair is highlighted.

Accurate modeling of the cellular microenvironment is important for improving studies of cell biology in vitro. Here, we demonstrate a flexible method for creating a cellular microenvironment in vitro that allows (i) controlled spatial distribution (patterning) of multiple types of cells within three-dimensional (3-D) matrices of a biologically derived, thermally curable hydrogel (Matrigel) and (ii) application of gradients of soluble factors, such as cytokines, across the hydrogel. The technique uses laminar flow to divide a microchannel into multiple subchannels separated by microslabs of hydrogel. It does not require the use of UV light or photoinitiators and is compatible with cell culture in the hydrogel. This technique makes it possible to design model systems to study cellular communication mediated by the diffusion of soluble factors within 3-D matrices. Such factors can originate either from secretions of neighboring cells patterned within the microchannel, or from an external source -- e.g., a solution of growth factors injected into a subchannel. This method is particularly useful for studying cells such as those of the immune system, which are often weakly adherent and difficult to position precisely with standard systems for cell culture. We demonstrated this application by co-culturing two types of macrophage-like cells (BAC1.2F5 and LADMAC cell lines) within spatially separated regions of a slab of hydrogel. This pair of cell lines represents a simple model system for intercellular communication: the LADMAC cells produce colony-stimulating factor 1 (CSF-1), which is required by the BAC cells for survival.

Emphysema is a chronic lung disease characterized by alveolar enlargement and tissue loss. Tissue engineering represents an attractive potential for regeneration of several organ systems. The complex three-dimensional architectural structure of lung parenchyma requiring connections of alveolar units to airways and the pulmonary circulation makes this strategy less optimistic. In the present study, we used Gelfoam sponge as a scaffold material, supplemented with fetal rat lung cells as progenitors, to explore the potential application of cell-based tissue engineering for lung regeneration in adult rats. After injection into lung parenchyma, the sponge showed porous structures similar to alveolar units. It did not induce severe local inflammatory response. Fetal lung cells in the sponge were able to survive in the adult lung for at least 35 days, determined by CMTMR [5-(and-6)-{[(4-chloromethyl)benzoyl]amino}tetramethylrhodamine] labeling. Proliferation of cells within the sponge was demonstrated in vivo by bromodeoxyuridine (BrdU) labeling. Cells formed “alveolar-like structures” at the border between the sponge and the surrounding lung tissue with positive immunohistochemical staining for epithelial and endothelial cells. Neovascularization of the sponge was demonstrated with India ink perfusion. The sponge degraded after several months. This study suggests that cell-based tissue engineering possesses the potential to regenerate alveolar-like structures, an important step towards our ultimate goal of lung regeneration.

Background— Disintegrin metalloproteinases (ADAMs) may contribute to structural cardiac remodeling by altering cell-surface matrix receptors (integrins) and activating potent biomolecules. We compared expression of ADAMs, their endogenous inhibitor tissue inhibitor of metalloproteinases (TIMP)-3, and integrins in human heart tissue with varied patterns of structural remodeling. Methods and Results— Myocardium was obtained from patients with dilated cardiomyopathy (n=20), hypertrophic obstructive cardiomyopathy (n=5), and nonfailing donor hearts (n=7). Paired samples (n=10) were obtained before left ventricular assist device insertion and at transplantation. The expressions of ADAM10, ADAM12, ADAM15, and ADAM17, TIMP-3, and integrin receptors β1D and β3 were determined by quantitative immunoblotting. Integrin shedding was assessed by the ratio of integrin cleavage products to intact protein abundance. Confocal microscopy was performed. Dilated cardiomyopathy was characterized by increased ADAM10 and ADAM15 expression and reduced TIMP-3 expression. The integrin β1D cleavage ratio was elevated, indicating receptor shedding. ADAM10 and ADAM15 expressions correlated with the cleavage ratio. ADAM10 colocalized with integrin β1D by confocal microscopy. ADAM10 expression correlated with clinical indices of chamber dilatation and systolic dysfunction. Hemodynamic unloading reduced ADAM10 and ADAM12 expressions and increased integrin β1D expression. ADAM12 and integrin β1D expressions were increased in HOCM. ADAM17 was increased in both dilated cardiomyopathy and hypertrophic obstructive cardiomyopathy. Conclusions— Disintegrin metalloproteinases are differentially expressed in human myocardium, reflecting the underlying pattern of structural remodeling. ADAM10 and ADAM15 may contribute to cardiac dilatation by reducing cell-matrix interactions via integrin shedding. Targeting disintegrin metalloproteinases, perhaps by restoring deficient TIMP-3 levels with gene or cell-based therapies, may prevent progressive chamber dilatation in human dilated cardiomyopathy.

This article is based on the plenary address given by George M. Whitesides of Harvard University on March 30, 2005, at the Materials Research Society Spring Meeting in San Francisco. Materials science and biomedicine are arguably two of the most exciting fields in science today. Research at the border between them will inevitably be a major focus, and the applications of materials science to problems in biomedicine—that is, biomaterials science—will bud into an important new branch of materials science. Accelerating the growth of this area requires an understanding of two very different fields, and being both thoughtful and entrepreneurial in considering “Why?” “How?” and “Where?” to put them together. In this fusion, biomedicine will, we believe, set the agenda; materials science will follow, and materials scientists must learn biology to be effective.

Objective : We analyzed the phenotypic and functional differences between venous and arterial smooth muscle cells (SMC) and the role of decorin in modulating these differences. Methods and results : SMC were isolated from the jugular veins and carotid arteries of male white New Zealand rabbits. Venous SMC demonstrated increased proliferation (2-fold, p

This report shows that the direction of polarization of attached mammalian cells determines the direction in which they move. Surfaces micropatterned with appropriately functionalized self-assembled monolayers constrain individual cells to asymmetric geometries (for example, a teardrop); these geometries polarize the morphology of the cell. After electrochemical desorption of the self-assembled monolayers removes these constraints and allows the cells to move across the surface, they move toward their blunt ends.

With lipid bilayer-bilayer junctions in mind as model systems for the study of cell-cell junctions, we have examined adhesion between simple lipid membranes using fluorescence resonance energy transfer (FRET) and fluorescence interference contrast microscopy (FLIC) to map the interfacial topography. The contact can take the form of uniform adhesion, with an intermembrane separation of nanometers, or nonuniform adhesion, in which blisters hundreds of nanometers in height coexist with tight adhesion zones. We find that blisters can result as a consequence of rapid interbilayer contact induced by osmotic shock. We develop a model for the formation and stabilization of the blisters, confirming that these phenomena are governed mainly by hydrodynamic flow in the intermembrane space.

Eukaryotic organisms have dynamic genomes, with transposable elements (TEs) as a major contributing factor. Although the large autonomous TEs can significantly shape genomic structures during evolution, genomes often harbor more miniature nonautonomous TEs that can infest genomic niches where large TEs are rare. In spite of their cut-and-paste transposition mechanisms that do not inherently favor copy number increase, miniature inverted-repeat transposable elements (MITEs) are abundant in eukaryotic genomes and exist in high copy numbers. Based on the large number of MITE families revealed in previous studies, accurate annotation of MITEs, particularly in newly sequenced genomes, will identify more genomes highly rich in these elements. Novel families identified from these analyses, together with the currently known families, will further deepen our understanding of the origins, transposase sources, and dramatic amplification of these elements.

The understanding of key processes and signaling mechanisms in lung development has been mainly demonstrated through gain and loss of function studies in mice, while human lung development remains largely unexplored due to inaccessibility. Several recent reports have exploited the identification of key signaling mechanisms that regulate lineage commitment and restriction in mouse lung development, to direct differentiation of both mouse and human pluripotent stem cells towards lung epithelial cells. In this review, we discuss the recent advances in the generation of respiratory epithelia from pluripotent stem cells and the potential of these engineered cells for novel scientific discoveries in lung diseases and future translation into regenerative therapies.

Cell replacement therapy is a promising approach for treatment of lung disease such as cystic fibrosis, although rates of engraftment need to be improved. We previously showed improved cell retention in the lung using transtracheal delivery compared to intravenous injection. Here, we optimized other parameters of cell delivery using 7-day cultured bone marrow cells (BMCs). Retention of BMC in the lung was dose-dependent. Naphthalene treatment had maximal effects on BMC retention when given 2 days before cell delivery. Naphthalene treatment of the donor amplified a CCSP(+) population and increased retention efficiency in the recipient. Repeated naphthalene treatment and repeated cell delivery both resulted in greater retention. The contribution of the second cell dose was minimal suggesting that a second delivery of BMC promotes proliferation of the first. Busulfan-induced myelosuppression augmented retention of exogenous BMC by up to 20-fold. These BMC helped CCSP reconstitution. Using the optimal delivery techniques and cytokeratin-18-driven green fluorescent protein (GFP) reporter mice, we detected threefold more GFP suggesting more BMC differentiated to epithelial cells. We propose that improved engraftment in the lung will increase cell replacement and thus be a more efficient therapeutic approach for various lung diseases.

Rationale: The contribution of bone marrow (BM) to repair of lung injury is controversial, in part due to the robust restorative capacity of endogenous lung progenitor cells. We have previously shown that a subpopulation of bone marrow cells express Clara cell secretory protein (CCSP); a marker of airway progenitor cells. To study how endogenous bone marrow CCSP-expressing cells affect local progenitor regeneration, we used the CCtk transgenic mouse model in which the Herpes simplex thymidine kinase suicide gene is expressed under the control of the CCSP promoter. Ganciclovir treatment results in elimination of CCSP-expressing cells. Methods: Wild type female mice were lethally irradiated and transplanted with male BM cells from either CCtk or wild type mice. After two months to establish bone marrow engraftment, animals were treated with ganciclovir 7 mg/day for 20 days to eliminate bone marrow CCSP-expressing cells in the CCtk BM group with no effects in lung. Mice were treated with naphthalene 200 mg/kg to injure airway epithelium and were sacrificed after 2, 5 and 10 days of naphthalene treatment. Airway recovery was assessed using CCSP immunofluorescence staining. Inflammatory cells were estimated by total and differential cell counts of bronchoalveolar lavage (BAL) cells. Quantitative real-time PCR analysis was done to compare expression of epithelial and secretory cell markers. Arterial blood gases (PaO2/FIO2 ratio) were analyzed for each time point. Results: Mice showed significant differences between groups only at day 5. The CCtk BM group showed less CCSP+ cells lining the airway epithelium (score of 2.75±0.17 vs. 4±0; p

Inadequate invasion of the uterus by cytotrophoblasts is speculated to result in pregnancy-induced disorders such as preeclampsia. However, the molecular mechanisms that govern appropriate invasion of cytotrophoblasts are unknown. Here, we demonstrate that under low-oxygen conditions (2.5% oxygen), 2-methoxyestradiol (2-ME), which is a metabolite of estradiol and is generated by catechol-o-methyltransferase (COMT), induces invasion of cytotrophoblasts into a naturally-derived, extracellular matrix. Neither low-oxygen conditions nor 2-ME alone induces the invasion of cytotrophoblasts in this system; however, low-oxygen conditions combined with 2-ME result in the appropriate invasion of cytotrophoblasts into the extracellular matrix. Cytotrophoblast invasion under these conditions is also associated with a decrease in the expression of hypoxia-inducible factor-1alpha (HIF-1alpha), transforming growth factor-beta3 (TGF-beta3), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Pregnant COMT-deficient mice with hypoxic placentas and preeclampsia-like features demonstrate an up-regulation of HIF-1alpha, TGF-beta3, and TIMP-2 when compared with wild-type mice; normal levels are restored on administration of 2-ME, which also results in the resolution of preeclampsia-like features in these mice. Indeed, placentas from patients with preeclampsia reveal lower levels of COMT and higher levels of HIF-1alpha, TGF-beta3, and TIMP-2 when compared with those from normal pregnant women. We demonstrate that low-oxygen conditions of the placenta are a critical co-stimulator along with 2-ME for the proper invasion of cytotrophoblasts to facilitate appropriate vascular development and oxygenation during pregnancy.

The bone marrow compartment is enriched in stem and progenitor cells, and an unidentified subpopulation of these cells can contribute to lung epithelial repair. Here we identify this subpopulation and quantitate its relative contribution to injured airway epithelium. A subpopulation of adherent human and murine bone marrow cells that expresses Clara cell secretory protein (CCSP) was identified using flow cytometry. When cultured at the air-liquid interface in ex vivo cultures, Ccsp+ cells expressed type I and type II alveolar markers as well as basal cell markers and active epithelial sodium channels. Ccsp+ cells preferentially homed to naphthalene-damaged airways when delivered transtracheally or intravenously, with the former being more efficient than the latter. Interestingly, naphthalene-induced lung damage transiently increased Ccsp expression in bone marrow and peripheral circulation. Furthermore, lethally irradiated Ccsp-null mice that received tagged wild-type bone marrow contained donor-derived epithelium in both normal and naphthalene-damaged airways. This study therefore identifies what we believe to be a newly discovered cell in the bone marrow that might have airway reconstitution potential in the context of cell-based therapies for lung disease. Additionally, these data could reconcile previous controversies regarding the contribution of bone marrow to lung regeneration.

This paper demonstrates that a hand-powered egg beater can be modified to serve as a centrifuge for separating plasma from human whole blood. Immunoassays used to diagnose infectious diseases often require plasma from whole blood, and obtaining plasma typically requires electrically-powered centrifuges, which are not widely available in resource-limited settings. Human whole blood was loaded into polyethylene (PE) tubing, and the tubing was attached to the paddle of an egg beater. Spinning the paddle pelleted the blood cells to the distal end of the PE tubing; the plasma remained as the supernatant. A cholesterol assay (run on patterned paper) demonstrated the suitability of this plasma for use in diagnostic assays. The physics of the system was also analyzed as a guide for the selection of other rotating systems for use in centrifugation. Egg beaters, polyethylene tubing, and paper are readily available devices and supplies that can facilitate the use of point-of-care diagnostics at sites far from centralized laboratory facilities.

It has been suggested that some adult bone marrow cells (BMC) can localize to the lung and develop tissue-specific characteristics including those of pulmonary epithelial cells. Here, we show that the combination of mild airway injury (naphthalene-induced) as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype. Confocal analysis of airway and alveolar-localized BMC (fluorescently labeled) with epithelial markers shows expression of the pulmonary epithelial proteins, Clara cell secretory protein, and surfactant protein C. To confirm epithelial gene expression by BMC, we generated transgenic mice expressing green fluorescent protein (GFP) driven by the epithelial-specific cytokeratin-18 promoter and injected BMC from these mice transtracheally into wild-type recipients after naphthalene-induced airway injury. BMC retention in the lung was observed for at least 120 days following cell delivery with increasing GFP transgene expression over time. Some BMC cultured in vitro over time also expressed GFP transgene, suggesting epithelial transdifferentiation of the BMC. The results indicate that targeted delivery of BMC can promote airway regeneration.

We establish two methods to deposit native biomembranes (human erythrocyte membranes and sarcoplasmic reticulum membranes) selectively onto biocompatible microtemplates. The first method utilizes UV photolithography to micropattern the regenerated cellulose, while the second uses the “stamping” of protein barriers onto homogeneous cellulose supports. The relatively simple methods established here allow for the position selective spreading of three-dimensional native cells into two-dimensional films, retaining the orientation and lateral density of transmembrane proteins in their native state.

Although the application of cold energy, cryotherapy, has been shown to cause selective damage to cellular components with preservation of matrix structure resulting in less fibrosis in a variety of tissues, the effects of intravascular cryotherapy on vessel wall repair after balloon angioplasty are unknown. We sought to characterize the effects of cryotherapy application on vessel wall repair after balloon angioplasty and study the relationship between collagen accumulation in the vessel wall and late lumen loss as assessed by serial intravascular ultrasound.The immediate, early (72 h) and late (10 weeks) effects of three intravascular cryotherapy application time periods (60, 120 and 240 s) after iliac artery balloon angioplasty ('cryotherapy') were compared with balloon angioplasty alone ('control') in 59 rabbits. Arterial lumen area was measured by intravascular ultrasound immediately after the procedure, at 72 h and at 10 weeks. Collagen content was calculated separately for intima and media/adventitia layers and correlated with late lumen loss.Cryotherapy produced average vessel wall temperature of -26 degrees C (range, -20 to -45 degrees C) and resulted in significantly larger lumen cross-sectional area (CSA) immediately after application (5.74+/-1.18 vs. 4.14+/-0.75 mm(2), P=0.008) but was not different than control arteries at 10 weeks. At 72 h, there was extensive cell loss in the medial and adventitial layers accompanied by increased macrophage infiltration in cryotherapy treated arteries compared to control. At 10 weeks, intimal hyperplasia was increased 2-fold in cryotherapy treated arteries. Collagen content was increased 2-fold in the medial/adventitial layers, and nearly 3-fold in the intima of cryotherapy treated arteries. Collagen content in arterial intima (P=0.01) as well as media/adventitia (P=0.005) positively correlated with late lumen loss. Foci of chondro- and osseous metaplasia and calcification were evident at the medial-adventitial junction in cryotherapy treated arteries at 10 weeks.Intravascular cryotherapy induced early arterial wall cell loss and late intimal hyperplasia, vascular fibrosis and chondro- and osseous metaplastic changes with no late beneficial effects on lumen area compared to balloon angioplasty alone. Collagen accumulation in all three layers of the vessel wall contributes to the development of late inward remodeling after balloon angioplasty.

Fluorescence resonance energy transfer (FRET) between lipid-linked donor and acceptor molecules in two apposing lipid bilayer membranes is used to resolve topographical features at an intermembrane junction. Efficient energy transfer occurs when the membranes are apposed closely, which creates an image, or footprint, that maps the contact zone and reveals nanometer-scale topographical structures. We experimentally characterize intermembrane FRET by using a supported membrane junction consisting of a glass-supported lipid membrane, onto which a second membrane is deposited by rupture of a giant vesicle. A series of membrane junctions containing different glycolipids (phosphatidylinositol and ganglioside G M1 ), protein (cholera toxin), and lipid-linked polyethylene glycol are studied. The carbohydrate and protein components influence the intermembrane separation. Differential FRET efficiency is clearly distinguishable for each case. Quantitative analysis of the FRET efficiency yields measurements of intermembrane-separation distances that agree precisely with structural data on G M1 and cholera toxin. The lateral arrangement of molecular species on the membrane surface thus can be discerned by their influence on membrane spacing without the need for direct labeling of the molecule of interest. In the case of polyethylene glycol lipid-containing membrane junctions, imaging by intermembrane FRET reveals spontaneously forming patterns that are not visible in conventional fluorescence images.