Embryonic Stem Cells Cultured in Microfluidic Chambers Take Control of Their Fate by Producing Endogenous Signals Including LIF.

It is important to understand the role played by endogenous signals in shaping stem cell fate decisions to develop better culture systems and to improve understanding of development processes. In this study, we describe the behavior of mouse embryonic stem cells (mESCs) inside microfluidic chambers (microchambers) operated under conditions of minimal perfusion. mESCs inside microchambers formed colonies and expressed markers of pluripotency in the absence of feeders or pluripotency-inducing signals such as leukemia inhibitory factor (LIF), while mESCs in standard cultureware differentiated rapidly. In a series of experiments, we demonstrate that remarkable differences in stem cell phenotype are due to endogenous production of LIF and other growth factors brought upon by cultivation in confines of a microchamber in the absence of perfusion (dilution). At the protein level, mESCs produced ∼140 times more LIF inside microchambers than under standard culture conditions. In addition, we demonstrate that pluripotent phenotype of stem cells could be degraded by increasing the height (volume) of the microchamber. Furthermore, we show that inhibition of LIF in microchambers, via the JAK/STAT3 pathway, leads to preferential differentiation into mesoderm that is driven by bone morphogenetic protein (BMP)-4. Collectively, we demonstrate for the first time that it is possible to design a cell culture system where stem cell fate is controlled solely by the endogenous signals. Our study may help shift the paradigm of stem cell cultivation away from relying on expensive exogenous molecules such as growth factors and toward designing culture chambers for harnessing endogenous signals. Stem Cells 2016;34:1501-1512.

Growth Factor-Bearing Polymer Brushes--Versatile Bioactive Substrates Influencing Cell Response.

In this study we present the development of responsive nanoscale substrates exhibiting cell-guiding properties based on incorporated bioactive signaling cues. The investigative approach considered the effect of two different surface-bound growth factors (GFs) on cell behavior and response: hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF). Two surface biofunctionalization strategies were explored in order to conceive versatile, bioactive thin polymer brush films. Polymer brushes made of tethered poly(acrylic)acid (PAA) polymer layers with a high grafting density of polymer chains were biofunctionalized with GFs either by physisorption or chemisorption. Both GFs showed high binding efficiencies to PAA brushes based on their initial loading concentrations. The GF release kinetics can be distinguished depending on the applied biofunctionalization method. Specifically, a high initial burst followed by a constant slow release was observed in the case of both physisorbed HGF and bFGF. In contrast, the release kinetics of chemisorbed GFs were quite different. Remarkably, chemisorbed HGF remained bound to the brush surface for over 1 week, whereas 50% of chemisorbed bFGF was released slowly. Furthermore, the effect of these GF-biofunctionalized PAA brushes on different cells was investigated. A human hepatoma cell line (HepG2) was used to analyze the bioactivity of HGF-modified PAA brushes by measuring cell growth inhibition and scattering effects. Additionally, the differentiation of mouse embryonic stem cells (mESCs) toward endoderm was studied on bFGF-modified PAA brush surfaces. Finally, the results illustrate that PAA brushes, particularly those biofunctionalized with chemisorbed GFs, produce an expected measurable effect on both cell types. Therefore, PAA polymer brushes biofunctionalized with GFs can be used as bioactive cell culture substrates with tuned efficiency.

Electrochemical release of hepatocyte-on-hydrogel microstructures from ITO substrates.

This paper describes a novel platform that utilizes micropatterning and electrochemistry to release cells-on-hydrogel microstructures from conductive indium tin oxide (ITO) substrates. In this approach, UV photopolymerization was employed to micropattern heparin-based hydrogels onto glass substrates containing ITO electrodes. ITO/glass substrates were first functionalized with acrylated silane to promote attachment of hydrogel structures. The surfaces containing hydrogel micropatterns were further functionalized with poly(ethylene glycol) thiol, rendering the regions around the hydrogel structures non-fouling to proteins and cells. After incubating surfaces with collagen (I), primary rat hepatocytes were shown to selectively attach on top of the hydrogel and not on surrounding glass/ITO regions. Electrical activation of specific ITO electrodes (-1.8 V vs. Ag/AgCl reference) was then used to release cells-on-hydrogel microstructures from the substrate. Immunostaining and reverse transcription polymerase chain reaction analysis of albumin, an important indicator of hepatic function, showed that the hepatocyte-on-hydrogel microstructures released from the surface maintained their function at levels similar to hepatocytes remaining on the culture substrate. In the future, switchable conductive substrates described here may be to collect cell samples at different time points and may also be used for harvesting cell-carrying vehicles for transplantation studies.

Real-world impact of vaccination on coronavirus disease 2019 (COVID-19) incidence in healthcare personnel at an academic medical center.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) vaccination effectiveness in healthcare personnel (HCP) has been established. However, questions remain regarding its performance in high-risk healthcare occupations and work locations. We describe the effect of a COVID-19 HCP vaccination campaign on SARS-CoV-2 infection by timing of vaccination, job type, and work location. METHODS: We conducted a retrospective review of COVID-19 vaccination acceptance, incidence of postvaccination COVID-19, hospitalization, and mortality among 16,156 faculty, students, and staff at a large academic medical center. Data were collected 8 weeks prior to the start of phase 1a vaccination of frontline employees and ended 11 weeks after campaign onset. RESULTS: The COVID-19 incidence rate among HCP at our institution decreased from 3.2% during the 8 weeks prior to the start of vaccinations to 0.38% by 4 weeks after campaign initiation. COVID-19 risk was reduced among individuals who received a single vaccination (hazard ratio [HR], 0.52; 95% confidence interval [CI], 0.40-0.68; P < .0001) and was further reduced with 2 doses of vaccine (HR, 0.17; 95% CI, 0.09-0.32; P < .0001). By 2 weeks after the second dose, the observed case positivity rate was 0.04%. Among phase 1a HCP, we observed a lower risk of COVID-19 among physicians and a trend toward higher risk for respiratory therapists independent of vaccination status. Rates of infection were similar in a subgroup of nurses when examined by work location. CONCLUSIONS: Our findings show the real-world effectiveness of COVID-19 vaccination in HCP. Despite these encouraging results, unvaccinated HCP remain at an elevated risk of infection, highlighting the need for targeted outreach to combat vaccine hesitancy.

Derivation and Validation of a Diagnostic Prediction Tool for Interstitial Lung Disease.

BACKGROUND: Interstitial lung disease (ILD) results in high morbidity and health-care utilization. Diagnostic delays remain common and often occur in nonpulmonology settings. Screening for ILD in these settings has the potential to reduce diagnostic delays and improve patient outcomes. RESEARCH QUESTION: This study sought to determine whether a pulmonary function test (PFT)-derived diagnostic prediction tool (ILD-Screen) could accurately identify incident ILD cases in patients undergoing PFT in nonpulmonology settings. STUDY DESIGN AND METHODS: Clinical and physiologic PFT variables predictive of ILD were identified by using iterative multivariable logistic regression models. ILD status was determined by using a multi-reader approach. An ILD-Screen score was generated by using final regression model coefficients, with a score ≥ 8 considered positive. ILD-Screen test performance was validated in an independent external cohort and applied prospectively to PFTs over 1 year to identify incident ILD cases at our institution. RESULTS: Variables comprising the ILD-Screen were age, height, total lung capacity, FEV1, diffusion capacity, and PFT indication. The ILD-Screen showed consistent test performance across cohorts, with a sensitivity of 0.79 and a specificity of 0.83 when applied prospectively. A positive ILD-Screen strongly predicted ILD (OR, 18.6; 95% CI, 9.4-36.9) and outperformed common ILD clinical features, including cough, dyspnea, lung crackles, and restrictive lung physiology. Prospective ILD-Screen application resulted in a higher proportion of patients undergoing chest CT imaging compared with a historical control cohort (74% vs 56%, respectively; P = .003), with a significantly shorter median time to chest CT imaging (5.6 vs 21.1 months; P < .001). INTERPRETATION: The ILD-Screen showed good test performance in predicting ILD across diverse geographic settings and when applied prospectively. Systematic ILD-Screen application has the potential to reduce diagnostic delays and facilitate earlier intervention in patients with ILD.

Interstitial lung abnormality is prevalent and associated with worse outcome in patients undergoing transcatheter aortic valve replacement.

BACKGROUND: Interstitial lung abnormality (ILA) is found in 5-10% of the general population and is associated with increased mortality risk. Risk factors for ILA, including advanced age and smoking history also increase the risk for aortic stenosis (AS). Transcatheter aortic valve replacement (TAVR) has become an increasingly utilized intervention for patients with severe AS, and requires a high-resolution computed tomography (HRCT) of the chest to assess aortic valve dimensions. OBJECTIVES: To determine the prevalence and clinical significance of ILA on HRCT performed in patients referred for TAVR. METHODS: Consecutive pre-TAVR HRCTs performed over a 5-year period were reviewed. ILA was defined as bilateral, nondependent reticular opacities. All-cause mortality among TAVR recipients was compared between ILA cases and non-ILA controls matched 2:1 by age and gender using Cox proportional hazards regression and the Kaplan Meier estimator. RESULTS: Of 623 HRCTs screened, ILA was detected in 92 (14.7%), including 62 patients that underwent TAVR. Among ILA cases, 17 (27.4%) had a typical or probable usual interstitial pneumonia pattern, suggesting a diagnosis of idiopathic pulmonary fibrosis. Survival was worse in ILA cases compared to non-ILA controls (p = 0.008) and ILA was an independent predictor of mortality after multivariable adjustment (HR 3.29, 95% CI 1.34-8.08; p = 0.009). CONCLUSIONS: ILA is a common finding among patients with severe AS and is associated with increased mortality in those undergoing TAVR. Further research is needed to elucidate the biology underpinning this observation and determine whether ILA evaluation and risk stratification modulates this mortality risk.

Cell biology is different in small volumes: endogenous signals shape phenotype of primary hepatocytes cultured in microfluidic channels.

The approaches for maintaining hepatocytes in vitro are aimed at recapitulating aspects of the native liver microenvironment through the use of co-cultures, surface coatings and 3D spheroids. This study highlights the effects of spatial confinement-a less studied component of the in vivo microenvironment. We demonstrate that hepatocytes cultured in low-volume microfluidic channels (microchambers) retain differentiated hepatic phenotype for 21 days whereas cells cultured in regular culture plates under identical conditions de-differentiate after 7 days. Careful consideration of nutrient delivery and oxygen tension suggested that these factors could not solely account for enhanced cell function in microchambers. Through a series of experiments involving microfluidic chambers of various heights and inhibition of key molecular pathways, we confirmed that phenotype of hepatocytes in small volumes was shaped by endogenous signals, both hepato-inductive growth factors (GFs) such as hepatocyte growth factor (HGF) and hepato-disruptive GFs such as transforming growth factor (TGF)-ß1. Hepatocytes are not generally thought of as significant producers of GFs-this role is typically assigned to nonparenchymal cells of the liver. Our study demonstrates that, in an appropriate microenvironment, hepatocytes produce hepato-inductive and pro-fibrogenic signals at the levels sufficient to shape their phenotype and function.

Local control of hepatic phenotype with growth factor-encoded surfaces.

The goal of the present study was to modulate the phenotype expression of hepatocytes in vitro on surfaces imprinted with growth factors (GFs). Hepatocyte growth factor (HGF) or transforming-growth factor-ß1 (TGF-ß1) were mixed with collagen (I) and robotically printed onto standard glass slides to create arrays of 300 µm or 500 µm diameter spots. Primary rat hepatocytes were seeded on top of the arrays, forming clusters corresponding in size to the underlying protein spots. The TGF-ß1 spots appeared to downregulate markers of hepatic (epithelial) phenotype while upregulating expression of mesenchymal markers. Conversely, hepatocytes cultured on HGF spots maintained high level of epithelial markers. When hepatocytes were seeded onto alternating spots of HGF and TGF-ß1, their phenotype was found to depend on center-to-center distance between the spots. At shorter distances cross-expression of epithelial and mesenchymal markers was observed while at distances exceeding 1.25 mm divergence of phenotypes, epithelial on HGF and mesenchymal on TGF-ß was seen. Overall, our results demonstrate that GF-encoded surfaces can modulate phenotype within groups of cells cultured on the same surface. Given the importance of phenotype switching in development, fibrosis and cancer, this platform may be used to gain useful insights into the mechanisms of processes such as epithelial-to-mesenchymal transition or stem cell fate selections.