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Objectives The purpose of the present study was to explore Muslim Arab American women's intersecting identities, the types of discrimination they experience, and the psychosocial effects of those experiences on identity and well-being. Method Thirteen Muslim Arab American women were interviewed utilizing the consensual qualitative research (CQR) approach. Results Final domains that emerged from the data included conceptualization of racial, ethnic, and religious identity; experiences and effect of microaggressions; and coping strategies. Conclusions The results document discrimination at the intersection of their multiple identities, a perceived shift in stereotypes from weak and oppressed to anti-American and violent, and within-group discrimination in the form of colorism and judgment. Reported psychological effects of discrimination include internalization of stereotypes, burden to represent all Muslims, rejection of the White racial label, and increased activism. The findings highlight resilience and coping through active self-acceptance and advocacy. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Efficient generation of human induced pluripotent stem cell (hiPSC)-derived human intestinal organoids (HIOs) would facilitate the development of in vitro models for a variety of diseases that affect the gastrointestinal tract, such as inflammatory bowel disease or Cystic Fibrosis. Here, we report a directed differentiation protocol for the generation of mesenchyme-free HIOs that can be primed towards more colonic or proximal intestinal lineages in serum-free defined conditions. Using a CDX2eGFP iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors and robust generation of mesenchyme-free organoids expressing characteristic markers of small intestinal or colonic epithelium. We employ HIOs generated in this way to measure CFTR function using cystic fibrosis patient-derived iPSC lines before and after correction of the CFTR mutation, demonstrating their future potential for disease modeling and therapeutic screening applications., Human induced pluripotent stem cell-derived intestinal organoids (HIOs) are powerful tools to study development and diseases of the gastrointestinal tract. Here, the authors develop a directed differentiation protocol to generate mesenchyme-free HIOs that can be patterned towards proximal small intestine or colonic epithelium, and demonstrated their utility in modeling CFTR function.

Summary Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway., Graphical Abstract, Highlights • Reporter lines allow detailed profiling of pluripotent stem cell-derived airway • PSC-derived airway spheres contain basal and secretory cells as well as non-lung • Single-cell RNA-seq reveals heterogeneity and potential plasticity, In this article, Kotton and colleagues show that human pluripotent stem cell-derived airway epithelial spheres contain basal and secretory airway cells. Using reporter lines for the secretory airway lineage alongside transcriptomic and functional analyses, they demonstrate that these cells have characteristic features of this population but are susceptible to Wnt-dependent phenotypic drift toward an alveolar type II cell-like program.

It has been postulated that during human fetal development, all cells of the lung epithelium derive from embryonic, endodermal, NK2 homeobox 1–expressing (NKX2-1+) precursor cells. However, this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity, these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support, this NKX2-1+ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively, when recombined with fetal mouse lung mesenchyme, the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved, stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted, patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine.

Alveolar epithelial type II cells (AEC2s) are the facultative progenitors of lung alveoli and serve as the surfactant-producing cells of air-breathing organisms. Although primary human AEC2s are difficult to maintain stably in cell cultures, recent advances have facilitated the derivation of AEC2-like cells from human pluripotent stem cells (hPSCs) in vitro. Here, we provide a detailed protocol for the directed differentiation of hPSCs into self-renewing AEC2-like cells that can be maintained for up to 1 year in culture as epithelial-only spheres without the need for supporting mesenchymal feeder cells. The month-long protocol requires recapitulation of the sequence of milestones associated with in vivo development of the distal lung, beginning with differentiation of cells into anterior foregut endoderm, which is followed by their lineage specification into NKX2–1(+) lung progenitors and then distal/alveolar differentiation to produce progeny that express transcripts and possess functional properties associated with AEC2s.

Alveolar epithelial type 2 cells (AEC2s) are the facultative progenitors responsible for maintaining lung alveoli throughout life, yet are difficult to access from patients for biomedical research or lung regeneration applications. Here we engineer AEC2s from human induced pluripotent stem cells (iPSCs) in vitro and use single cell RNA sequencing (scRNA-seq) to profile the detailed kinetics of their differentiation over time. We focus on both the desired target cells as well as those that appear to diverge to alternative endodermal fates. By combining scRNA-seq with lentiviral barcoding to trace differentiating clones, we reveal the bifurcating cell fate trajectories followed as primordial lung progenitors differentiate into mature AEC2s. We define the global transcriptomic signatures of primary developing human AEC2s from fetal through adult stages in order to identify the subset of in vitro differentiating cells that appear to recapitulate the path of in vivo development. In addition, we develop computational methods based on Continuous State Hidden Markov Models (CSHMM) to identify the precise timing and type of signals, such as over-exuberant Wnt responses, that induce some early multipotent NKX2-1+ progenitors to lose lung fate as they clonally diverge into a variety of non-lung endodermal lineages. Finally, we find that this initial developmental plasticity is regulatable via Wnt modulation, and subsides over time, ultimately resulting in iPSC-derived AEC2s that exhibit a stable phenotype and nearly limitless self-renewal capacity in vitro. Our methods and computational approaches can be widely applied to study and control directed differentiation, producing an inexhaustible supply of mature lineages, exemplified here by the generation of AEC2s.

Background Cystic fibrosis (CF) is one of the most common lethal genetic diseases and is caused by mutations in the CFTR gene where a deletion of phenylalanine at position 508 (F508del) accounts for the most prevalent mutation worldwide. MicroRNA (miRNA) are small non-coding RNAs that can regulate up to two thirds of gene expression in human cells and have been shown to be dysregulated in the CF airway and implicated in the direct control of CFTR Induced Pluripotent stem cells (iPSC) can be routinely generated from any patient and contain that person’s unique genetic background, CF mutation and can form all the cell types affected by CF representing an attractive tool to advance the understanding of CF. Recent advances in iPSC technology have led to an in vitro platform of airway epithelial organoids derived from CF and CF-corrected cells for modelling CF lung disease however this model requires further molecular characterisation. We hypothesise that specific miRNA play a key role in the pathogenesis of CF lung disease and airway organoids generated from patient derived iPSCs can better elucidate this role. Methods Cystic fibrosis patient specific iPSC derived airway organoids were generated from an individual homozygous for the F508del mutation (ΔF508/ΔF508) and its gene corrected counterpart (ΔF508/wild-type [WT]) using established protocols. Expression of a miRNA panel we have previously shown to regulate CFTR (miR-145, miR-223 and miR-494) were examined in CF versus CF corrected iPSC lines at Day 0 and Day 32 following directed differentiation of iPSC into proximal airway organoids. Results In preliminary work we have observed that miRNA known to target CFTR are detectable in CF and gene-corrected iPSC lines and differential expression is observed between cells at the pluripotent stage (iPSC) at Day 0 and cells that have been differentiated in 3D as organoids to airway-like cells (bronchospheres) at Day 32. Importantly, there is differential expression of key CFTR related miRNA when we compare CF patient-derived bronchospheres to those where the F508del mutation has been corrected. Increased expression of miR-494 was observed in CF vs. CF corrected cells whereas levels of miR-145 and -223 appeared to be reduced. Conclusions We have demonstrated for the first time that miRNA are detectable in CF iPSC derived bronchospheres with altered expression following a directed differentiation approach of iPSC into proximalised airway epithelium. Expression patterns in iPSC may be different to patient bronchial epithelial cells previously described and warrants further investigation.

Summary Alveolar epithelial type 2 cells (AEC2s) are the facultative progenitors responsible for maintaining lung alveoli throughout life but are difficult to isolate from patients. Here, we engineer AEC2s from human pluripotent stem cells (PSCs) in vitro and use time-series single-cell RNA sequencing with lentiviral barcoding to profile the kinetics of their differentiation in comparison to primary fetal and adult AEC2 benchmarks. We observe bifurcating cell-fate trajectories as primordial lung progenitors differentiate in vitro, with some progeny reaching their AEC2 fate target, while others diverge to alternative non-lung endodermal fates. We develop a Continuous State Hidden Markov model to identify the timing and type of signals, such as overexuberant Wnt responses, that induce some early multipotent NKX2-1+ progenitors to lose lung fate. Finally, we find that this initial developmental plasticity is regulatable and subsides over time, ultimately resulting in PSC-derived AEC2s that exhibit a stable phenotype and nearly limitless self-renewal capacity.

Although immunosuppressive drugs improve short term survival, rejection incidence and long term survival remain largely unchanged in heart transplant patients. While Human Leukocyte Antigen (HLA) is a common initiator of graft-specific immune responses, non-HLA antibody mediated rejection (AMR) also commonly occurs. Furthermore, non-HLA antibodies have been implicated in cell mediated rejection and cardiac allograft vasculopathy. Therefore, there is a need to identify the role of non-HLA antigens in heart transplant rejection. Serum from heart transplant patients lacking donor specific anti-HLA antibodies was collected longitudinally; pre-transplantation, acute rejection, and post-rejection for patients with AMR and matched controls. Serum IgG was used to form anti-human heart affinity columns and antigenic proteins from donor heart extracts captured. Antigens were eluted from the columns and subjected to LC-MS/MS. Longitudinal graft-specific changes in immune response were calculated. Response toward non-HLA antigens increased longitudinally, with 41 high prevalence antigens found in ≥50% of the cohort. Co-culturing cardiomyocyte and endothelial cells with patient IgG indicated cell function changes correlated with specific antigens (e.g., Prohibitin). Immunofluorescence staining of patient biopsies revealed that longitudinal increase in antigen expression correlated with non-HLA antibody response. The non-HLA antigens identified in this study elicit acute and chronic graft-specific adaptive immune responses in heart transplant recipients. Antibodies against these non-HLA antigens induced cellular dysfunction by changing expression of their target protein in the donor heart.

The in vitro-directed differentiation of pluripotent stem cells (PSCs) through stimulation of developmental signaling pathways can generate mature somatic cell types for basic laboratory studies or regenerative therapies. However, there has been significant uncertainty regarding a method to separately derive lung versus thyroid epithelial lineages, as these two cell types each originate from Nkx2-1+ foregut progenitors and the minimal pathways claimed to regulate their distinct lineage specification in vivo or in vitro have varied in previous reports. Here, we employ PSCs to identify the key minimal signaling pathways (Wnt+BMP versus BMP+FGF) that regulate distinct lung- versus thyroid-lineage specification, respectively, from foregut endoderm. In contrast to most previous reports, these minimal pathways appear to be evolutionarily conserved between mice and humans, and FGF signaling, although required for thyroid specification, unexpectedly appears to be dispensable for lung specification. Once specified, distinct Nkx2-1+ lung or thyroid progenitor pools can now be independently derived for functional 3D culture maturation, basic developmental studies or future regenerative therapies.

Performance testing is vital for all types of systems and applications, especially in critical fields like Medical, Drug discovery systems and Healthcare, and also for Flight control, defense, Automotive etc. which are mission critical. The testing has to be started in the early stages of the product development cycle for better performance and quality. The performance testing tools tests the system or application rigorously before delivering it to customers and its efficiency builds confidence in users so that they always view the statistical results as accurate and genuine. This paper presents a comparison on the commonly used performance testing tools and concepts, and also the methodologies of testing for a variety of existing and emerging applications is explained.

Effective derivation of functional airway organoids from induced pluripotent stem cells (iPSCs) would provide valuable models of lung disease and facilitate precision therapies for airway disorders such as cystic fibrosis. However, limited understanding of human airway patterning has made this goal challenging. Here, we show that cyclical modulation of the canonical Wnt signaling pathway enables rapid directed differentiation of human iPSCs via an NKX2-1+ progenitor intermediate into functional proximal airway organoids. We find that human NKX2-1+ progenitors have high levels of Wnt activation but respond intrinsically to decreases in Wnt signaling by rapidly patterning into proximal airway lineages at the expense of distal fates. Using this directed approach, we were able to generate cystic fibrosis patient-specific iPSC-derived airway organoids with a defect in forskolin-induced swelling that is rescued by gene editing to correct the disease mutation. Our approach has many potential applications in modeling and drug screening for airway diseases.

The in vitro-directed differentiation of pluripotent stem cells (PSCs) through stimulation of developmental signaling pathways can generate mature somatic cell types for basic laboratory studies or regenerative therapies. However, there has been significant uncertainty regarding a method to separately derive lung versus thyroid epithelial lineages, as these two cell types each originate from Nkx2-1+ foregut progenitors and the minimal pathways claimed to regulate their distinct lineage specification in vivo or in vitro have varied in previous reports. Here, we employ PSCs to identify the key minimal signaling pathways (Wnt+BMP versus BMP+FGF) that regulate distinct lung- versus thyroid-lineage specification, respectively, from foregut endoderm. In contrast to most previous reports, these minimal pathways appear to be evolutionarily conserved between mice and humans, and FGF signaling, although required for thyroid specification, unexpectedly appears to be dispensable for lung specification. Once specified, distinct Nkx2-1+ lung or thyroid progenitor pools can now be independently derived for functional 3D culture maturation, basic developmental studies or future regenerative therapies.

Introduction Alpha-1 antitrypsin (AAT) deficiency (AATD) is the most frequent form of genetically determined emphysema. Type II alveolar cells (AEC2) are capable of producing AAT but little is known about their role in AATD pathogenesis. Aims and objectives We hypothesise that AEC2s play an intrinsic role in AATD pathogenesis and therefore we sought to engineer a novel in vitro model to enable the derivation of lung epithelial cells to define the developmental timing of AAT expression in emerging AEC2s. Methods We targeted 2 fluorescent reporters into the endogenous NKX2.1 and surfactant protein C loci of a human induced pluripotent stem cell (iPSC) line from an individual without AATD. Using a directed differentiation approach that recapitulates embryonic development we generated lung progenitors followed by putative AEC2s. A microarray RNA expression profile was produced and RT-qPCR was used to further quantify mRNA expression. Results Sorted lung progenitors demonstrate significant expression of AAT at day 15 of differentiation and subsequently increase up to day 34. Sorting more mature cells that co-express both reporters demonstrates expression of several AEC2-specific genes and increased expression of AAT mRNA. Flow cytometry for intracellular AAT reveals increased protein expression in AEC2s. Conclusions We demonstrate that differentiation of a novel dual reporter iPSC line can generate early lung progenitors and later AEC2s and that these cells express AAT in significant quantities at both the mRNA and protein levels. We intend to target fluorescent reporters into iPSC from individuals with AATD therefore generating a novel model of disease in AEC2s.

Today's world witnesses increased number of mine accidents caused due to explosion and fire. When the methane gas concentration goes high, it causes fire leading to explosion. In this paper, an IoT based system is proposed to ensure safety to the mine workers in underground collieries. The proposed system consists of DHT-11 sensor to monitor the temperature and humidity of coal mines. When the gas sensor detects high methane gas level, blower is activated so that the atmospheric air can be pumped in from outside to dilute the gas concentration. The smoke sensor is also used to detect the fire. In case of any abnormality in any of these parameters the buzzer sounds. All these parameters are uploaded to the cloud directly so that the people at the control station can be well informed of the underground mines.

Summary Lung alveoli, which are unique to air-breathing organisms, have been challenging to generate from pluripotent stem cells (PSCs) in part because there are limited model systems available to provide the necessary developmental roadmaps for in vitro differentiation. Here we report the generation of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, from human PSCs. Using multicolored fluorescent reporter lines, we track and purify human SFTPC+ alveolar progenitors as they emerge from endodermal precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells form monolayered epithelial "alveolospheres" in 3D cultures without the need for mesenchymal support, exhibit self-renewal capacity, and display additional AEC2 functional capacities. Footprint-free CRISPR-based gene correction of PSCs derived from patients carrying a homozygous surfactant mutation (SFTPB 121ins2 ) restores surfactant processing in AEC2s. Thus, PSC-derived AEC2s provide a platform for disease modeling and future functional regeneration of the distal lung.