Biomechanical Modeling of Cell Chirality and Symmetry Breaking of Biological Systems.

Accumulating evidence strongly suggests that cell chirality plays a pivotal role in driving left-right (LR) symmetry breaking, a widespread phenomenon in living organisms. Whole embryos and excised organs have historically been employed to investigate LR symmetry breaking and have yielded exciting findings. In recent years, in vitro engineered platforms have emerged as powerful tools to reveal cellular chiral biases and led to uncovering molecular and biophysical insights into chiral morphogenesis, including the significant role of the actin cytoskeleton. Establishing a link between observed in vivo tissue chiral morphogenesis and the determined chiral bias of cells in vitro has become increasingly important. In this regard, computational mathematical models hold immense value as they can explain and predict tissue morphogenic behavior based on the chiral biases of individual cells. Here, we present the formulations and discoveries achieved using various computational models spanning different biological scales, from the molecular and cellular levels to tissue and organ levels. Furthermore, we offer insights into future directions and the role of such models in advancing the study of asymmetric cellular mechanobiology.

Engineered platforms for mimicking cardiac development and drug screening.

Congenital heart defects are associated with significant health challenges, demanding a deep understanding of the underlying biological mechanisms and, thus, better devices or platforms that can recapitulate human cardiac development. The discovery of human pluripotent stem cells has substantially reduced the dependence on animal models. Recent advances in stem cell biology, genetic editing, omics, microfluidics, and sensor technologies have further enabled remarkable progress in the development of in vitro platforms with increased fidelity and efficiency. In this review, we provide an overview of advancements in in vitro cardiac development platforms, with a particular focus on technological innovation. We categorize these platforms into four areas: two-dimensional solid substrate cultures, engineered substrate architectures that enhance cellular functions, cardiac organoids, and embryos/explants-on-chip models. We conclude by addressing current limitations and presenting future perspectives.

Asymmetrical positioning of cell organelles reflects the cell chirality of mouse myoblast cells.

Cell chirality is crucial for the chiral morphogenesis of biological tissues, yet its underlying mechanism remains unclear. Cell organelle polarization along multiple axes in a cell body, namely, apical-basal, front-rear, and left-right, is known to direct cell behavior such as orientation, rotation, and migration. Among these axes, the left-right bias holds significant sway in determining the chiral directionality of these behaviors. Normally, mouse myoblast (C2C12) cells exhibit a strong counterclockwise chirality on a ring-shaped micropattern, whereas they display a clockwise dominant chirality under Latrunculin A treatment. To investigate the relationship between multicellular chirality and organelle positioning in single cells, we studied the left-right positioning of cell organelles under distinct cell chirality in single cells via micropatterning technique, fluorescent microscopy, and imaging analysis. We found that on a "T"-shaped micropattern, a C2C12 cell adopts a triangular shape, with its nucleus-centrosome axis pointing toward the top-right direction of the "T." Several other organelles, including the Golgi apparatus, lysosomes, actin filaments, and microtubules, showed a preference to polarize on one side of the axis, indicating the universality of the left-right asymmetrical organelle positioning. Interestingly, upon reversing cell chirality with Latrunculin A, the organelles correspondingly reversed their left-right positioning bias, as suggested by the consistently biased metabolism and contractile properties at the leading edge. This left-right asymmetry in organelle positioning may help predict cell migration direction and serve as a potential marker for identifying cell chirality in biological models.

Helical vasculogenesis driven by cell chirality.

The cellular helical structure is well known for its crucial role in development and disease. Nevertheless, the underlying mechanism governing this phenomenon remains largely unexplored, particularly in recapitulating it in well-controlled engineering systems. Leveraging advanced microfluidics, we present compelling evidence of the spontaneous emergence of helical endothelial tubes exhibiting robust right-handedness governed by inherent cell chirality. To strengthen our findings, we identify a consistent bias toward the same chirality in mouse vascular tissues. Manipulating endothelial cell chirality using small-molecule drugs produces a dose-dependent reversal of the handedness in engineered vessels, accompanied by non-monotonic changes in vascular permeability. Moreover, our three-dimensional cell vertex model provides biomechanical insights into the chiral morphogenesis process, highlighting the role of cellular torque and tissue fluidity in its regulation. Our study unravels an intriguing mechanism underlying vascular chiral morphogenesis, shedding light on the broader implications and distinctive perspectives of tubulogenesis within biological systems.

Cell jamming regulates epithelial chiral morphogenesis.

Internal organs such as the heart demonstrate apparent left-right (LR) asymmetric morphology and positioning. Cellular chirality and associated LR biased mechanical behavior such as cell migration have been attributed to LR symmetry breaking during embryonic development. Mathematical models have shown that chiral directional migration can be driven by cellular intrinsic torque. Tissue jamming state (i.e., solid-like vs fluid-like state) strongly regulates collective migratory behavior, but how it might affect chiral morphogenesis is still unknown. Here, we develop a cell vertex model to study the role of tissue rigidity or jamming state on chiral morphogenesis of the cells on a patterned ring-shaped tissue, simulating a previously reported experimental setup for measuring cell chirality. We simulate chirality as torsional forces acting on cell vertices. As expected, the cells undergo bidirectional migration at the opposing (inner and outer) boundaries of the ring-shaped tissue. We discover that more fluid-like tissues (unjammed) demonstrate a stronger chiral cell alignment and elongation than more solid-like (jammed) tissues and maintain a bigger difference in migration velocity between opposing tissue boundaries. Finally, we find that fluid-like tissues undergo more cell-neighbor exchange events. This study reveals that chiral torque is sufficient to achieve a biased cellular alignment as seen in vitro. It further sheds light on the mechanical regulation of chiral morphogenesis of tissues and reveals a role of cell density-independent tissue rigidity in this process.

The Actin Crosslinker Fascin Regulates Cell Chirality.

The left-right (L-R) asymmetry of the cells, or cell chirality, is a well-known intrinsic property derived from the dynamic organization of the actin cytoskeleton. Cell chirality can be regulated by actin-binding proteins such as α-actinin-1 and can also be mediated by certain signaling pathways, such as protein kinase C (PKC) signaling. Fascin, an actin crosslinker known to mediate parallel bundling of actin filaments, appears as a prominent candidate in cell chirality regulation, given its role in facilitating cell migration as an important PKC substrate. Here, it is shown that the chirality of NIH/3T3 cells can be altered by PKC activation and fascin manipulation. With either small-molecule drug inhibition or genetic knockdown of fascin, the chirality of 3T3 cells is reversed from a clockwise (CW) bias to a counterclockwise (CCW) bias on ring-shaped micropatterns, accompanied by the reversal in cell directional migration. The Ser-39 fascin-actin binding sites are further explored in cell chirality regulation. The findings of this study reveal the critical role of fascin as an important intermediator in cell chirality, shedding novel insights into the mechanisms of L-R asymmetric cell migration and multicellular morphogenesis.

Interacting with tumor cells weakens the intrinsic clockwise chirality of endothelial cells.

Endothelial cells (ECs) possess a strong intrinsic clockwise (CW, or rightward) chirality under normal conditions. Enervating this chirality of ECs significantly impairs the function of the endothelial barrier. Malignant tumor cells (TCs) undergo metastasis by playing upon the abnormal leakage of blood vessels. However, the impact of TCs on EC chirality is still poorly understood. Using a transwell model, we co-cultured the human umbilical vein endothelial cells or human lung microvascular endothelial cells and breast epithelial tumor cell lines to simulate the TC-EC interaction. Using a micropatterning method, we assessed the EC chirality changes induced by paracrine signaling of and physical contact with TCs. We found that the intrinsic clockwise chirality of ECs was significantly compromised by the TC's physical contact, while the paracrine signaling (i.e., without physical contact) of TCs causes minimal changes. In addition, ECs neighboring TCs tend to possess a left bias, while ECs spaced apart from TCs are more likely to preserve the intrinsic right bias. Finally, we found the chirality change of ECs could result from physical binding between CD44 and E-selectin, which activates protein kinase C alpha (PKCα) and induces pseudopodial movement of EC toward TC. Our findings together suggest the crucial role of EC-TC physical interaction in EC chirality and that weakening the EC chirality could potentially compromise the overall endothelial integrity which increases the probability of metastatic cancer spread.

A Micropatterning Assay for Measuring Cell Chirality.

Chirality is an intrinsic cellular property, which depicts the asymmetry in terms of polarization along the left-right axis of the cell. As this unique property attracts increasing attention due to its important roles in both development and disease, a standardized quantification method for characterizing cell chirality would advance research and potential applications. In this protocol, we describe a multicellular chirality characterization assay that utilizes micropatterned arrays of cells. Cellular micropatterns are fabricated on titanium/gold-coated glass slides via microcontact printing. After seeding on the geometrically defined (e.g., ring-shaped), protein-coated islands, cells directionally migrate and form a biased alignment toward either the clockwise or the counterclockwise direction, which can be automatically analyzed and quantified by a custom-written MATLAB program. Here we describe in detail the fabrication of micropatterned substrates, cell seeding, image collection, and data analysis and show representative results obtained using the NIH/3T3 cells. This protocol has previously been validated in multiple published studies and is an efficient and reliable tool for studying cell chirality in vitro.

Cell Chirality as a Novel Measure for Cytotoxicity.

Cytotoxicity assessment has great importance in both research and pharmaceutical development. The mainstream in vitro cytotoxicity assays are mostly biochemical assays that evaluate a specific cellular activity such as proliferation and apoptosis. Few assays assess toxicity by characterizing overall functional outcomes in cellular physiology such as multicellular morphogenesis. The intrinsic cellular chiral bias (also known as cell chirality, left-right asymmetry, or handedness), which determines cellular polarization along the left-right axis, is demonstrated to play important roles in development and disease. This chiral property of cells gives insights not only into functions of individual cells, such as motility and polarity but also into emerging behaviors of cell clusters, such as collective cell migration. Therefore, cell chirality characterization can be potentially used as a biomarker for assessing the overall effects of pharmaceutical drugs and environmental factors on the health of the cell. In this review article, the current in vitro techniques for cell chirality characterization and their applications are discussed and the advantages and limitations of these cell chirality assays as potential tools for detecting cytotoxicity are discussed.

Effects of Alzheimer's Disease-Related Proteins on the Chirality of Brain Endothelial Cells.

INTRODUCTION: Cell chirality is an intrinsic cellular property that determines the directionality of cellular polarization along the left-right axis. We recently show that endothelial cell chirality can influence intercellular junction formation and alter trans-endothelial permeability, depending on the uniformity of the chirality of adjacent cells, which suggests a potential role for cell chirality in neurodegenerative diseases with blood-brain barrier (BBB) dysfunctions, such as Alzheimer's disease (AD). In this study, we determined the effects of AD-related proteins amyloid-ß (Aß), tau, and apolipoprotein E4 (ApoE4) on the chiral bias of the endothelial cell component in BBB. METHODS: We first examined the chiral bias and effects of protein kinase C (PKC)-mediated chiral alterations of human brain microvascular endothelial cells (hBMECs) using the ring micropattern chirality assay. We then investigated the effects of Aß, tau, and ApoE4 on hBMEC chirality using chirality assay and biased organelle positions. RESULTS: The hBMECs have a strong clockwise chiral bias, which can be reversed by protein kinase C (PKC) activation. Treatment with tau significantly disrupted the chiral bias of hBMECs with altered cellular polarization. In contrast, neither ApoE4 nor Aß-42 caused significant changes in cell chirality. CONCLUSIONS: We conclude that tau might cause BBB dysfunction by disrupting cell polarization and chiral morphogenesis, while the effects of ApoE4 and Aß-42 on BBB integrity might be chirality-independent. The potential involvement of chiral morphogenesis in tau-mediated BBB dysfunction in AD provides a novel perspective in vascular dysfunction in tauopathies such as AD, chronic traumatic encephalopathy, progressive supranuclear palsy, and frontotemporal dementia. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00669-w.

Recent Advances in Cellular and Molecular Bioengineering for Building and Translation of Biological Systems.

In January of 2020, the Biomedical Engineering Society (BMES)- Cellular and Molecular Bioengineering (CMBE) conference was held in Puerto Rico and themed "Vision 2020: Emerging Technologies to Elucidate the Rule of Life." The annual BME-CMBE conference gathered worldwide leaders and discussed successes and challenges in engineering biological systems and their translation. The goal of this report is to present the research frontiers in this field and provide perspectives on successful engineering and translation towards the clinic. We hope that this report serves as a constructive guide in shaping the future of research and translation of engineered biological systems.

Hyperosmolar Ionic Solutions Modulate Inflammatory Phenotype and sGAG Loss in a Cartilage Explant Model.

OBJECTIVE: The objective of this study was to compare the effects of hyperosmolar sodium (Na+), lithium (Li+) and potassium (K+) on catabolic and inflammatory osteoarthritis (OA) markers and sulfated glycosaminoglycan (sGAG) loss in TNF-α-stimulated cartilage explants. METHODS: Explants from bovine stifle joints were stimulated with TNF-α for 1 day to induce cartilage degradation followed by supplementation with 50 mM potassium chloride (KCl), 50 mM lithium chloride (LiCl), 50 mM sodium chloride (NaCl), or 100 nM dexamethasone for an additional 6 days. We assessed the effect of TNF-α stimulation and hyperosmolar ionic treatment on sGAG loss and expression of OA-associated proteins: ADAMTS-5, COX-2, MMP-1, MMP-13, and VEGF. RESULTS: TNF-α treatment increased sGAG loss (P < 0.001) and expression of COX-2 (P = 0.018), MMP-13 (P < 0.001), and VEGF (P = 0.017) relative to unstimulated controls. Relative to activated controls, LiCl and dexamethasone treatment attenuated sGAG loss (P = 0.008 and P = 0.042, respectively) and expression of MMP-13 (P = 0.005 and P = 0.036, respectively). In contrast, KCl treatment exacerbated sGAG loss (P = 0.032) and MMP-1 protein expression (P = 0.010). NaCl treatment, however, did not alter sGAG loss or expression of OA-related proteins. Comparing LiCl and KCl treatment shows a potent reduction (P < 0.05) in catabolic and inflammatory mediators following LiCl treatment. CONCLUSION: These results suggest that these ionic species elicit varying responses in TNF-α-stimulated explants. Cumulatively, these findings support additional studies of hyperosmolar ionic solutions for potential development of novel intraarticular injections targeting OA.

Cell chirality in cardiovascular development and disease.

The cardiovascular system demonstrates left-right (LR) asymmetry: most notably, the LR asymmetric looping of the bilaterally symmetric linear heart tube. Similarly, the orientation of the aortic arch is asymmetric as well. Perturbations to the asymmetry have been associated with several congenital heart malformations and vascular disorders. The source of the asymmetry, however, is not clear. Cell chirality, a recently discovered and intrinsic LR asymmetric cellular morphological property, has been implicated in the heart looping and vascular barrier function. In this paper, we summarize recent advances in the field of cell chirality and describe various approaches developed for studying cell chirality at multi- and single-cell levels. We also examine research progress in asymmetric cardiovascular development and associated malformations. Finally, we review evidence connecting cell chirality to cardiac looping and vascular permeability and provide thoughts on future research directions for cell chirality in the context of cardiovascular development and disease.

Cardiomyocyte orientation modulated by the Numb family proteins-N-cadherin axis is essential for ventricular wall morphogenesis.

The roles of cellular orientation during trabecular and ventricular wall morphogenesis are unknown, and so are the underlying mechanisms that regulate cellular orientation. Myocardial-specific Numb and Numblike double-knockout (MDKO) hearts display a variety of defects, including in cellular orientation, patterns of mitotic spindle orientation, trabeculation, and ventricular compaction. Furthermore, Numb- and Numblike-null cardiomyocytes exhibit cellular behaviors distinct from those of control cells during trabecular morphogenesis based on single-cell lineage tracing. We investigated how Numb regulates cellular orientation and behaviors and determined that N-cadherin levels and membrane localization are reduced in MDKO hearts. To determine how Numb regulates N-cadherin membrane localization, we generated an mCherry:Numb knockin line and found that Numb localized to diverse endocytic organelles but mainly to the recycling endosome. Consistent with this localization, cardiomyocytes in MDKO did not display defects in N-cadherin internalization but rather in postendocytic recycling to the plasma membrane. Furthermore, N-cadherin overexpression via a mosaic model partially rescued the defects in cellular orientation and trabeculation of MDKO hearts. Our study unravels a phenomenon that cardiomyocytes display spatiotemporal cellular orientation during ventricular wall morphogenesis, and its disruption leads to abnormal trabecular and ventricular wall morphogenesis. Furthermore, we established a mechanism by which Numb modulates cellular orientation and consequently trabecular and ventricular wall morphogenesis by regulating N-cadherin recycling to the plasma membrane.

Cell organelle-based analysis of cell chirality.

The maintenance of tight endothelial junctions requires the establishment of proper cell polarity, which includes not only the apicobasal and front-rear polarity but also the left-right (L-R) polarity. The cell possesses an intrinsic mechanism of orienting the L-R axis with respect to the other axes, following a left-hand or right-hand rule, termed cell chirality. We have previously reported that endothelial cells exhibit a clockwise or rightward bias on ring-shaped micropatterns. Now we further characterize the chirality of individual endothelial cells on micropatterns by analyzing the L-R positioning of the cell centroid relative to the nucleus-centrosome axis. Our results show that the centroids of endothelial cells preferably polarized towards the right side of the nucleus-centrosome axis. This bias is consistent with cell chirality characterized by other methods. These results suggest that the positioning of cell organelles is intrinsically L-R biased inside individual cells. This L-R bias provides an opportunity for determining cell chirality in situ, even in vivo, without the limitations of using isolated cells in in vitro engineered platforms.

Identification of Chondrocyte Genes and Signaling Pathways in Response to Acute Joint Inflammation.

Traumatic joint injuries often result in elevated proinflammatory cytokine (such as IL-1ß) levels in the joint cavity, which can increase the catabolic activities of chondrocytes and damage cartilage. This study investigated the early genetic responses of healthy in situ chondrocytes under IL-1ß attack with a focus on cell cycle and calcium signaling pathways. RNA sequencing analysis identified 2,232 significantly changed genes by IL-1ß, with 1,259 upregulated and 973 downregulated genes. Catabolic genes related to ECM degeneration were promoted by IL-1ß, consistent with our observations of matrix protein loss and mechanical property decrease during 24-day in vitro culture of cartilage explants. IL-1ß altered the cell cycle (108 genes) and Rho GTPases signaling (72 genes) in chondrocytes, while chondrocyte phenotypic shift was observed with histology, cell volume measurement, and MTT assay. IL-1ß inhibited the spontaneous calcium signaling in chondrocytes, a fundamental signaling event in chondrocyte metabolic activities. The expression of 24 genes from 6 calcium-signaling related pathways were changed by IL-1ß exposure. This study provided a comprehensive list of differentially expressed genes of healthy in situ chondrocytes in response to IL-1ß attack, which represents a useful reference to verify and guide future cartilage studies related to the acute inflammation after joint trauma.

Temporal effects of cytokine treatment on lubricant synthesis and matrix metalloproteinase activity of fibroblast-like synoviocytes.

Fibroblast-like synoviocytes (FLS) are major contributors to the composition and function of synovial fluid (SF). In disease, changes to important SF molecules such as hyaluronic acid (HA), lubricin, and numerous inflammatory markers contribute to a loss of SF functional properties. Previous studies characterized the ability of FLS to produce SF molecules in short-term cultures using continuous cytokine supplementation. This study assessed the HA, lubricin, and matrix metalloproteinase-2 (MMP-2) secretion profile of FLS over 12 days of culture. FLS were subjected to continuous, intermittent, and sequential cytokine treatments of interleukin-1 beta (IL-1ß), tumour necrosis factor-alpha (TNF-α), and transforming growth factor-beta 1 (TGF-ß1). HA was assessed by an enzyme-linked immunosorbent assay (ELISA) for content and agarose gel electrophoresis for molecular weight distribution. Relative lubricin content was determined by western blot. Pro MMP-2 and active MMP-2 were quantified by gelatin zymography. All intermittent and sequential treatments significantly increased secretion of high-molecular-weight (>3 MDa) HA for the duration of the culture. Sequentially treated groups elevated lubricin synthesis, whereas only groups receiving IL-1ß and TNF-α for 2 days followed by TGF-ß1 for 1 day reduced active MMP-2 to unstimulated control levels. These data provide important information on the long-term functional potential of cytokine-stimulated FLS and suggest that temporal regulation of cytokine exposure can be a powerful tool to guide healthy synovial secretions.

In Vitro Microscale Models for Embryogenesis.

Embryogenesis is a highly regulated developmental process requiring complex mechanical and biochemical microenvironments to give rise to a fully developed and functional embryo. Significant efforts have been taken to recapitulate specific features of embryogenesis by presenting the cells with developmentally relevant signals. The outcomes, however, are limited partly due to the complexity of this biological process. Microtechnologies such as micropatterned and microfluidic systems, along with new emerging embryonic stem cell-based models, could potentially serve as powerful tools to study embryogenesis. The aim of this article is to review major studies involving the culturing of pluripotent stem cells using different geometrical patterns, microfluidic platforms, and embryo/embryoid body-on-a-chip modalities. Indeed, new research opportunities have emerged for establishing in vitro culture for studying human embryogenesis and for high-throughput pharmacological testing platforms and disease models to prevent defects in early stages of human development.

Cell chirality regulates intercellular junctions and endothelial permeability.

Cell chirality is a newly discovered intrinsic property of the cell, reflecting the bias of the cell to polarize in the left-right axis. Despite increasing evidence on its substantial role in the asymmetric development of embryos, little is known about implications of cell chirality in physiology and disease. We demonstrate that cell chirality accounts for the nonmonotonic, dose-response relationship between endothelial permeability and protein kinase C (PKC) activation. The permeability of the endothelial cell layer is tightly controlled in our body, and dysregulation often leads to tissue inflammation and diseases. Our results show that low-level PKC activation is sufficient to reverse cell chirality through phosphatidylinositol 3-kinase/AKT signaling and alters junctional protein organization between cells with opposite chirality, leading to an unexpected substantial change in endothelial permeability. Our findings suggest that cell chirality regulates intercellular junctions in important ways, providing new opportunities for drug delivery across tightly connected semipermeable cellular sheets.

Intrinsic cellular chirality regulates left-right symmetry breaking during cardiac looping.

The vertebrate body plan is overall symmetrical but left-right (LR) asymmetric in the shape and positioning of internal organs. Although several theories have been proposed, the biophysical mechanisms underlying LR asymmetry are still unclear, especially the role of cell chirality, the LR asymmetry at the cellular level, on organ asymmetry. Here with developing chicken embryos, we examine whether intrinsic cell chirality or handedness regulates cardiac C looping. Using a recently established biomaterial-based 3D culture platform, we demonstrate that chick cardiac cells before and during C looping are intrinsically chiral and exhibit dominant clockwise rotation in vitro. We further show that cells in the developing myocardium are chiral as evident by a rightward bias of cell alignment and a rightward polarization of the Golgi complex, correlating with the direction of cardiac tube rotation. In addition, there is an LR polarized distribution of N-cadherin and myosin II in the myocardium before the onset of cardiac looping. More interestingly, the reversal of cell chirality via activation of the protein kinase C signaling pathway reverses the directionality of cardiac looping, accompanied by a reversal in cellular biases on the cardiac tube. Our results suggest that myocardial cell chirality regulates cellular LR symmetry breaking in the heart tube and the resultant directionality of cardiac looping. Our study provides evidence of an intrinsic cellular chiral bias leading to LR symmetry breaking during directional tissue rotation in vertebrate development.