Modeling long QT syndrome type 2 on-a-chip via in-depth assessment of isogenic gene-edited 3D cardiac tissues.

Long QT syndrome (LQTS) is a cardiovascular disease characterized by QT interval prolongation that can lead to sudden cardiac death. Many mutations with heterogeneous mechanisms have been identified in KCNH2, the gene that encodes for hERG (Kv11.1), which lead to onset of LQTS type 2 (LQTS2). In this work, we developed a LQTS2-diseased tissue-on-a-chip model, using 3D coculture of isogenic stem cell-derived cardiomyocytes (CMs) and cardiac fibroblasts (CFs) within an organotypic microfluidic chip technology. Primarily, we created a hiPSC line with R531W mutation in KCNH2 using CRISPR-Cas9 gene-editing technique and characterized the resultant differentiated CMs and CFs. A deficiency in hERG trafficking was identified in KCNH2-edited hiPSC-CMs, revealing a possible mechanism of R531W mutation in LQTS2 pathophysiology. Following creation of a 3D LQTS2 tissue-on-a-chip, the tissues were extensively characterized, through analysis of calcium handling and response to ß-agonist. Furthermore, attempted phenotypic rescue via pharmacological intervention of LQTS2 on a chip was investigated.

Investigating the Interactions of Glioma Stem Cells in the Perivascular Niche at Single-Cell Resolution using a Microfluidic Tumor Microenvironment Model.

The perivascular niche (PVN) is a glioblastoma tumor microenvironment (TME) that serves as a safe haven for glioma stem cells (GSCs), and acts as a reservoir that inevitably leads to tumor recurrence. Understanding cellular interactions in the PVN that drive GSC treatment resistance and stemness is crucial to develop lasting therapies for glioblastoma. The limitations of in vivo models and in vitro assays have led to critical knowledge gaps regarding the influence of various cell types in the PVN on GSCs behavior. This study developed an organotypic triculture microfluidic model as a means to recapitulate the PVN and study its impact on GSCs. This triculture platform, comprised of endothelial cells (ECs), astrocytes, and GSCs, is used to investigate GSC invasion, proliferation and stemness. Both ECs and astrocytes significantly increased invasiveness of GSCs. This study futher identified 15 ligand-receptor pairs using single-cell RNAseq with putative chemotactic mechanisms of GSCs, where the receptor is up-regulated in GSCs and the diffusible ligand is expressed in either astrocytes or ECs. Notably, the ligand-receptor pair SAA1-FPR1 is demonstrated to be involved in chemotactic invasion of GSCs toward PVN. The novel triculture platform presented herein can be used for therapeutic development and discovery of molecular mechanisms driving GSC biology.

Cardiac ischemia on-a-chip to investigate cellular and molecular response of myocardial tissue under hypoxia.

Tissue engineering has enabled the development of advanced and physiologically relevant models of cardiovascular diseases, with advantages over conventional 2D in vitro assays. We have previously demonstrated development of a heart on-a-chip microfluidic model with mature 3D anisotropic tissue formation that incorporates both stem cell-derived cardiomyocytes and cardiac fibroblasts within a collagen-based hydrogel. Using this platform, we herein present a model of myocardial ischemia on-a-chip, that recapitulates ischemic insult through exposure of mature 3D cardiac tissues to hypoxic environments. We report extensive validation and molecular-level analyses of the model in its ability to recapitulate myocardial ischemia in response to hypoxia, demonstrating the 1) induction of tissue fibrosis through upregulation of contractile fibers, 2) dysregulation in tissue contraction through functional assessment, 3) upregulation of hypoxia-response genes and downregulation of contractile-specific genes through targeted qPCR, and 4) transcriptomic pathway regulation of hypoxic tissues. Further, we investigated the complex response of ischemic myocardial tissues to reperfusion, identifying 5) cell toxicity, 6) sustained contractile irregularities, as well as 7) re-establishment of lactate levels and 8) gene expression, in hypoxic tissues in response to ischemia reperfusion injury.

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium's cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling. Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

Engineering anisotropic human stem cell-derived three-dimensional cardiac tissue on-a-chip.

Despite significant efforts in the study of cardiovascular diseases (CVDs), they persist as the leading cause of mortality worldwide. Considerable research into human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has highlighted their immense potential in the development of in vitro human cardiac tissues for broad mechanistic, therapeutic, and patient-specific disease modeling studies in the pursuit of CVD research. However, the relatively immature state of hPSC-CMs remains an obstacle in enhancing clinical relevance ofengineered cardiac tissue models. In this study, we describe development of a microfluidic platform for 3D modeling of cardiac tissues, derived from both rat cells and hPSC-CMs, to better recapitulate the native myocardium through co-culture with interstitial cells (specifically cardiac fibroblasts), biomimetic collagen hydrogel encapsulation, and induction of highly anisotropic tissue architecture. The presented platform is precisely engineered through incorporation of surface topography in the form of staggered microposts to enable long-term culture and maturation of cardiac cells, resulting in formation of physiologically relevant cardiac tissues with anisotropy that mimics native myocardium. After two weeks of culture, hPSC-derived cardiac tissues exhibited well-defined sarcomeric striations, highly synchronous contractions, and upregulation of several maturation genes, including HCN1, KCNQ1, CAV1.2, CAV3.1, PLN, and RYR2. These findings demonstrate the ability of the proposed engineered platform to mature animal- as well as human stem cell-derived cardiac tissues over an extended period of culture, providing a novel microfluidic chip with the capability for cardiac disease modeling and therapeutic testing.

Three-dimensional scaffold-free microtissues engineered for cardiac repair.

Cardiovascular diseases, including myocardial infarction (MI), persist as the leading cause of mortality and morbidity worldwide. The limited regenerative capacity of the myocardium presents significant challenges specifically for the treatment of MI and, subsequently, heart failure (HF). Traditional therapeutic approaches mainly rely on limiting the induced damage or the stress on the remaining viable myocardium through pharmacological regulation of remodeling mechanisms, rather than replacement or regeneration of the injured tissue. The emerging alternative regenerative medicine-based approaches have focused on restoring the damaged myocardial tissue with newly engineered functional and bioinspired tissue units. Cardiac regenerative medicine approaches can be broadly categorized into three groups: cell-based therapies, scaffold-based cardiac tissue engineering, and scaffold-free cardiac tissue engineering. Despite significant advancements, however, the clinical translation of these approaches has been critically hindered by two key obstacles for successful structural and functional replacement of the damaged myocardium, namely: poor engraftment of engineered tissue into the damaged cardiac muscle and weak electromechanical coupling of transplanted cells with the native tissue. To that end, the integration of micro- and nanoscale technologies along with recent advancements in stem cell technologies have opened new avenues for engineering of structurally mature and highly functional scaffold-based (SB-CMTs) and scaffold-free cardiac microtissues (SF-CMTs) with enhanced cellular organization and electromechanical coupling for the treatment of MI and HF. In this review article, we will present the state-of-the-art approaches and recent advancements in the engineering of SF-CMTs for myocardial repair.

The role of tumor-stroma interactions on desmoplasia and tumorigenicity within a microengineered 3D platform.

The tumor microenvironment has been demonstrated to play a crucial role in modulating cancer progression. Amongst various cell types within the tumor microenvironment, cancer associated fibroblasts (CAFs) are in abundance, serving to modulate the biophysical properties of the stromal matrix, through excessive deposition of extracellular matrix (ECM) proteins that leads to enhanced tumor progression. There is still a critical need to develop a fundamental framework on the role of tumor-stromal cell interactions on desmoplasia and tumorigenicity. Herein, we developed a 3D microengineered organotypic tumor-stroma model incorporated with breast cancer cells surrounded by CAF-embedded collagen matrix. We further integrated our platform with atomic force microscopy (AFM) to study the dynamic changes in stromal stiffness during active tumor invasion. Our findings primarily demonstrated enhanced tumor progression in the presence of CAFs. Furthermore, we highlighted the crucial role of crosstalk between tumor cells and CAFs on stromal desmoplasia, where we identified the role of tumor-secreted PDGF-AA/-BB on elevated matrix stiffness. Inhibition of the activity of PDGFRs in CAFs led to attenuation of stromal stiffness. Overall, our work presents a well-controlled tumor microenvironment model capable of dissecting specific biophysical and biochemical signaling cues which lead to stromal desmoplasia and tumor progression.

Endothelial Immune Activation by Medin: Potential Role in Cerebrovascular Disease and Reversal by Monosialoganglioside-Containing Nanoliposomes.

Background The function of medin, one of the most common human amyloid proteins that accumulates in the vasculature with aging, remains unknown. We aim to probe medin's role in cerebrovascular disease by comparing cerebral arterial medin content between cognitively normal and vascular dementia (VaD) patients and studying its effects on endothelial cell (EC) immune activation and neuroinflammation. We also tested whether monosialoganglioside-containing nanoliposomes could reverse medin's adverse effects. Methods and Results Cerebral artery medin and astrocyte activation were measured and compared between VaD and cognitively normal elderly brain donors. ECs were exposed to physiologic dose of medin (5 µmol/L), and viability and immune activation (interleukin-8, interleukin-6, intercellular adhesion molecule-1, and plasminogen activator inhibitor-1) were measured without or with monosialoganglioside-containing nanoliposomes (300 µg/mL). Astrocytes were exposed to vehicle, medin, medin-treated ECs, or their conditioned media, and interleukin-8 production was compared. Cerebral collateral arterial and parenchymal arteriole medin, white matter lesion scores, and astrocyte activation were higher in VaD versus cognitively normal donors. Medin induced EC immune activation (increased interleukin-8, interleukin-6, intercellular adhesion molecule-1, and plasminogen activator inhibitor-1) and reduced EC viability, which were reversed by monosialoganglioside-containing nanoliposomes. Interleukin-8 production was augmented when astrocytes were exposed to medin-treated ECs or their conditioned media. Conclusions Cerebral arterial medin is higher in VaD compared with cognitively normal patients. Medin induces EC immune activation that modulates astrocyte activation, and its effects are reversed by monosialoganglioside-containing nanoliposomes. Medin is a candidate novel risk factor for aging-related cerebrovascular disease and VaD.

Three-dimensional microengineered models of human cardiac diseases.

In vitro three-dimensional (3D) microengineered tissue models have been the recent focus of pathophysiological studies, particularly in the field of cardiovascular research. These models, as classified by 3D biomimetic tissues within micrometer-scale platforms, enable precise environmental control on the molecular- and cellular-levels to elucidate biological mechanisms of disease progression and enhance efficacy of therapeutic research. Microengineered models also incorporate directed stem cell differentiation and genome modification techniques that warrant derivation of patient-specific and genetically-edited human cardiac cells for precise recapitulation of diseased tissues. Additionally, integration of added functionalities and/or structures into these models serves to enhance the capability to further extract disease-specific phenotypic, genotypic, and electrophysiological information. This review highlights the recent progress in the development of in vitro 3D microengineered models for study of cardiac-related diseases (denoted as CDs). We will primarily provide a brief overview on currently available 2D assays and animal models for studying of CDs. We will further expand our discussion towards currently available 3D microengineered cardiac tissue models and their implementation for study of specific disease conditions.

Upper-Extremity Dual-Task Function: An Innovative Method to Assess Cognitive Impairment in Older Adults.

BACKGROUND: Difficulties in orchestrating simultaneous tasks (i.e., dual-tasking) have been associated with cognitive impairments in older adults. Gait tests have been commonly used as the motor task component for dual-task assessments; however, many older adults have mobility impairments or there is a lack of space in busy clinical settings. We assessed an upper-extremity function (UEF) test as an alternative motor task to study the dual-task motor performance in older adults. METHODS: Older adults (≥65 years) were recruited, and cognitive ability was measured using the Montreal cognitive assessment (MoCA). Participants performed repetitive elbow flexion with their maximum pace, once single-task, and once while counting backward by one (dual-task). Single- and dual-task gait tests were also performed with normal speed. Three-dimensional kinematics was measured both from upper-extremity and lower-extremity using wearable sensors to determine UEF and gait parameters. Parameters were compared between the cognitively impaired and healthy groups using analysis of variance tests, while controlling for age, gender, and body mass index (BMI). Correlations between UEF and gait parameters for dual-task and dual-task cost were assessed using linear regression models. RESULTS: Sixty-seven older adults were recruited (age = 83 ± 10 years). Based on MoCA, 10 (15%) were cognitively impaired. While no significant differences were observed in the single-task condition, within the dual-task condition, the cognitively impaired group showed significantly less arm flexion speed (62%, d = 1.51, p = 0.02) and range of motion (27%, d = 0.93, p = 0.04), and higher speed variability (88%, d = 1.82, p < 0.0001) compared to the cognitively intact group, when adjusted with age, gender, and BMI. Significant correlations were observed between UEF speed parameters and gait stride velocity for dual-task condition (r = 0.55, p < 0.0001) and dual-task cost (r = 0.28, p = 0.03). CONCLUSION: We introduced a novel test for assessing dual-task performance in older adults that lasts 20 s and is based on upper-extremity function. Our results confirm significant associations between upper-extremity speed, range of motion, and speed variability with both the MoCA score and the gait performance within the dual-task condition.