Chenyu Huang

Biomaterials as carrier, barrier and reactor for cell-based regenerative medicine.

Cell therapy has achieved tremendous success in regenerative medicine in the past several decades. However, challenges such as cell loss, death and immune-rejection after transplantation still persist. Biomaterials have been designed as carriers to deliver cells to desirable region for local tissue regeneration; as barriers to protect transplanted cells from host immune attack; or as reactors to stimulate host cell recruitment, homing and differentiation. With the assistance of biomaterials, improvement in treatment efficiency has been demonstrated in numerous animal models of degenerative diseases compared with routine free cell-based therapy. Emerging clinical applications of biomaterial assisted cell therapies further highlight their great promise in regenerative therapy and even cure for complex diseases, which have been failed to realize by conventional therapeutic approaches.

Engineering EMT using 3D micro-scaffold to promote hepatic functions for drug hepatotoxicity evaluation.

Accompanied by decreased hepatic functions, epithelial-mesenchymal transition (EMT) was observed in two dimensional (2D) cultured hepatocytes with elongated morphology, loss of polarity and weakened cell-cell interaction, while upgrading to 3D culture has been considered as significant improvement of its 2D counterpart for hepatocyte maintenance. Here we hypothesize that 3D culture enhances hepatic functions through regulating the EMT status. Biomaterial-engineered EMT was achieved by culturing HepaRG as 3D spheroids (SP-3D) or 3D stretched cells (ST-3D) in non-adherent and adherent micro-scaffold respectively. In SP-3D, constrained EMT of HepaRG, a hepatic stem cell line, as represented by increased epithelial markers and decreased mesenchymal markers, was echoed by improved hepatic functions. To investigate the relationship between EMT status and hepatic functions, time-series RNA-Seq and gene network analysis were used for comparing different cell culture models, which identified histone deacetylases (HDACs) as key mediating factors. Protein analysis confirmed that high HDAC activity was correlated with high expression of Cadherin-1 (CDH1) and hepatic function genes, which were decreased upon HDAC inhibitor treatment in SP-3D, suggesting HDACs may play positive role in regulating EMT and hepatic functions. To illustrate the application of 3D micro-scaffold culture in drug safety evaluation, hepatotoxicity and metabolism assays of two hepatotoxins (i.e. N-acetyl-p-aminophenol and Doxorubicin) were performed and SP-3D showed more biomimetic toxicity response, indicating regulation of EMT as a vital consideration in designing 3D hepatocyte culture configuration.

Substrate stiffness orchestrates epithelial cellular heterogeneity with controlled proliferative pattern via E-cadherin/β-catenin mechanotransduction

UNLABELLED Epithelial cellular heterogeneity has been observed in pathological tissues with abnormal matrix stiffness and cells cultured on rigid substrates. However, it remains unclear how matrix stiffness influences cellular heterogeneity formation in multi-cellular population. Here, we demonstrated that cellular heterogeneity regulated by substrate stiffness is evident starting from the initial single-cell stage (indicated by cellular Youngs modulus and morphology) until the resulting multi-cellular stage (indicated by cellular functions) through distinguished proliferative patterns. Epithelial cells on soft substrate proliferated in a neighbor-dependent manner with stronger E-cadherin expression and more homogeneous E-cadherin/β-catenin localization compared to those on coverslips, which resulted in reduced heterogeneity in downstream cellular functions of the multi-cellular population. In particular, decreased heterogeneity in human embryonic stem cells upon expansion and endodermal induction was achieved on soft substrate. Overall, our work provides new insights on mechanotransduction during epithelial proliferation which regulates the formation of cellular heterogeneity and potentially provides a highly efficient approach to regulate stem cell fate by fine-tuning substrate stiffness. STATEMENT OF SIGNIFICANCE This study demonstrates that cellular heterogeneity regulated by substrate stiffness is evident starting from the initial single-cell stage until the resulting multi-cellular stage through distinguished proliferative patterns. During this process, E-cadherin/β-catenin mechanotransduction is found to play important role in substrate stiffness-regulated epithelial cellular heterogeneity formation. In particular, decreased heterogeneity in human embryonic stem cells upon expansion and endodermal induction is achieved on soft substrate. Hence, we believe that this work not only provides new insights on mechanotransduction of E-cadherin/β-catenin which regulates the formation of cellular heterogeneity during proliferation, but also potentially provides a highly efficient approach to regulate stem cell fate by fine-tuning substrate stiffness.

Preconditioning of mesenchymal stromal cells toward nucleus pulposus-like cells by microcryogels-based 3D cell culture and syringe-based pressure loading system.

To precondition mesenchymal stromal/stem cells (MSCs) with mechanical stimulation may enhance cell survival and functions following implantation in load bearing environment such as nucleus pulposus (NP) in intervertebral disc (IVD). In this study, preconditioning of MSCs toward NP-like cells was achieved in previously developed poly (ethylene glycol) diacrylate (PEGDA) microcryogels (PMs) within a syringe-based three-dimensional (3D) culture system which provided a facile and cost-effective pressure loading approach. PMs loaded with alginate and MSCs could be incubated in a sealable syringe which could be air-compressed to apply pressure loading through a programmable syringe pump. Expression levels of chondrogenic marker genes SOX9, COL II, and ACAN were significantly upregulated in MSCs when pressure loading of 0.2 MPa or 0.8 MPa was implemented. Expression levels of COL I and COL X were downregulated when pressure loading was applied. In a nude mouse model, MSCs loaded in PMs mechanically stimulated for three days were subcutaneously injected using the same culture syringe. Three weeks postinjection, more proteoglycans (PGs) were deposited and more SOX9 and COL II but less COL I and COL X were stained in 0.2 MPa group. Furthermore, injectable MSCs-loaded PMs were utilized in an ex vivo rabbit IVD organ culture model that demonstrated the leak-proof function and enhanced cell retention of PMs assisted cell delivery to a load bearing environment for potential NP regeneration. This microcryogels-based 3D cell culture and syringe-based pressure loading system represents a novel method for 3D cell culture with mechanical stimulation for better function.

Keloid progression: a stiffness gap hypothesis.

Keloids are fibroproliferative skin disorders characterised clinically by continuous horizontal progression and post‐surgical recurrence and histologically by the accumulation of collagen and fibroblast ingredients. Till now, their aetiology remains clear, which may cover genetic, environmental and metabolic factors. Evidence in the involvement of local mechanics (e.g. predilection site and typical shape) and the progress in mechanobiology have incubated our stiffness gap hypotheses in illustrating the chronic but constant development in keloid. We put forward that the enlarged gap between extracellular matrix (ECM) stiffness and cellular stiffness potentiates keloid progression. Matrix stiffness itself provides organisational guidance cues to regulate the mechanosensitive resident cells (e.g. proliferation, migration and apoptosis). During this dynamic process, the ECM stiffness and cell stiffness are not well balanced, and the continuously enlarged stiffness gap between them potentiates keloid progression. The cushion factors, such as prestress for cell stiffness and topology for ECM stiffness, serve as compensations, the decompensation of which aggravates keloid development. It can well explain the typical shape of keloids, their progression in a horizontal but not vertical direction and the post‐surgical recurrence, which were evidenced by our clinical cases. Such a stiffness gap hypothesis might be bridged to mechanotherapeutic approaches for keloid progression.

Endothelial dysfunction and mechanobiology in pathological cutaneous scarring: lessons learned from soft tissue fibrosis

Hypertrophic scars/keloids are pathogenic scars that are characterized by collagen and fibroblast accumulation. The endothelia in the lesions are mechanosensitive and participate actively in the pathogenesis of these scars. The present review summarizes how endothelium responds to mechanical stimuli such as shear, stretch and hydrostatic pressure. It also shows that in heart, liver, kidney and lung fibrosis, endothelial dysfunctions (EDs) associate with changes in vascular tone, endothelial permeability, coagulation and vasomodulation that result in inflammation, hypertension and coagulation. Pathological scars exhibit similar EDs during their development and progression. Mechanopharmaceutical or mechanotherapeutic interventions that rescue EDs may be useful scar treatments.

Gene Therapy in Pathologic Scars

Hypertrophic scars (HSs) and keloids, as the commonly seen pathological scars, are characteristic of rampant proliferation of fibroblasts, accumulation of collagens in the ECM, and altered regulation of growth factors/cytokines. Though their etiology still remains unclear and the postsurgical recurrence keeps a high profile, great efforts have been made on potential gene therapies for more specific, long-lived and hopefully cheaper results, thanks to the update progress in gene therapy techniques. Here we classify the current gene therapy strategies for HSs and keloids on their targets of cellular behavior, extracellular collagen production, or growth factors/chemokines involved. Namely, (1) gene therapies that alter the balance between cellular proliferation and apoptosis by inducing fibroblast apoptosis or decreasing its proliferation, using suicide gene tools or manipulating miRNA-21 levels respectively; (2) gene therapies that alter the balance between collagen synthesis and degradation focusing on directly reducing collagen production through miRNA-29b or inducing collagen degradation through TIMP siRNAs, or decreasing collagen accumulation through PAI-1-targeting siRNAs or HSP47-siRNAs. In particular, the interfibrillar bonding of collagens is also successfully regulated by antisense oligonucleotides of the non-collagen matrix ingredients (e.g. decorin) and thereby interfering in collagen remodeling; and (3) gene therapies that alter the balance between pro-fibrotic and anti-fibrotic growth factors/cytokines. Different TGF-β subtypes, their receptors, and the TGF-β signaling pathway molecules demonstrate to be potential gene therapy candidates in scar treatments. A better understanding of the current progress in gene therapies for pathological scars will lead to the development of novel interventions that can prevent, reduce, or even reverse the formation and/or progression of HSs or keloids.

Engineering Embryonic Stem Cell Microenvironments for Tailored Cellular Differentiation

The rapid progress of embryonic stem cell (ESCs) research offers great promise for drug discovery, tissue engineering, and regenerative medicine. However, a major limitation in translation of ESCs technology to pharmaceutical and clinical applications is how to induce their differentiation into tailored lineage commitment with satisfactory efficiency. Many studies indicate that this lineage commitment is precisely controlled by the ESC microenvironment in vivo. Engineering and biomaterial-based approaches to recreate a biomimetic cellular microenvironment provide valuable strategies for directing ESCs differentiation to specific lineages in vitro. In this review, we summarize and examine the recent advances in application of engineering and biomaterial-based approaches to control ESC differentiation. We focus on physical strategies (e.g., geometrical constraint, mechanical stimulation, extracellular matrix (ECM) stiffness, and topography) and biochemical approaches (e.g., genetic engineering, soluble bioactive factors, coculture, and synthetic small molecules), and highlight the three-dimensional (3D) hydrogel-based microenvironment for directed ESC differentiation. Finally, future perspectives in ESCs engineering are provided for the subsequent advancement of this promising research direction.