Large-Scale Preparation of Hair Follicle Germs Using a Microfluidic Device.

Hair follicle morphogenesis during embryonic development is driven by the formation of hair follicle germs (HFGs) via interactions between epithelial and mesenchymal cells. Bioengineered HFGs are potential tissue grafts for hair regenerative medicine because they can replicate interactions and hair follicle morphogenesis after transplantation. However, a mass preparation approach for HFGs is necessary for clinical applications, given that thousands of de novo hair follicles are required to improve the appearance of a single patient with alopecia. In this study, we developed a microfluidics-based approach for the large-scale preparation of HFGs. A simple flow-focusing microfluidic device allowed collagen solutions containing epithelial and mesenchymal cells to flow and generate collagen microbeads with distinct Janus structures. During the 3 days of culture, the collagen beads contracted owing to cellular traction forces, resulting in collagen- and cell-dense HFGs. The transplantation of HFGs into nude mice resulted in highly efficient de novo hair follicle regeneration. This method provides a scalable and robust tissue graft preparation approach for hair regeneration.

Biomimetic Vasculatures by 3D-Printed Porous Molds.

Despite recent advances in biofabrication, recapitulating complex architectures of cell-laden vascular constructs remains challenging. To date, biofabricated vascular models have not yet realized four fundamental attributes of native vasculatures simultaneously: freestanding, branching, multilayered, and perfusable. In this work, a microfluidics-enabled molding technique combined with coaxial bioprinting to fabricate anatomically relevant, cell-laden vascular models consisting of hydrogels is developed. By using 3D porous molds of poly(ethylene glycol) diacrylate as casting templates that gradually release calcium ions as a crosslinking agent, freestanding, and perfusable vascular constructs of complex geometries are fabricated. The bioinks can be tailored to improve the compatibility with specific vascular cells and to tune the mechanical modulus mimicking native blood vessels. Crucially, the integration of relevant vascular cells (such as smooth muscle cells and endothelial cells) in a multilayer and biomimetic configuration is highlighted. It is also demonstrated that the fabricated freestanding vessels are amenable for testing percutaneous coronary interventions (i.e., drug-eluting balloons and stents) under physiological mechanical states such as stretching and bending. Overall, a versatile fabrication technique with multifaceted possibilities of generating biomimetic vascular models that can benefit future research in mechanistic understanding of cardiovascular diseases and the development of therapeutic interventions is introduced.

A Micropatterned Human-Specific Neuroepithelial Tissue for Modeling Gene and Drug-Induced Neurodevelopmental Defects.

The generation of structurally standardized human pluripotent stem cell (hPSC)-derived neural embryonic tissues has the potential to model genetic and environmental mediators of early neurodevelopmental defects. Current neural patterning systems have so far focused on directing cell fate specification spatio-temporally but not morphogenetic processes. Here, the formation of a structurally reproducible and highly-organized neuroepithelium (NE) tissue is directed from hPSCs, which recapitulates morphogenetic cellular processes relevant to early neurulation. These include having a continuous, polarized epithelium and a distinct invagination-like folding, where primitive ectodermal cells undergo E-to-N-cadherin switching and apical constriction as they acquire a NE fate. This is accomplished by spatio-temporal patterning of the mesoendoderm, which guides the development and self-organization of the adjacent primitive ectoderm into the NE. It is uncovered that TGFß signaling emanating from endodermal cells support tissue folding of the prospective NE. Evaluation of NE tissue structural dysmorphia, which is uniquely achievable in the model, enables the detection of apical constriction and cell adhesion dysfunctions in patient-derived hPSCs as well as differentiating between different classes of neural tube defect-inducing drugs.

Substrate stiffness modulates the multipotency of human neural crest derived ectomesenchymal stem cells via CD44 mediated PDGFR signaling.

Mesenchymal stem cells (MSCs) have been isolated from various mesodermal and ectodermal tissues. While the phenotypic and functional heterogeneity of MSCs stemming from their developmental origins has been acknowledged, the genetic and environmental factors underpinning these differences are not well-understood. Here, we investigated whether substrate stiffness mediated mechanical cues can directly modulate the development of ectodermal MSCs (eMSCs) from a precursor human neural crest stem cell (NCSC) population. We showed that NCSC-derived eMSCs were transcriptionally and functionally distinct from mesodermal bone marrow MSCs. eMSCs derived on lower substrate stiffness specifically increased their expression of the MSC marker, CD44 in a Rho-ROCK signaling dependent manner, which resulted in a concomitant increase in the eMSCs' adipogenic and chondrogenic differentiation potential. This mechanically-induced effect can only be maintained for short-term upon switching back to a stiff substrate but can be sustained for longer-term when the eMSCs were exclusively maintained on soft substrates. We also discovered that CD44 expression modulated eMSC self-renewal and multipotency via the downregulation of downstream platelet-derived growth factor receptor beta (PDGFRß) signaling. This is the first instance demonstrating that substrate stiffness not only influences the differentiation trajectories of MSCs but also their derivation from upstream progenitors, such as NCSCs.

GSK3ß negatively regulates TRAX, a scaffold protein implicated in mental disorders, for NHEJ-mediated DNA repair in neurons.

Translin-associated protein X (TRAX) is a scaffold protein with various functions and has been associated with mental illnesses, including schizophrenia. We have previously demonstrated that TRAX interacts with a Gsα protein-coupled receptor, the A2A adenosine receptor (A2AR), and mediates the function of this receptor in neuritogenesis. In addition, stimulation of the A2AR markedly ameliorates DNA damage evoked by elevated oxidative stress in neurons derived from induced pluripotent stem cells (iPSCs). Here, we report that glycogen synthase kinase 3 beta (GSK3ß) and disrupted-in-schizophrenia 1 (DISC1) are two novel interacting proteins of TRAX. We present evidence to suggest that the stimulation of A2AR markedly facilitated DNA repair through the TRAX/DISC1/GSK3ß complex in a rat neuronal cell line (PC12), primary mouse neurons, and human medium spiny neurons derived from iPSCs. A2AR stimulation led to the inhibition of GSK3ß, thus dissociating the TRAX/DISC1/GSK3ß complex and facilitating the non-homologous end-joining pathway (NHEJ) by enhancing the activation of a DNA-dependent protein kinase via phosphorylation at Thr2609. Similarly, pharmacological inhibition of GSK3ß by SB216763 also facilitated the TRAX-mediated repair of oxidative DNA damage. Collectively, GSK3ß binds with TRAX and negatively affects its ability to facilitate NHEJ repair. The suppression of GSK3ß by A2AR activation or a GSK3ß inhibitor releases TRAX for the repair of oxidative DNA damage. Our findings shed new light on the molecular mechanisms underlying diseases associated with DNA damage and provides a novel target (i.e., the TRAX/DISC1/GSK3ß complex) for future therapeutic development for mental disorders.