Airway disease decreases the therapeutic potential of epithelial stem cells.

BACKGORUND: Tissue-engineered tracheal grafts (TETG) can be recellularized by the host or pre-seeded with host-derived cells. However, the impact of airway disease on the recellularization process is unknown. METHODS: In this study, we determined if airway disease alters the regenerative potential of the human tracheobronchial epithelium (hTBE) obtained by brushing the tracheal mucosa during clinically-indicated bronchoscopy from 48 pediatric and six adult patients. RESULTS: Our findings revealed that basal cell recovery and frequency did not vary by age or region. At passage 1, all samples produced enough cells to cellularize a 3.5 by 0.5 cm2 graft scaffold at low cell density (~ 7000 cells/cm2), and 43.75% could cellularize a scaffold at high cell density (~ 100,000 cells/cm2). At passage 2, all samples produced the number of cells required for both recellularization models. Further evaluation revealed that six pediatric samples (11%) and three (50%) adult samples contained basal cells with a squamous basal phenotype. These cells did not form a polarized epithelium or produce differentiated secretory or ciliated cells. In the pediatric population, the squamous basal cell phenotype was associated with degree of prematurity (< 28 weeks, 64% vs. 13%, p = 0.02), significant pulmonary history (83% vs. 34%, p = 0.02), specifically with bronchopulmonary dysplasia (67% vs. 19%, p = 0.01), and patients who underwent previous tracheostomy (67% vs. 23%, p = 0.03). CONCLUSIONS: In summary, screening high-risk pediatric or adult population based on clinical risk factors and laboratory findings could define appropriate candidates for airway reconstruction with tracheal scaffolds. LEVEL OF EVIDENCE: Level III Cohort study.

Regulation of Human Airway Epithelial Tissue Stem Cell Differentiation by ß-Catenin, P300, and CBP.

The wingless/integrase-1 (WNT)/ß-catenin signaling pathway is active in several chronic lung diseases including idiopathic pulmonary fibrosis, asthma, and chronic obstructive pulmonary disease. Although this WNT/ß-catenin pathway activity is associated with an increase in mucus cell frequency and a decrease in ciliated cell frequency, a cause and consequence relationship between signaling and cell frequency has not been established. We previously demonstrated that genetic stabilization of ß-catenin inhibited differentiation of mouse bronchiolar tissue stem cells (TSC). This study determined the effect of ß-catenin and its co-factors P300 (E1A-binding protein, 300 kDa) and cAMP response element binding (CREB)-binding protein (CBP) on human bronchial epithelial TSC differentiation to mucus and ciliated cells. We developed a modified air-liquid interface (ALI) culture system in which mucus and ciliated cell frequency is similar. These cultures were treated with the ß-catenin agonist CHIR99021 (CHIR) and antagonists to ß-catenin (XAV939), P300 (IQ1), and CBP (ICG001). We report that human TSC differentiation to mucus and ciliated cells can be divided into two stages, specification and commitment. CHIR treatment inhibited mucus and ciliated cell commitment while XAV939 treatment demonstrated that ß-catenin was necessary for mucus and ciliated cell specification. Additional studies demonstrate that a ß-catenin/P300 complex promotes mucus cell specification and that ß-catenin interacts with either P300 or CBP to inhibit ciliated cell commitment. These data indicate that activation of ß-catenin-dependent signaling in chronic lung disease leads to changes in mucus and ciliated cell frequency and that P300 and CBP tune the ß-catenin signal to favor mucus cell differentiation. Stem Cells 2018;36:1905-12.

Thioredoxin Reductase Inhibition Attenuates Neonatal Hyperoxic Lung Injury and Enhances Nuclear Factor E2-Related Factor 2 Activation.

Oxygen toxicity and antioxidant deficiencies contribute to the development of bronchopulmonary dysplasia. Aurothioglucose (ATG) and auranofin potently inhibit thioredoxin reductase-1 (TrxR1), and TrxR1 disruption activates nuclear factor E2-related factor 2 (Nrf2), a regulator of endogenous antioxidant responses. We have shown previously that ATG safely and effectively prevents lung injury in adult murine models, likely via Nrf2-dependent mechanisms. The current studies tested the hypothesis that ATG would attenuate hyperoxia-induced lung developmental deficits in newborn mice. Newborn C3H/HeN mice were treated with a single dose of ATG or saline within 12 hours of birth and were exposed to either room air or hyperoxia (85% O2). In hyperoxia, ATG potently inhibited TrxR1 activity in newborn murine lungs, attenuated decreases in body weight, increased the transcription of Nrf2-regulated genes nicotinamide adenine dinucleotide phosphate reduced quinone oxidoreductase-1 (NQO1) and heme oxygenase 1, and attenuated alterations in alveolar development. To determine the impact of TrxR1 inhibition on Nrf2 activation in vitro, murine alveolar epithelial-12 cells were treated with auranofin, which inhibited TrxR1 activity, enhanced Nrf2 nuclear levels, and increased NQO1 and heme oxygenase 1 transcription. Our novel data indicate that a single injection of the TrxR1 inhibitor ATG attenuates hyperoxia-induced alterations in alveolar development in newborn mice. Furthermore, our data support a model in which the effects of ATG treatment likely involve Nrf2 activation, which is consistent with our findings in other lung injury models. We conclude that TrxR1 represents a novel therapeutic target to prevent oxygen-mediated neonatal lung injury.

Airway Progenitor Clone Formation Is Enhanced by Y-27632-Dependent Changes in the Transcriptome.

The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 10(10) cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.

Type III transforming growth factor beta receptor regulates vascular and osteoblast development during palatogenesis.

BACKGROUND: Cleft palate occurs in up to 1:1,000 live births and is associated with mutations in multiple genes. Palatogenesis involves a complex choreography of palatal shelf elongation, elevation, and fusion. Transforming growth factor ß (TGFß) and bone morphogenetic protein 2 (BMP2) canonical signaling is required during each stage of palate development. The type III TGFß receptor (TGFßR3) binds all three TGFß ligands and BMP2, but its contribution to palatogenesis is unknown. RESULTS: The role of TGFßR3 during palate formation was found to be during palatal shelf elongation and elevation. Tgfbr3(-) (/) (-) embryos displayed reduced palatal shelf width and height, changes in proliferation and apoptosis, and reduced vascular and osteoblast differentiation. Abnormal vascular plexus organization as well as aberrant expression of arterial (Notch1, Alk1), venous (EphB4), and lymphatic (Lyve1) markers was also observed. Decreased osteoblast differentiation factors (Runx2, alk phos, osteocalcin, col1A1, and col1A2) demonstrated poor mesenchymal cell commitment to the osteoblast lineage within the maxilla and palatal shelves in Tgfbr3(-) (/) (-) embryos. Additionally, in vitro bone mineralization induced by osteogenic medium (OM+BMP2) was insufficient in Tgfbr3(-) (/) (-) palatal mesenchyme, but mineralization was rescued by overexpression of TGFßR3. CONCLUSIONS: These data reveal a critical, previously unrecognized role for TGFßR3 in vascular and osteoblast development during palatogenesis.

Cranial neural crest deletion of VEGFa causes cleft palate with aberrant vascular and bone development.

Cleft palate is among the most common craniofacial congenital anomalies. Up to 30% of patients with cleft palate also have associated cardiac and vascular defects. VEGFa, a critical growth factor involved in multiple developmental processes including angiogenesis and ossification, is also required for palate development. Conditional deletion of VEGFa in cranial neural crest (CNC) cells using Wnt1-Cre (VEGFaCKO) resulted in cleft palate in mice. The phenotype included reduced proliferation of cells within the palatal shelves, abnormal palatal shelf elongation and elevation, and the inability to undergo fusion. Vascularization of the VEGFaCKO palatal shelves was greatly reduced, suggesting a non-cell autonomous role of VEGFa signaling from the CNC-derived cells to the endothelium during vessel formation. Defective vascular development was coupled with deficient intramembranous ossification of maxillary and palatal mesenchyme. In vitro assessment of CNC-derived palatal mesenchymal cells from VEGFaCKO mice demonstrated normal ossification after BMP2 stimulation, suggesting that inadequate expression of Bmp2 in VEGFaCKO mice was, in part, responsible for reduced ossification. Taken together, these data demonstrate that VEGFa produced in the CNC-derived mesenchyme drives proliferation, vascularization, and ossification, all of which are critical for palate development.

Implementing a Critical Thinking Tool to Evaluate Educational Needs for Inpatient Rehabilitation Nurses.

ABSTRACT: BACKGROUND: Most critical thinking assessment tools are resource intensive and require significant time and money to administer. Moreover, these tools are not tailored to evaluate critical thinking skills among inpatient rehabilitation facility (IRF) nurses. This pilot study explores the efficacy of using short videos to evaluate critical thinking for nurses working in an IRF. METHODS: We developed and filmed 3 clinical scenarios representative of common IRF events that require critical thinking on behalf of the nurse. Thirty-one IRF nurses participated in the study and independently scored their own critical thinking skills using a visual analog scale. Using the same scale, nurse managers and assistant managers who worked closely with the nurses also rated the critical thinking ability of each nurse. The nurse then viewed and responded in narrative form to each of the 3 videos. A scoring rubric was used to independently evaluate the critical thinking skills for each nurse based on the nurses' responses. RESULTS: Nurses rated their own critical thinking skills higher than mangers rated them (m = 85.23 vs 62.89). There was high interrater reliability for scoring video 1k (0.65), video 2k (0.90), and video 3k (0.84). CONCLUSION: The results demonstrate efficacy for further study of low-cost alternatives to evaluate critical thinking among neuroscience nurses providing IRF care.

The cytoplasmic domain of TGFßR3 through its interaction with the scaffolding protein, GIPC, directs epicardial cell behavior.

The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor ß Receptor (TGFßR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFßR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFßR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially derived cell invasion in Tgfbr3(-/-) hearts. Tgfbr3(-/-) epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFß1 and TGFß2. Unexpectedly, loss of TGFßR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFßR3 in Tgfbr3(-/-) cells rescued deficits in invasion in vitro in response TGFß1 and TGFß2 as well as FGF2 and HMW-HA. Expression of TGFßR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3(-/-) cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3(+/+) cells decreased invasion in response to TGFß2, FGF2, and HMW-HA. We conclude that TGFßR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3(-/-) mice.

Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate in mucosecretory lung disease.

Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. Goblet and ciliated cell types are derived from tracheobronchial stem/progenitor cells via a Notch-dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and JAG2 is unclear. This study examined JAG1 and JAG2 function using human air-liquid-interface cultures that were treated with γ-secretase complex (GSC) inhibitors, neutralizing peptides/antibodies, or WNT/ß-catenin pathway antagonists/agonists. These experiments revealed that JAG1 and JAG2 regulated cell fate determination in the tracheobronchial epithelium; however, their roles did not adhere to simple necessity and sufficiency rules. Biochemical studies indicated that JAG1 and JAG2 underwent posttranslational modifications that resulted in generation of a JAG1 C-terminal peptide and regulated the abundance of full-length JAG2 on the cell surface. GSC and glycogen synthase kinase 3 were implicated in these posttranslational events, but WNT agonist/antagonist studies and RNA-Seq indicated a WNT-independent mechanism. Collectively, these data suggest that posttranslational modifications create distinct assemblies of JAG1 and JAG2, which regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

Assessment of Beta-2 Microglobulin Gene Edited Airway Epithelial Stem Cells as a treatment for Sulfur Mustard Inhalation.

Respiratory system damage is the primary cause of mortality in individuals who are exposed to vesicating agents including sulfur mustard (SM). Despite these devastating health complications, there are no fielded therapeutics that are specific for such injuries. Previous studies reported that SM inhalation depleted the tracheobronchial airway epithelial stem cell (TSC) pool and supported the hypothesis, TSC replacement will restore airway epithelial integrity and improve health outcomes for SM-exposed individuals. TSC express Major Histocompatibility Complex (MHC-I) transplantation antigens which increases the chance that allogeneic TSC will be rejected by the patient's immune system. However, previous studies reported that Beta-2 microglobulin (B2M) knockout cells lacked cell surface MHC-I and suggested that B2M knockout TSC would be tolerated as an allogeneic graft. This study used a Cas9 ribonucleoprotein (RNP) to generate B2M-knockout TSC, which are termed Universal Donor Stem Cells (UDSC). Whole genome sequencing identified few off-target modifications and demonstrated the specificity of the RNP approach. Functional assays demonstrated that UDSC retained their ability to self-renew and undergo multilineage differentiation. A preclinical model of SM inhalation was used to test UDSC efficacy and identify any treatment-associated adverse events. Adult male Sprague-Dawley rats were administered an inhaled dose of 0.8 mg/kg SM vapor which is the inhaled LD50 on day 28 post-challenge. On recovery day 2, vehicle or allogeneic Fisher rat UDSC were delivered intravenously (n = 30/group). Clinical parameters were recorded daily, and planned euthanasia occurred on post-challenge days 7, 14, and 28. The vehicle and UDSC treatment groups exhibited similar outcomes including survival and a lack of adverse events. These studies establish a baseline which can be used to further develop UDSC as a treatment for SM-induced airway disease.

BMP-2 and TGFß2 shared pathways regulate endocardial cell transformation.

Valvular heart disease is a major cause of mortality and morbidity. Revealing the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. A key step in valve formation during heart development is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endocardial cells in the atrioventricular cushion (AVC). The type III transforming growth factor-ß receptor (TGFßR3) regulates AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Little is known concerning the signaling mechanisms downstream of TGFßR3. Here we use endocardial cell EMT in vitro to determine the role of 2 well-characterized downstream TGFß signaling pathways in TGFßR3-dependent endocardial cell EMT. Targeting of Smad4, the common mediator Smad, demonstrated that Smad signaling is required for EMT in the AVC and TGFßR3-dependent EMT stimulated by TGFß2 or BMP-2. Although we show that Smads 1, 2, 3, and 5 are required for AVC EMT, overexpression of Smad1 or Smad3 is not sufficient to induce EMT. Consistent with the activation of the Par6/Smurf1 pathway downstream of TGFßR3, targeting ALK5, Par6, or Smurf1 significantly inhibited EMT in response to either TGFß2 or BMP-2. The requirement for ALK5 activity, Par6, and Smurf1 for TGFßR3-dependent endocardial cell EMT is consistent with the documented role of this pathway in the dissolution of tight junctions. Taken together, our data demonstrate that TGFßR3-dependent endocardial cell EMT stimulated by either TGFß2 or BMP-2 requires Smad4 and the activation of the Par6/Smurf1 pathway.

Implementation of the MATRIX Staffing Grid Improves Nurse Satisfaction With Rehabilitation Unit Staffing.

ABSTRACT: BACKGROUND: Information on nurse satisfaction and unit acuity is scarce in the literature. The purpose of this study is to evaluate the effect of the MATRIX Staffing Grid (MSG) on nurse assignment satisfaction in a 20-bed inpatient rehabilitation facility. METHODS: Prospective systematic implementation study of the MSG occurred in 5 phases: development, baseline, run-in, implementation, and sustainability. Pretest/posttest nursing satisfaction data were analyzed using Wilcoxon-Mann-Whitney tests. RESULTS: Analysis of 128 satisfaction surveys demonstrated that the median total satisfaction score increased by 35% after MSG implementation (P < .05), with no change in patient satisfaction or adverse event rates. CONCLUSION: A systematic approach to implementation of the MSG evidence-based practice significantly improved nursing satisfaction with patient assignment in a way that addressed specific needs. The MSG has now been adopted into practice at our institution. The MSG may be feasible for implementation in inpatient rehabilitation units to improve staffing satisfaction.

Repeated injury promotes tracheobronchial tissue stem cell attrition.

Chronic lung disease has been attributed to stem cell aging and/or exhaustion. We investigated these mechanisms using mouse and human tracheobronchial tissue-specific stem cells (TSC). In mouse, chromatin labeling and flow cytometry demonstrated that naphthalene (NA) injury activated a subset of TSC. These activated TSC continued to proliferate after the epithelium was repaired and a clone study demonstrated that ~96% of activated TSC underwent terminal differentiation. Despite TSC attrition, epithelial repair after a second NA injury was normal. The second injury accelerated proliferation of previously activated TSC and a nucleotide-label retention study indicated that the second injury recruited TSC that were quiescent during the first injury. These mouse studies indicate that (a) injury causes selective activation of the TSC pool; (b) activated TSC are predisposed to further proliferation; and (c) the activated state leads to terminal differentiation. In human TSC, repeated proliferation also led to terminal differentiation and depleted the TSC pool. A clone study identified long- and short-lived TSC and showed that short-lived TSC clones had significantly shorter telomeres than their long-lived counterparts. The TSC pool was significantly depleted in dyskeratosis congenita donors, who harbor mutations in telomere biology genes. The remaining TSC had short telomeres and short lifespans. Collectively, the mouse and human studies support a model in which epithelial injury increases the biological age of the responding TSC. When applied to chronic lung disease, this model suggests that repeated injury accelerates the biological aging process resulting in abnormal repair and disease initiation.

Airway epithelial stem cell chimerism in cystic fibrosis lung transplant recipients.

BACKGROUND: The conducting airway epithelium is repaired by tissue specific stem cells (TSC). In response to mild/moderate injury, each TSC repairs a discrete area of the epithelium. In contrast, severe epithelial injury stimulates TSC migration and expands the stem cell's reparative domain. Lung transplantation (LTx) can cause a moderate/severe airway injury and the remodeled airway contains a chimeric mixture of donor and recipient cells. These studies supported the hypothesis, LTx stimulates TSC migration resulting in epithelial chimerism. We tested this hypothesis in cystic fibrosis (CF) LTx patients. METHODS: Airway mucosal injury was quantified using bronchoscopic imaging and a novel grading system. Bronchial brushing was used to recover TSC from 10 sites in the recipient and allograft airways. TSC chimerism was quantified by short tandem repeat analysis. TSC self-renewal and differentiation potential were assayed using the clone forming cell frequency and air-liquid-interface methods. Electrophysiology was used to determine if TSC chimerism altered epithelial ion channel activity. RESULTS: LTx caused a mild to moderate airway mucosal injury. Donor and recipient TSC were identified in 91% of anastomotic sites and 93% of bronchial airways. TSC chimerism did not alter stem cell self-renewal or differentiation potential. The frequency of recipient TSC was proportional to CF Transmembrane Conductance Regulator (CFTR)-dependent ion channel activity and 33% of allograft regions were at risk for abnormal CFTR activity. CONCLUSIONS: LTx in CF patients stimulates bidirectional TSC migration across the anastomoses. TSC chimerism may alter ion homeostasis and compromise the host defense capability of the allograft airway epithelium.

Testing the test: Are exams measuring understanding?

Grades in undergraduate science, technology, engineering, and mathematics (STEM) courses are distributed under the assumption that high-performing students have a strong understanding of the material. Similarly, the STEM education literature often presents exam performance as equivalent to understanding. Despite these assumptions, we have little knowledge regarding student thinking in relation to exam scores. To investigate this relationship, we asked undergraduate students to complete a series of written and verbal tasks. Twenty-two participants were presented with biology questions and were instructed to write exam-like responses along with their thought process. More than half of the participants then took part in retrospective interviews. We graded the exam-like responses to award a performance score and coded the entirety of participants' writing for their understanding. We found a discrepancy between performance and understanding for over one quarter of our data. Furthermore, interviews allowed for a more complete picture of participant understanding than written responses. These results contribute to calls for re-evaluating our course assessments and for questioning the understanding those assessments value. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):296-302, 2019.

Protein Abundance Determination: An Optimized Western Blot Workflow.

We report that the quantitative western blot (qWB) analysis requires a target protein-specific approach, and we provide a workflow that streamlines development of this process. First, the optimal primary antibody dilution is determined. Blots containing 15 µg total protein per lane are probed with the primary antibody at three concentrations and a secondary antibody concentration that is defined by the manufacturer. The lowest primary antibody concentration that detects a discrete band at the correct molecular weight is used in the remaining two steps. Secondly, the optimal protein load is determined. Blots containing 3.75 to 60 µg protein per lane are probed using the antibody concentrations defined in step 1. A target protein band intensity vs. protein load plot is used to determine the linear dynamic range (LDR) for the target protein. The midpoint of the LDR is defined as the optimal protein load. Finally, an appropriate loading control (LC) is identified. We found that the LDR for ß-actin, a commonly used LC, exhibited a narrow range, 3.75 to 15 µg. In contrast, the total protein assessed by a Ponceau staining method exhibited a broader LDR, 3.75 to 60 µg. Thus, the total protein is used as a LC. We conclude that the sensitivity and accuracy of the qWB method is dependent on the use of an optimal: 1) primary antibody dilution; 2) total protein load; 3) and LC. Our workflow simplifies the identification of these values.

Electrospun scaffolds limit the regenerative potential of the airway epithelium.

OBJECTIVE: Significant morbidity and mortality are associated with clinical use of synthetic tissue-engineered tracheal grafts (TETG). Our previous work focused on an electrospun polyethylene terephthalate and polyurethane (PET/PU) TETG that was tested in sheep using a long-segment tracheal defect model. We reported that graft stenosis and limited epithelialization contributed to graft failure. The present study determined if the epithelialization defect could be attributed to: 1) postsurgical depletion of native airway basal stem/progenitor cells; 2) an inability of the PET/PU-TETG to support epithelial migration; or 3) compromised basal stem/progenitor cell proliferation within the PET/PU environment. STUDY DESIGN: Experimental. METHODS: Basal stem/progenitor cell frequency in sheep that underwent TETG implantation was determined using the clone-forming cell frequency (CFCF) method. A novel migration model that mimics epithelial migration toward an acellular scaffold was developed and used to compare epithelial migration toward a control polyester scaffold and the PET/PU scaffold. Basal stem/progenitor cell proliferation within the PET/PU scaffold was evaluated using the CFCF assay, doubling-time analysis, and mitotic cell quantification. RESULTS: We report that TETG implantation did not decrease basal stem/progenitor cell frequency. In contrast, we find that epithelial migration toward the PET/PU scaffold was significantly less extensive than migration toward a polyester scaffold and that the PET/PU scaffold did not support basal stem/progenitor cell proliferation. CONCLUSIONS: We conclude that epithelialization of a PET/PU scaffold is compromised by poor migration of native tissue-derived epithelial cells and by a lack of basal stem/progenitor cell proliferation within the scaffold. LEVEL OF EVIDENCE: NA.

Cell Therapy for Cystic Fibrosis Lung Disease: Regenerative Basal Cell Amplification.

The human airway epithelium is regenerated by basal cells. Thus, basal cell therapy has the potential to cure cystic fibrosis (CF) lung disease. We previously reported that the human basal cells repopulated the mouse airway epithelium after transplantation, and we estimated that 60 million cells would be needed to treat a human patient. To further develop cell therapy, we compared the proliferation potential of non-CF and CF tissue-derived bronchial basal cells. Three methods were used: regenerative cell frequency, burst size, and cell division frequency. Second, we used a serial passage strategy to determine if CF basal cells could be amplified to the estimated therapeutic dose. These studies evaluated that tissue-derived bronchial basal cells and the basal cells that were recovered by brushing bronchial airways or the nasal respiratory epithelium. Finally, we used the limiting dilution method to isolate non-CF and CF basal cell clones. The proliferation assays and the air-liquid-interface differentiation method were used to determine if cell amplification altered the proliferation and/or differentiation potential of clonal isolates. We demonstrate that: (a) non-CF and CF basal cell proliferation is similar, (b) CF basal cells can be amplified to a therapeutic cell dose, and (c) amplified non-CF and CF basal cell clones differentiate normally. Despite these encouraging findings, we also find that the cell amplification process depletes the regenerative basal cell pool. Analysis of basal cell clones indicates that serial passage selects for long-lived basal cells and raise the possibility that prospective isolation of these stem-like cells will improve the efficacy of cell replacement therapy. Stem Cells Translational Medicine 2019;8:225&235.

Teaching Assistant Attention and Responsiveness to Student Reasoning in Written Work.

Instructors communicate what they value about students' written work through their comments and feedback, and this feedback has the potential to direct how students approach writing assignments. In this study, we examined how graduate student teaching assistants (TAs) attended and responded to students' written lab reports in an introductory biology course. We collected and analyzed marked lab reports from five TAs and interviewed them about their marking decisions. The results show that TAs attended mainly to writing style and form in their markings and comments on lab reports. However, there were occasions when they attended to students' scientific reasoning in their markings and during interviews. We provide evidence that TAs' understanding of the purpose of the laboratory course and assessment structure influenced their attention. We also provide evidence that TAs could shift their attention from style and form to reasoning in response to moment-to-moment contextual cues. Building on these results, we discuss course design and professional development that reframes labs and reports to focus on students' biological reasoning.

The wound healing capacity of undifferentiated and differentiated airway epithelial cells in vitro.

INTRODUCTION: Congenital or acquired tracheal lesions alter airway epithelial structure and can lead to long-segment tracheal defects. Tissue engineered tracheal grafts (TETG) have the potential to cure such defects; however, clinical applications have been plagued with numerous complications including delayed graft epithelialization. The knowledge that epithelial cells migrate from native tissue to the TETG raises the possibility that TETG performance can be improved by increasing the rate of epithelialization. OBJECTIVES: We developed a model that can be used quantify epithelial migration in clinically-relevant conditions. METHODS: Existing histological analyses determined the differentiation status of the normal and injured human tracheal epithelium and were used to identify in vitro culture conditions that mimic these parameters. The classical scratch assay was adapted to permit analysis of migratory velocity as a function of differentiation status. Migration of undifferentiated (UD), partially-differentiated (PD), and well-differentiated (WD) epithelia was quantified. RESULTS: The normal and injured epithelium can be modeled using human cells that are cultured using a modified air-liquid-interface culture system. PD cell cultures are similar to the remodeled epithelium; whereas; WD cultures are similar to the normal epithelium. Preliminary results indicate that PD cells migrate more rapidly than WD cells and that PD and WD cells migrate more rapidly than UD cells. CONCLUSION: Pending verification of these results, we suggest that epithelial migration is significantly altered by differentiation status. Thus, efforts to improve TETG epithelialization should use model systems that faithfully-represent the differentiation state of the native tissue.