Diversity, Equity, and Inclusion in the Pediatric Pulmonary Workforce: An Official American Thoracic Society Workshop Report.

Despite growing recognition of the need for increased diversity among students, trainees, and faculty in health care, the medical workforce still lacks adequate representation from groups historically underrepresented in medicine (URiM). The subspecialty field of pediatric pulmonology is no exception. Although there have been efforts to address issues of diversity, equity, and inclusion (DEI) in our own field, gaps persist. To address these gaps, the members of the Diversity, Equity, and Inclusion Advisory Group (DEI-AG) of the American Thoracic Society Pediatrics Assembly created and distributed a Needs Assessment Survey in the United States and Canada to better understand the racial and ethnic demographics of the pediatric pulmonary workforce and to learn more about successes, gaps, and opportunities to enhance how we recruit, train, and retain a diverse workforce. The DEI-AG leadership cochairs convened a workshop to review the findings of the DEI Needs Assessment Survey and to develop strategies to improve the recruitment and retention of URiM fellows and faculty. This Official ATS Workshop Report aims to identify barriers and opportunities for recruitment, training, and career development within the field of pediatric pulmonology. Additionally, we offer useful strategies and resources to improve the recruitment of URiM residents, the mentorship of trainees and junior faculty, and the career development of URiM faculty in academic centers. This Workshop Report is an important first deliverable by the DEI-AG. We hope that this work, originating from within the Pediatrics Assembly, will serve as a model for other Assemblies, disciplines across the ATS, and other fields in Pediatrics.

An unwitnessed case of foreign body aspiration of barium from an unknown source.

Foreign body (FB) aspiration is potentially life-threatening in children. A variety of sources and objects have been noted in aspiration events with possible complications ranging from mild to life-threatening. While rare, barium aspiration can cause severe complications, and removal is particularly challenging. Complications of retained barium include acute respiratory distress syndrome, pneumonitis, sepsis, even death. Regardless of the foreign body's identity, substance removal is critical in management. Resourcefulness of removal techniques and an interdisciplinary approach may allow for maximally effective management. We present a case of a pediatric barium aspiration from an unknown source and review evaluation and management strategies.

Correction: Policy threats to maternal and child nutrition: putting the unborn child at a lifelong disadvantage.

Following publication of this article the authors noticed that affiliations were incorrectly assigned. The original article has now been updated so that the author "Joyce R. Javier" is associated with the "Children's Hospital Los Angeles, University of Southern California Keck School of Medicine, Department of Pediatrics, Division of General Pediatrics, Los Angeles, CA, USA", and the author "Vivek Balasubramaniam" is associated with the "Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, USA". This has been corrected in both the PDF and HTML versions of the article.

Intraperitoneal injection of MSC-derived exosomes prevent experimental bronchopulmonary dysplasia.

Mesenchymal stromal cell (MSC) derived exosomes mediate tissue protection and regeneration in many injuries and diseases by modulating cell protein production, protecting from apoptosis, inhibiting inflammation, and increasing angiogenesis. In the present study, daily intraperitoneal injection of MSC-derived exosomes protected alveolarization and angiogenesis in a newborn rat model of bronchopulmonary dysplasia (BPD) induced by 14 days of neonatal hyperoxia exposure (85% O2). Exosome treatment during hyperoxia prevented disruption of alveolar growth, increased small blood vessel number, and inhibited right heart hypertrophy at P14, P21, and P56. In vitro, exosomes significantly increased tube-like network formation by HUVEC, in part through a VEGF mediated mechanism. In summary, daily intraperitoneal injection of exosomes increased blood vessel number and size in the lung through pro-angiogenic mechanisms. MSC-derived exosomes therefore have both anti-inflammatory and pro-angiogenic mechanism to protect the lung from hyperoxia induced lung and heart disease associated with BPD.

Epithelial-mesenchymal crosstalk influences cellular behavior in a 3D alveolus-fibroblast model system.

Interactions between lung epithelium and interstitial fibroblasts are increasingly recognized as playing a major role in the progression of several lung pathologies, including cancer. Three-dimensional in vitro co-culture systems offer tissue-relevant platforms to study the signaling interplay between diseased and healthy cell types. Such systems provide a controlled environment in which to probe the mechanisms involved in epithelial-mesenchymal crosstalk. To recapitulate the native alveolar tissue architecture, we employed a cyst templating technique to culture alveolar epithelial cells on photodegradable microspheres and subsequently encapsulated the cell-laden spheres within poly (ethylene glycol) (PEG) hydrogels containing dispersed pulmonary fibroblasts. A fibroblast cell line (CCL-210) was co-cultured with either healthy mouse alveolar epithelial primary cells or a cancerous alveolar epithelial cell line (A549) to probe the influence of tumor-stromal interactions on proliferation, migration, and matrix remodeling. In 3D co-culture, cancerous epithelial cells and fibroblasts had higher proliferation rates. When examining fibroblast motility, the fibroblasts migrated faster when co-cultured with cancerous A549 cells. Finally, a fluorescent peptide reporter for matrix metalloproteinase (MMP) activity revealed increased MMP activity when A549s and fibroblasts were co-cultured. When MMP activity was inhibited or when cells were cultured in gels with a non-degradable crosslinker, fibroblast migration was dramatically suppressed, and the increase in cancer cell proliferation in co-culture was abrogated. Together, this evidence supports the idea that there is an exchange between the alveolar epithelium and surrounding fibroblasts during cancer progression that depends on MMP activity and points to potential signaling routes that merit further investigation to determine targets for cancer treatment.

Umbilical Cord Blood Circulating Progenitor Cells and Endothelial Colony-Forming Cells Are Decreased in Preeclampsia.

Preeclampsia (PE) is a pregnancy-specific disease characterized by the new onset of hypertension and proteinuria. Mothers with PE are known to develop endothelial dysfunction, but its effect on infants has been understudied, as newborns are often asymptomatic. Recent studies indicate that infants born from preeclamptic pregnancies develop endothelial dysfunction including higher blood pressure during childhood and an increased risk of stroke later in life. We hypothesize that PE reduces the number and function of fetal angiogenic progenitor cells and may contribute to this increased risk. We quantified 2 distinct types of angiogenic progenitors, pro-angiogenic circulating progenitor cells (CPCs) and endothelial colony-forming cells (ECFCs), from the umbilical cord blood of preeclamptic pregnancies and normotensive controls. Pro-angiogenic and nonangiogenic CPCs were enumerated via flow cytometry and ECFCs by cell culture. Additionally, we studied the growth, migration, and tube formation of ECFCs from PE and gestational age-matched normotensive control pregnancies. We found that PE resulted in decreased cord blood pro-angiogenic CPCs and ECFCs. Nonangiogenic CPCs were also decreased. Preeclamptic ECFCs demonstrated decreased growth and migration but formed tube-like structures in vitro similar to controls. Our results suggest that the preeclamptic environment alters the number and function of angiogenic progenitor cells and may increase the risk of later vascular disease.

Protective Polymer Coatings for High-Throughput, High-Purity Cellular Isolation.

Cell-based therapies are emerging as the next frontier of medicine, offering a plausible path forward in the treatment of many devastating diseases. Critically, current methods for antigen positive cell sorting lack a high throughput method for delivering ultrahigh purity populations, prohibiting the application of some cell-based therapies to widespread diseases. Here we show the first use of targeted, protective polymer coatings on cells for the high speed enrichment of cells. Individual, antigen-positive cells are coated with a biocompatible hydrogel which protects the cells from a surfactant solution, while uncoated cells are immediately lysed. After lysis, the polymer coating is removed through orthogonal photochemistry, and the isolate has >50% yield of viable cells and these cells proliferate at rates comparable to control cells. Minority cell populations are enriched from erythrocyte-depleted blood to >99% purity, whereas the entire batch process requires 1 h and <$2000 in equipment. Batch scale-up is only contingent on irradiation area for the coating photopolymerization, as surfactant-based lysis can be easily achieved on any scale.

In vitro model alveoli from photodegradable microsphere templates.

Recreating the 3D cyst-like architecture of the alveolar epithelium in vitro has been challenging to achieve in a controlled fashion with primary lung epithelial cells. Here, we demonstrate model alveoli formed within a tunable synthetic biomaterial platform using photodegradable microspheres as templates to create physiologically relevant, cyst structures. Poly(ethylene glycol) (PEG)-based hydrogels were polymerized in suspension to form microspheres on the order of 120 µm in diameter. The gel chemistry was designed to allow erosion of the microspheres with cytocompatible light doses (≤15 min exposure to 10 mW cm(-2) of 365 nm light) via cleavage of a photolabile nitrobenzyl ether crosslinker. Epithelial cells were incubated with intact microspheres, modified with adhesive peptide sequences to facilitate cellular attachment to and proliferation on the surface. A tumor-derived alveolar epithelial cell line, A549, completely covered the microspheres after only 24 hours, whereas primary mouse alveolar epithelial type II (ATII) cells took ∼3 days. The cell-laden microsphere structures were embedded within a second hydrogel formulation at user defined densities; the microsphere templates were subsequently removed with light to render hollow epithelial cysts that were cultured for an additional 6 days. The resulting primary cysts stained positive for cell-cell junction proteins (ß-catenin and ZO-1), indicating the formation of a functional epithelial layer. Typically, primary ATII cells differentiated in culture to the alveolar epithelial type I (ATI) phenotype; however, each cyst contained ∼1-5 cells that stained positive for an ATII marker (surfactant protein C), which is consistent with ATII cell numbers in native mouse alveoli. This biomaterial-templated alveoli culture system should be useful for future experiments to study lung development and disease progression, and is ideally suited for co-culture experiments where pulmonary fibroblasts or endothelial cells could be presented in the hydrogel surrounding the epithelial cysts.

Interfacial polymerization for colorimetric labeling of protein expression in cells.

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

High proliferative potential endothelial colony-forming cells contribute to hypoxia-induced pulmonary artery vasa vasorum neovascularization.

Angiogenic expansion of the vasa vasorum (VV) is an important contributor to pulmonary vascular remodeling in the pathogenesis of pulmonary hypertension (PH). High proliferative potential endothelial progenitor-like cells have been described in vascular remodeling and angiogenesis in both systemic and pulmonary circulations. However, their role in hypoxia-induced pulmonary artery (PA) VV expansion in PH is not known. We hypothesized that profound PA VV neovascularization observed in a neonatal calf model of hypoxia-induced PH is due to increased numbers of subsets of high proliferative cells within the PA adventitial VV endothelial cells (VVEC). Using a single cell clonogenic assay, we found that high proliferative potential colony-forming cells (HPP-CFC) comprise a markedly higher percentage in VVEC populations isolated from the PA of hypoxic (VVEC-Hx) compared with control (VVEC-Co) calves. VVEC-Hx populations that comprised higher numbers of HPP-CFC also demonstrated markedly higher expression levels of CD31, CD105, and c-kit than VVEC-Co. In addition, significantly higher expression of CD31, CD105, and c-kit was observed in HPP-CFC vs. the VVEC of the control but not of hypoxic animals. HPP-CFC exhibited migratory and tube formation capabilities, two important attributes of angiogenic phenotype. Furthermore, HPP-CFC-Co and some HPP-CFC-Hx exhibited elevated telomerase activity, consistent with their high replicative potential, whereas a number of HPP-CFC-Hx exhibited impaired telomerase activity, suggestive of their senescence state. In conclusion, our data suggest that hypoxia-induced VV expansion involves an emergence of HPP-CFC populations of a distinct phenotype with increased angiogenic capabilities. These cells may serve as a potential target for regulating VVEC neovascularization.

Cord blood endothelial colony-forming cells from newborns with congenital diaphragmatic hernia.

Endothelial colony-forming cells (ECFCs) are decreased in the cord blood of preterm infants with moderate-to-severe bronchopulmonary dysplasia. We quantified ECFCs from infants with congenital diaphragmatic hernia, a neonatal disorder with severe lung hypoplasia. Unlike newborns who develop bronchopulmonary dysplasia, those with congenital diaphragmatic hernia had increased and highly-proliferative cord blood ECFCs.

Endothelial colony-forming cell conditioned media promote angiogenesis in vitro and prevent pulmonary hypertension in experimental bronchopulmonary dysplasia.

Late-outgrowth endothelial colony-forming cells (ECFCs), a type of circulating endothelial progenitor cell (EPC), may contribute to pulmonary angiogenesis during development. Cord blood ECFCs from preterm newborns proliferate more rapidly than term ECFCs but are more susceptible to the adverse effects of hyperoxia. Recent studies suggest that bone marrow-derived EPCs protect against experimental lung injury via paracrine mechanisms independent of vascular engraftment. To determine whether human umbilical cord blood ECFCs from preterm and term newborns have therapeutic benefit in experimental neonatal lung injury, we isolated cord blood ECFCs from full-term and preterm newborns and prepared ECFC-conditioned medium (CM) to test its therapeutic benefit on fetal pulmonary artery endothelial cell (PAEC) proliferation and function as well as alveolar type 2 (AT2) cell growth. PAECs and AT2 cells were isolated from late-gestation fetal sheep. Additionally, we administered both ECFCs and ECFC-CM to bleomycin-exposed newborn rats, an experimental model of bronchopulmonary dysplasia (BPD). Both term ECFC-CM and preterm ECFC-CM promoted cell growth and angiogenesis in vitro. However, when ECFC-CM was collected during exposure to mild hyperoxia, the benefit of preterm ECFC-CM was no longer observed. In the bleomycin model of BPD, treatment with ECFC-CM (or CM from mature EC) effectively decreased right ventricular hypertrophy but had no effect on alveolar septation. We conclude that term ECFC-CM is beneficial both in vitro and in experimental BPD. During oxidative stress, preterm ECFC-CM, but not term ECFC-CM, loses its benefit. The inability of term ECFC-CM to promote alveolarization may limit its therapeutic potential.