Cadherin-11 Influences Differentiation in Human Mesenchymal Stem Cells by Regulating the Extracellular Matrix Via the TGFβ1 Pathway

For regenerative medicine, directing stem cell fate is one of the key aims. Human mesenchymal stem cells (hMSCs) are versatile adult stem cells that have been proposed for several clinical applications, making directing their fate of utmost importance. For most clinical applications, their differentiation toward the adipogenic lineage is an undesired outcome. Understanding the mechanisms that regulate hMSC commitment toward the adipogenic lineage might help open up new avenues for fine-tuning implanted hMSCs for regenerative medicine applications. We know that cadherin-11 is required for hMSC commitment to the adipogenic lineage; therefore, we sought to investigate the mechanisms through which cadherin-11 regulates adipogenic differentiation. We observed that hMSCs lacking cadherin-11 had decreased expression of type VI collagen and increased expression of fibronectin. We provide evidence of increased transforming growth factor beta 1 and the subsequent translocation of phosphorylated SMAD2/3 into the nucleus by cells that lack cadherin-11, which could be attributed to the changes in extracellular matrix composition. Taken together, our study implicates cadherin-11 in regulating extracellular matrix production and thereby helping improve cell- and material-based regenerative medicine approaches

A single-cell RNA-seq analysis unravels the heterogeneity of primary cultured human corneal endothelial cells

Abstract The cornea is a transparent and avascular tissue located in front of the eye. Its inner surface is lined by a monolayer of corneal endothelial cells (CECs), which maintain the cornea transparency. CECs remain arrested in a non-proliferative state and damage to these cells can compromise their function leading to corneal opacity. The primary culture of donor-derived CECs is a promising cell therapy. It confers the potential to treat multiple patients from a single donor, alleviating the global donor shortage. Nevertheless, this approach has limitations preventing its adoption, particularly culture protocols allow limited expansion of CECs and there is a lack of clear parameters to identify therapy-grade CECs. To address this limitation, a better understanding of the molecular changes arising from the primary culture of CECs is required. Using single-cell RNA sequencing on primary cultured CECs, we identify their variable transcriptomic fingerprint at the single cell level, provide a pseudo-temporal reconstruction of the changes arising from primary culture, and suggest markers to assess the quality of primary CEC cultures. This research depicts a deep transcriptomic understanding of the cellular heterogeneity arising from the primary expansion of CECs and sets the basis for further improvement of culture protocols and therapies

Oxygen and nutrient delivery in tissue engineering:Approaches to graft vascularization

The field of tissue engineering is making great strides in developing replacement tissue grafts for clinical use, marked by the rapid development of novel biomaterials, their improved integration with cells, better-directed growth and differentiation of cells, and improved three-dimensional tissue mass culturing. One major obstacle that remains, however, is the lack of graft vascularization, which in turn renders many grafts to fail upon clinical application. With that, graft vascularization has turned into one of the holy grails of tissue engineering, and for the majority of tissues, it will be imperative to achieve adequate vascularization if tissue graft implantation is to succeed. Many different approaches have been developed to induce or augment graft vascularization, both in vitro and in vivo. In this review, we highlight the importance of vascularization in tissue engineering and outline various approaches inspired by both biology and engineering to achieve and augment graft vascularization

Corrigendum: Win, Lose or Tie: Mathematical Modeling of Ligand Competition at the Cell-Extracellular Matrix Interface:Win, Lose or Tie: Mathematical Modeling of Ligand Competition at the Cell-Extracellular Matrix Interface

[This corrects the article DOI: 10.3389/fbioe.2021.657244.]

Win, Lose, or Tie: Mathematical Modeling of Ligand Competition at the Cell–Extracellular Matrix Interface

Integrin transmembrane proteins conduct mechanotransduction at the cell–extracellular matrix (ECM) interface. This process is central to cellular homeostasis and therefore is particularly important when designing instructive biomaterials and organoid culture systems. Previous studies suggest that fine-tuning the ECM composition and mechanical properties can improve organoid development. Toward the bigger goal of fully functional organoid development, we hypothesize that resolving the dynamics of ECM–integrin interactions will be highly instructive. To this end, we developed a mathematical model that enabled us to simulate three main interactions, namely integrin activation, ligand binding, and integrin clustering. Different from previously published computational models, we account for the binding of more than one type of ligand to the integrin. This competition between ligands defines the fate of the system. We have demonstrated that an increase in the initial concentration of ligands does not ensure an increase in the steady state concentration of ligand-bound integrins. The ligand with higher binding rate occupies more integrins at the steady state than does the competing ligand. With cell type specific, quantitative input on integrin-ligand binding rates, this model can be used to develop instructive cell culture systems

Win, Lose, or Tie: Mathematical Modeling of Ligand Competition at the Cell-Extracellular Matrix Interface

Integrin transmembrane proteins conduct mechanotransduction at the cell-extracellular matrix (ECM) interface. This process is central to cellular homeostasis and therefore is particularly important when designing instructive biomaterials and organoid culture systems. Previous studies suggest that fine-tuning the ECM composition and mechanical properties can improve organoid development. Toward the bigger goal of fully functional organoid development, we hypothesize that resolving the dynamics of ECM-integrin interactions will be highly instructive. To this end, we developed a mathematical model that enabled us to simulate three main interactions, namely integrin activation, ligand binding, and integrin clustering. Different from previously published computational models, we account for the binding of more than one type of ligand to the integrin. This competition between ligands defines the fate of the system. We have demonstrated that an increase in the initial concentration of ligands does not ensure an increase in the steady state concentration of ligand-bound integrins. The ligand with higher binding rate occupies more integrins at the steady state than does the competing ligand. With cell type specific, quantitative input on integrin-ligand binding rates, this model can be used to develop instructive cell culture systems

Quantitative PCR established mRNA expression in hMSCs in three different chondrogenesis models (pellet culture, micromass culture, or a type II collagen hydrogel) in either growth or chondrogenic medium over a time-course of 21 days.

<p>A high expression of ACAN (A-C) and <i>HSPG2</i> (D-F) are markers of a hyaline cartilage ECM and pericellular matrix, respectively. RUNX2 (G-I) and SOX9 (J-L) are markers of osteogenesis and chondrogenesis, respectively. Data are transcript expression relative to GAPDH as an internal control (N.D.: not detected). Black squares: hMSCs cultured in growth medium, red circles: hMSCs cultured in chondrogenic medium. Data are the mean values of <i>N</i>=3 independent experiments performed in technical triplicate. Error bars indicate the range of values. Statistical significance is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082035#pone.0082035.s015" target="_blank">Figures S15-S16</a>.</p

To study the changing expression of integrins during chondrogenic differentiation, human mesenchymal stem cells (hMSCs) were cultured in three different chondrogenesis models: pellet culture, micromass, and type II collagen hydrogels under two different conditions: growth medium and chondrogenic medium.

<p>To study the changing expression of integrins during chondrogenic differentiation, human mesenchymal stem cells (hMSCs) were cultured in three different chondrogenesis models: pellet culture, micromass, and type II collagen hydrogels under two different conditions: growth medium and chondrogenic medium.</p

Lentiviral particles containing shRNA against ITGB8 were used to investigate its role during <i>in</i><i>vitro</i> chondrogenesis in the micromass model.

<p>A knockdown efficiency of 89% after puromycin selection was determined by qPCR (A) and confirmed by Western blot (B). All chondrogenic phenotype markers were affected by reduced ITGB8. In particular, hyaline cartilage ECM markers ACAN (C) and COL2A1 (D) were down-regulated, while pericellular matrix markers COL6A1 (E) and <i>HSPG2</i> (F) were up-regulated. Non-hyaline cartilage markers COL1A1 (G) and COL10A1 (H) were down-regulated. The transcription factors RUNX2 (I) and SOX9 (J) were also down-regulated. Black squares: growth medium, red circles: chondrogenic medium. Solid lines: wildtype, dashed line: ITGB8 knockdown. Data represent mean and range of values relative to GAPDH for <i>N</i>=2 or 3 experiments performed in technical triplicate. Statistical significance is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082035#pone.0082035.s018" target="_blank">Figure S18</a>.</p