EFFECTS OF FLUID SHEAR STRESS ON MESENCHYMAL STEM CELL CONTRACTILITY AND FATE

Ph.DPH.D. IN MECHANOBIOLOGY (FOS

Prepubertal Buffalo (Bubalus bubalis) Leydig Cells: Isolation, Culture and Characterization

Abstract: Water buffalo (Bubalus bubalis) is an economically important livestock species in India. Male buffaloes display delayed sexual maturity as compared to the bulls (Bos taurus). Serum testosterone level, the key regulator of sexual maturity of males, is reported to be low in male buffaloes in comparison to bulls. Testosterone secretion and progression of spermatogenesis is mediated essentially by Leydig cells in the males. Establishment of primary culture for buffalo Leydig cells can provide an excellent tool to investigate the factors which regulate testicular steroidogenesis. Therefore, the objectives of the present study were to isolate, culture and characterize buffalo Leydig cells. Immunohistological analysis revealed that cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) specifically mark the Leydig cells in prepubertal buffalo testis. Using enzymatic digestion and Percoll density gradient centrifugation, a cell population that consisted of approximately 95% pure Leydig cells was obtained as indicated by CYP11A1 staining. Purified Leydig cells were cultured in DMEM/F12 supplemented with 10% foetal bovine serum (FBS) for 72 h. The cultured Leydig cells proliferated, expressed Leydig-cell specific transcripts (STAR, HSD3B1, HSD3B6, and CYP17A1) and proteins (CYP11A1, HSD3B and LHCGR), and secreted testosterone. It was concluded from the present study that buffalo Leydig cells can be maintained in culture for 72 h. The primary culture of buffalo Leydig cells can be used for studying acute responses, biochemical properties and other factors regulating testicular steroidogenesis, independent of other testicular cell types

Editorial: Materials for mechanotransduction and beyond

No abstract available

Soft tubular microfluidics for 2D and 3D applications

Microfluidics has been the key component for many applications, including biomedical devices, chemical processors, microactuators, and even wearable devices. This technology relies on soft lithography fabrication which requires cleanroom facilities. Although popular, this method is expensive and labor-intensive. Furthermore, current conventional microfluidic chips precludes reconfiguration, making reiterations in design very time-consuming and costly. To address these intrinsic drawbacks of microfabrication, we present an alternative solution for the rapid prototyping of microfluidic elements such as microtubes, valves, and pumps. In addition, we demonstrate how microtubes with channels of various lengths and cross-sections can be attached modularly into 2D and 3D microfluidic systems for functional applications. We introduce a facile method of fabricating elastomeric microtubes as the basic building blocks for microfluidic devices. These microtubes are transparent, biocompatible, highly deformable, and customizable to various sizes and cross-sectional geometries. By configuring the microtubes into deterministic geometry, we enable rapid, low-cost formation of microfluidic assemblies without compromising their precision and functionality. We demonstrate configurable 2D and 3D microfluidic systems for applications in different domains. These include microparticle sorting, microdroplet generation, biocatalytic micromotor, triboelectric sensor, and even wearable sensing. Our approach, termed soft tubular microfluidics, provides a simple, cheaper, and faster solution for users lacking proficiency and access to cleanroom facilities to design and rapidly construct microfluidic devices for their various applications and needs. Keywords: flexible microfluidics, elastomeric microtubes, microfluidic assemblies, inertial focusing chip, microfluidic sensorSingapore-MIT Alliance for Research and Technology (SMART

Cellular and molecular characterization of the corneal epithelium in Xenopus frogs

The vertebrate cornea is a transparent, avascular tissue that forms the front window of the eye. Specifically, the corneal epithelium is exposed to detrimental conditions including infections, solar (Ultraviolet, UV) irradiation, mechanical injuries, and undergoes constant self-renewal. Corneal epithelial stem cells (CESCs) and their progeny, the transit amplifying cells (TACs), play a prominent role in the maintenance of corneal homeostasis, transparency, and wound repair processes. Towards this end, my thesis focuses on understanding the molecular signature of the corneal epithelium using the frog, Xenopus laevis, with the goal to explore novel markers that will reliably identify populations of CESCs and TACs. In Chapter 2, I start by examining the expression of known corneal biomarkers (previously reported in literature from corneal studies of various vertebrates) in the vertebrate species, the African clawed frog. Here, I used antibody-based immunohistochemical staining to molecularly characterize the expression of nine proteins in the corneas of both Xenopus larvae and post-metamorphic adults. I found that localization of some markers changes between tadpole and juvenile adult stages. Markers such as p63, Keratin19, and beta1-integrin are restricted to basal corneal epithelial cells of the larvae. After metamorphosis their expression is found in basal and intermediate layer cells of the adult frog cornea epithelium. Another protein, Pax6 was expressed in the larval corneas, but surprisingly it was not detectable in the adult corneal epithelium. For the first time we report that Tcf7l2 can be used as a marker to differentiate cornea vs. skin in frogs. Tcf7l2 is present only in the frog skin, which differs from reports indicating that the protein is expressed in the human cornea. Furthermore, I identified the transition between the inner, and the outer surface of the adult frog eyelid as a key boundary in terms of marker expression. Although these markers are useful to identify different regions and cellular layers of the frog corneal epithelium, none was unique to CESCs or TACs. The results of this study substantiate findings from other studies in the field indicating that there may not be a single conserved, specific CESC marker in vertebrates. To overcome the limitation of candidate-based biomarker characterization, however, I undertook single-cell genomics approaches to understand the temporal cell atlas of the frog cornea (Chapter 3). Using ~22,000 corneal cells isolated from two distinct developmental time-points, this work provides key insights about the amphibian corneal transcriptome. The data also reveals several novel genes expressed in corneal cells and spatiotemporal changes in gene expression during corneal differentiation. In addition, the data helps in understanding the developmental trajectory of corneal cells during development and differentiation, and identifies key gene regulatory networks that are involved in corneal maturation. In conclusion, this work provides a detailed molecular characterization of the Xenopus corneal epithelium and establishes distinct and newly identified biomarkers of corneal cellular layers. Although a single biomarker for CESCs was not identified in this work (and may not even exist), it helps identify a range of proteins that could be tested for their functional role in regulating the population of corneal stem cells in vertebrates, including humans. Furthermore, this work will be valuable for future studies to understand the critical factors that regulate cornea epithelial cells and lens regeneration, the latter phenomenon being unique to the larval stages of Xenopus frogs.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste

The yeast stress inducible Ssa Hsp70 reduces α-synuclein toxicity by promoting its degradation through autophagy.

The mechanism underlying the role of Hsp70s in toxicity associated with intracellular accumulation of toxic protein inclusions is under intense investigation. In current study, we examined the roles of all different isoforms of yeast cytosolic Ssa Hsp70 on α-synuclein mediated cellular toxicity. The study showed that yeast cells expressing stress-inducible Ssa3 or Ssa4 as sole Ssa Hsp70 isoforms, reduced α-synuclein toxicity better than those expressing a constitutive counterpart. The protective effect of stress-inducible Ssa Hsp70s was not α-syn specific, but more general to other inclusion forming proteins such as polyQ. We show that the protective effect is not by induction of a general stress response in Ssa3 cells rather by promoting α-synuclein degradation through autophagy. The present study revealed that effect of Hsp70s was isoform dependent, and that autophagy protects Ssa3 cells from the deleterious effects of toxic protein inclusions

Emergent patterns of collective cell migration under tubular confinement

Collective epithelial behaviours are studied in vitro in the context of flat sheets but a system to mimic tubular systems is lacking. Here, the authors develop a method to study collective behaviour in lumenal structures and show that several features depend on the extent of tubular confinement and/or curvature

Direct measurement of near‐nano‐Newton forces developed by self‐organizing actomyosin fibers bound α‐catenin

International audienceBackground Information: Actin cytoskeleton contractility plays a critical role in morphogenetic processes by generating forces that are then transmitted to cell-cell and cell-ECM adhesion complexes. In turn, mechanical properties of the environment are sensed and transmitted to the cytoskeleton at cell adhesion sites, influencing cellular processes such as cell migration, differentiation and survival. Anchoring of the actomyosin cytoskeleton to adhesion sites is mediated by adaptor proteins such as talin or α-catenin that link F-actin to transmembrane cell adhesion receptors, thereby allowing mechanical coupling between the intracellular and extracellular compartments. Thus, a key issue is to be able to measure the forces generated by actomyosin and transmitted to the adhesion complexes. Approaches developed in cells and those probing single molecule mechanical properties of α-catenin molecules allowed to identify α-catenin, an F-actin binding protein which binds to the cadherin complexes as a major player in cadherin-based mechanotransduction. However, it is still very difficult to bridge intercellular forces measured at cellular levels and those measured at the single-molecule level. Results: Here, we applied an intermediate approach allowing reconstruction of the actomyosin-α-catenin complex in acellular conditions to probe directly the transmitted forces. For this, we combined micropatterning of purified α-catenin and spontaneous actomyosin network assembly in the presence of G-actin and Myosin II with microforce sensor arrays used so far to measure cell-generated forces. Conclusions: Using this method, we show that self-organizing actomyosin bundles bound to micrometric α-catenin patches can apply near-nano-Newton forces. Significance: Our results pave the way for future studies on molecular/cellular mechanotransduction and mechanosensing

Bioengineering a Miniaturized In Vitro 3D Myotube Contraction Monitoring Chip For Modelization of Muscular Dystrophies

ABSTRACT Quantification of skeletal muscle functional strength is essential to assess the outcomes of therapeutic procedures for muscular disorders. Several muscle three-dimensional “Organ-on-chip” models have been developed to measure the generated force. Yet, these technologies require a substantial amount of biological material, which is problematic in the context of limited patient sample. Here we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity. Combination of light-induced molecular adsorption technology and optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled physical and chemical microenvironments. Spontaneous twitch contractions in 3D myotubes derived from primary human myoblasts were observed, the generated force was measured and the contraction pattern characterized. In addition, the impact of three-dimensional culture on nuclear morphology was analyzed, confirming the similarity in organization between the obtained 3D myotubes and in vivo myofibers. Our system enabled to model LMNA -related Congenital Muscular Dystrophy (L-CMD) with successful development of mutant 3D myotubes displaying contractile dysfunction. We anticipate that this technology shall be used to study contraction characteristics and evaluate how specific diseases affect muscle organization and force generation. Our downsized model system might allow to substantially improve drug screening capability for therapeutic oriented research