Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils.

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution.

RATIONALE: The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers. Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate because of inherent obstacles including small cell numbers, large cell-type heterogeneity, complex anatomy, and difficulty in isolation. Single-cell RNA-sequencing allows for genome-wide analysis of gene expression at single-cell resolution. OBJECTIVE: Assess the transcriptional landscape of the entire CCS at single-cell resolution by single-cell RNA-sequencing within the developing mouse heart. METHODS AND RESULTS: Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and 3 zones of microdissection were isolated, including: Zone I-sinoatrial node region; Zone II-atrioventricular node/His region; and Zone III-bundle branch/Purkinje fiber region. Tissue was digested into single-cell suspensions, cells isolated, mRNA reverse transcribed, and barcoded before high-throughput sequencing and bioinformatics analyses. Single-cell RNA-sequencing was performed on over 22 000 cells, and all major cell types of the murine heart were successfully captured including bona fide clusters of cells consistent with each major component of the CCS. Unsupervised weighted gene coexpression network analysis led to the discovery of a host of novel CCS genes, a subset of which were validated using fluorescent in situ hybridization as well as whole-mount immunolabeling with volume imaging (iDISCO+) in 3 dimensions on intact mouse hearts. Further, subcluster analysis unveiled isolation of distinct CCS cell subtypes, including the clinically relevant but poorly characterized transitional cells that bridge the CCS and surrounding myocardium. CONCLUSIONS: Our study represents the first comprehensive assessment of the transcriptional profiles from the entire CCS at single-cell resolution and provides a characterization in the context of development and disease.

Nkx2.5+ Cardiomyoblasts Contribute to Cardiomyogenesis in the Neonatal Heart.

During normal lifespan, the mammalian heart undergoes limited renewal of cardiomyocytes. While the exact mechanism for this renewal remains unclear, two possibilities have been proposed: differentiated myocyte replication and progenitor/immature cell differentiation. This study aimed to characterize a population of cardiomyocyte precursors in the neonatal heart and to determine their requirement for cardiac development. By tracking the expression of an embryonic Nkx2.5 cardiac enhancer, we identified cardiomyoblasts capable of differentiation into striated cardiomyocytes in vitro. Genome-wide expression profile of neonatal Nkx2.5+ cardiomyoblasts showed the absence of sarcomeric gene and the presence of cardiac transcription factors. To determine the lineage contribution of the Nkx2.5+ cardiomyoblasts, we generated a doxycycline suppressible Cre transgenic mouse under the regulation of the Nkx2.5 enhancer and showed that neonatal Nkx2.5+ cardiomyoblasts mature into cardiomyocytes in vivo. Ablation of neonatal cardiomyoblasts resulted in ventricular hypertrophy and dilation, supporting a functional requirement of the Nkx2.5+ cardiomyoblasts. This study provides direct lineage tracing evidence that a cardiomyoblast population contributes to cardiogenesis in the neonatal heart. The cell population identified here may serve as a promising therapeutic for pediatric cardiac regeneration.

Bioacoustic-enabled patterning of human iPSC-derived cardiomyocytes into 3D cardiac tissue.

The creation of physiologically-relevant human cardiac tissue with defined cell structure and function is essential for a wide variety of therapeutic, diagnostic, and drug screening applications. Here we report a new scalable method using Faraday waves to enable rapid aggregation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into predefined 3D constructs. At packing densities that approximate native myocardium (108-109 cells/ml), these hiPSC-CM-derived 3D tissues demonstrate significantly improved cell viability, metabolic activity, and intercellular connection when compared to constructs with random cell distribution. Moreover, the patterned hiPSC-CMs within the constructs exhibit significantly greater levels of contractile stress, beat frequency, and contraction-relaxation rates, suggesting their improved maturation. Our results demonstrate a novel application of Faraday waves to create stem cell-derived 3D cardiac tissue that resembles the cellular architecture of a native heart tissue for diverse basic research and clinical applications.

Contractile force generation by 3D hiPSC-derived cardiac tissues is enhanced by rapid establishment of cellular interconnection in matrix with muscle-mimicking stiffness.

Engineering 3D human cardiac tissues is of great importance for therapeutic and pharmaceutical applications. As cardiac tissue substitutes, extracellular matrix-derived hydrogels have been widely explored. However, they exhibit premature degradation and their stiffness is often orders of magnitude lower than that of native cardiac tissue. There are no reports on establishing interconnected cardiomyocytes in 3D hydrogels at physiologically-relevant cell density and matrix stiffness. Here we bioengineer human cardiac microtissues by encapsulating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in chemically-crosslinked gelatin hydrogels (1.25 × 108/mL) with tunable stiffness and degradation. In comparison to the cells in high stiffness (16 kPa)/slow degrading hydrogels, hiPSC-CMs in low stiffness (2 kPa)/fast degrading and intermediate stiffness (9 kPa)/intermediate degrading hydrogels exhibit increased intercellular network formation, α-actinin and connexin-43 expression, and contraction velocity. Only the 9 kPa microtissues exhibit organized sarcomeric structure and significantly increased contractile stress. This demonstrates that muscle-mimicking stiffness together with robust cellular interconnection contributes to enhancement in sarcomeric organization and contractile function of the engineered cardiac tissue. This study highlights the importance of intercellular connectivity, physiologically-relevant cell density, and matrix stiffness to best support 3D cardiac tissue engineering.

Validating Health Literacy and Numeracy Measures in Minority Groups.

BACKGROUND: Validation studies of existing health literacy or numeracy tools among racial/ethnic minorities are limited. OBJECTIVE: This study assessed the validity of the Subjective Numeracy Scale (SNS), the Diabetes Numeracy Test (DNT-5), the Brief Health Literacy Screen (BHLS), and the Short Test of Functional Health Literacy in Adults (S-TOFHLA) by trait (health literacy or numeracy) and by method (subjective or objective) among non-Hispanic white (NHW), non-Hispanic black (NHB), and Hispanic patients with type 2 diabetes mellitus (T2DM). METHODS: We conducted a secondary analysis of baseline data from the Partnering to Improve Diabetes Education (PRIDE) study, a clustered randomized controlled trial testing the efficacy of a health communication intervention on T2DM outcomes at state Department of Health clinics in middle Tennessee. PRIDE participants with race/ethnicity data available (n = 398) were included in this study. Most patients identified as NHW (59%), 18% identified as NHB, and 23% identified as Hispanic. Pearson correlations among the 4 measures were compared for each racial/ethnic group by trait and method. The convergent validity of each measure with education was also assessed using Pearson correlation analyses. KEY RESULTS: Significant correlations were observed across all 3 subgroups for the numeracy measures (SNS and DNT-5) and the objective measures (DNT-5 and S-TOFHLA). Nonsignificant correlations were observed among Hispanic participants for the health literacy measures (BHLS and S-TOFHLA, correlation coefficient = 0.13) and among NHB and Hispanic participants for the subjective measures (SNS and BHLS, correlations coefficients = 0.15 and 0.09, respectively). A significant positive correlation was noted between education and each measure across all 3 subgroups. CONCLUSIONS: Subjective and health literacy measures demonstrate weaker correlations than objective and numeracy measures, respectively, among minority patients in this study. Our findings highlight the need to further evaluate the appropriateness of these tools for use with minority populations, particularly the BHLS for Hispanic patients.

Stress-Related Changes in Attentional Bias to Social Threat in Young Adults: Psychobiological Associations with the Early Family Environment.

This study investigated the association of chronic childhood stress exposure with acute stress-related attentional alterations that have been previously linked to vulnerability to mental and physical illness in early adulthood. Participants were randomized in a crossover design to complete both a mild laboratory social stress task and a computerized task assessing attentional bias to socially threatening words. Salivary cortisol was measured throughout the study. Exposure to acute laboratory stress altered attentional processing, and this relationship was moderated by chronic childhood stress exposure. Also, a positive association between cortisol reactivity and attentional bias was observed, with cortisol reactivity negatively related to childhood chronic stress exposure. While previous work has supported a role for early chronic stress exposure in influencing acute stress reactivity, this work provides initial insight into how both prior chronic childhood stress and current acute stress together relate to the attentional gateway and may be associated with stress adaptation and psychological vulnerability into adulthood.

Role of cyclic nucleotide-dependent actin cytoskeletal dynamics:Ca(2+)](i) and force suppression in forskolin-pretreated porcine coronary arteries.

Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca(2+)]i) and phosphorylation of myosin light chains (MLC). However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA) prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca(2+)]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.