Nanofiber curvature with Rho GTPase activity increases mouse embryonic fibroblast random migration velocity

Mechanotransduction arises from information encoded in the shape of materials such as curvature. It induces activation of small GTPase signaling affecting cell phenotypes including differentiation. We carried out a set of preliminary experiments to test the hypothesis that curvature (1/radius) would also affect cell motility due to signal pathway crosstalk. High molecular weight poly (methyl methacrylate) straight nanofibers were electrospun with curvature ranging from 41 to 1 μm−1 and collected on a passivated glass substrate. The fiber curvature increased mouse mesenchymal stem cell aspect ratio (P < 0.02) and decreased cell area (P < 0.01). Despite little effect on some motility patterns such as polarity and persistence, we found selected fiber curvatures can increase normalized random fibroblastic mouse embryonic cell (MEF) migration velocity close to 2.5 times compared with a flat surface (P < 0.001). A maximum in the velocity curve occurred near 2.5 μm−1 and may vary with the time since initiation of attachment to the surface (range of 0–20 h). In the middle range of fiber curvatures, the relative relationship to curvature was similar regardless of treatment with Rho-kinase inhibitor (Y27632) or cdc42 inhibitor (ML141), although it was decreased on most curvatures (P < 0.05). However, below a critical curvature threshold MEFs may not be able to distinguish shallow curvature from a flat surface, while still being affected by contact guidance. The preliminary data in this manuscript suggested the large low curvature fibers were interpreted in a manner similar to a non-curved surface. Thus, curvature is a biomaterial construct design parameter that should be considered when specific biological responses are desired. Statement of integration, innovation, and insight Replacement of damaged or diseased tissues that cannot otherwise regenerate is transforming modern medicine. However, the extent to which we can rationally design materials to affect cellular outcomes remains low. Knowing the effect of material stiffness and diameter on stem cell differentiation, we investigated cell migration and signaling on fibrous scaffolds. By investigating diameters across orders of magnitude (50–2000 nm), we identified a velocity maximum of ~800 nm. Furthermore, the results suggest large fibers may not be interpreted by single cells as a curved surface. This work presents insight into the design of constructs for engineering tissues.

Shaping axial identity during human pluripotent stem cell differentiation to neural crest cells

The neural crest (NC) is a multipotent cell population which can give rise to a vast array of derivatives including neurons and glia of the peripheral nervous system, cartilage, cardiac smooth muscle, melanocytes and sympathoadrenal cells. An attractive strategy to model human NC development and associated birth defects as well as produce clinically relevant cell populations for regenerative medicine applications involves the in vitro generation of NC from human pluripotent stem cells (hPSCs). However, in vivo, the potential of NC cells to generate distinct cell types is determined by their position along the anteroposterior (A–P) axis and, therefore the axial identity of hPSC-derived NC cells is an important aspect to consider. Recent advances in understanding the developmental origins of NC and the signalling pathways involved in its specification have aided the in vitro generation of human NC cells which are representative of various A–P positions. Here, we explore recent advances in methodologies of in vitro NC specification and axis patterning using hPSCs.

Emilin-2 is a component of bone marrow extracellular matrix regulating mesenchymal stem cell differentiation and hematopoietic progenitors

Background Dissection of mechanisms involved in the regulation of bone marrow microenvironment through cell–cell and cell–matrix contacts is essential for the detailed understanding of processes underlying bone marrow activities both under physiological conditions and in hematologic malignancies. Here we describe Emilin-2 as an abundant extracellular matrix component of bone marrow stroma. Methods Immunodetection of Emilin-2 was performed in bone marrow sections of mice from 30 days to 6 months of age. Emilin-2 expression was monitored in vitro in primary and mesenchymal stem cell lines under undifferentiated and adipogenic conditions. Hematopoietic stem cells and progenitors in bone marrow of 3- to 10-month-old wild-type and Emilin-2 null mice were analyzed by flow cytometry. Results Emilin-2 is deposited in bone marrow extracellular matrix in an age-dependent manner, forming a meshwork that extends from compact bone boundaries to the central trabecular regions. Emilin-2 is expressed and secreted by both primary and immortalized bone marrow mesenchymal stem cells, exerting an inhibitory action in adipogenic differentiation. In vivo Emilin-2 deficiency impairs the frequency of hematopoietic stem/progenitor cells in bone marrow during aging. Conclusion Our data provide new insights in the contribution of bone marrow extracellular matrix microenvironment in the regulation of stem cell niches and hematopoietic progenitor differentiation.

G-quadruplex DNA structures in human stem cells and differentiation

The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.

Surface Modification of Polycaprolactone Scaffold With Improved Biocompatibility and Controlled Growth Factor Release for Enhanced Stem Cell Differentiation

Polycaprolactone (PCL) has been widely used as a scaffold material for tissue engineering. Reliable applications of the PCL scaffolds require overcoming their native hydrophobicity and obtaining the sustained release of signaling factors to modulate cell growth and differentiation. Here, we report a surface modification strategy for electrospun PCL nanofibers using an azide-terminated amphiphilic graft polymer. With multiple alkylation and pegylation on the side chains of poly-L-lysine, stable coating of the graft polymer on the PCL nanofibers was achieved in one step. Using the azide-alkyne “click chemistry”, we functionalized the azide-pegylated PCL nanofibers with dibenzocyclooctyne-modified nanocapsules containing growth factor, which rendered the nanofiber scaffold with satisfied cell adhesion and growth property. Moreover, by specific immobilization of pH-responsive nanocapsules containing bone morphogenetic protein 2 (BMP-2), controlled release of active BMP-2 from the PCL nanofibers was achieved within 21 days. When bone mesenchyme stem cells were cultured on this nanofiber scaffold, enhanced ossification was observed in correlation with the time-dependent release of BMP-2. The established surface modification can be extended as a generic approach to hydrophobic nanomaterials for longtime sustainable release of multiplex signaling proteins for tissue engineering.

Integrating Soft Hydrogel with Nanostructures Reinforces Stem Cell Adhesion and Differentiation

Biophysical cues can regulate stem cell behaviours and have been considered as critical parameters of synthetic biomaterials for tissue engineering. In particular, hydrogels have been utilized as promising biomimetic and biocompatible materials to emulate the microenvironment. Therefore, well-defined mechanical properties of a hydrogel are important to direct desirable phenotypes of cells. Yet, limited research pays attention to engineering soft hydrogel with improved cell adhesive property, which is crucial for stem cell differentiation. Herein, we introduce silica nanoparticles (SiO2 NPs) onto the surface of methacrylated hyaluronic (MeHA) hydrogel to manipulate the presentation of cell adhesive ligands (RGD) clusters, while remaining similar bulk mechanical properties (2.79 ± 0.31 kPa) to that of MeHA hydrogel (3.08 ± 0.68 kPa). RGD peptides are either randomly decorated in the MeHA hydrogel network or on the immobilized SiO2 NPs (forming MeHA–SiO2). Our results showed that human mesenchymal stem cells exhibited a ~1.3-fold increase in the percentage of initial cell attachment, a ~2-fold increase in cell spreading area, and enhanced expressions of early-stage osteogenic markers (RUNX2 and alkaline phosphatase) for cells undergoing osteogenic differentiation with the osteogenic medium on MeHA–SiO2 hydrogel, compared to those cultured on MeHA hydrogel. Importantly, the cells cultivated on MeHA–SiO2 expressed a ~5-fold increase in nuclear localization ratio of the yes-associated protein, which is known to be mechanosensory in stem cells, compared to the cells cultured on MeHA hydrogel, thereby promoting osteogenic differentiation of stem cells. These findings demonstrate the potential use of nanomaterials into a soft polymeric matrix for enhanced cell adhesion and provide valuable guidance for the rational design of biomaterials for implantation.

Hbxip (Lamtor5) is essential for embryogenesis and regulates embryonic stem cell differentiation through activating mTORC1

Hbxip, also named Lamtor5, has been well characterized as a transcriptional coactivator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5. Using Hbxip knockout embryonic stem cells (ESCs), we showed that depletion of Hbxip compromises the self-renewal of ESCs, with reduced expression of pluripotency genes, reduced cell proliferation, and decreased colony forming capacity. In addition, Hbxip-/- ESCs are defective in differentiation, particularly ectodermal and mesodermal differentiation. Consistently, Hbxip-/- epiblast fails to differentiate properly, indicated by sustained expression of Oct4 in E8.5 Hbxip-/- epiblast. Mechanistically, in ESCs, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, display differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/-, and p18-/- mice, lacking subunits of the Ragulator complex, also share similar phenotypes, embryonic lethality and retarded growth around E7-8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.

Autophagy Regulates the Effects of Adipose-Derived Stem Cell Small Extracellular Vesicles On Lipopolysaccharide-Induced Pulmonary Microvascular Barrier Damage

Background: Small extracellular vesicles (sEVs) have been recognized to be more effective than direct stem cell differentiation into functional target cells in preventing tissue injury and promoting tissue repair. Our previous study demonstrated the protective effect of adipose-derived stem cells (ADSCs) on lipopolysaccharide (LPS)-induced acute lung injury and the effect of autophagy on ADSC functions, but the role of ADSC-derived sEVs (ADSC-sEVs) and autophagy-mediated regulation of ADSC-sEVs in LPS-induced pulmonary microvascular barrier damage remains unclear. Methods: After treatment with sEVs from ADSCs with or without autophagy inhibition, LPS-induced human pulmonary microvascular endothelial cell (HPMVECs) barrier damage was detected. LPS-induced acute lung injury in mice was assessed in vivo after intravenous administration of sEVs from ADSCs with or without autophagy inhibition. The effects of autophagy on the bioactive miRNA components of ADSC-sEVs were assessed after prior inhibition of cell autophagy. Results: We found that ADSC-sEV effectively alleviated LPS-induced apoptosis, tight junction damage and high permeability of PMVECs. Moreover, in vivo administration of ADSC-sEV markedly inhibited LPS-triggered lung injury. However, autophagy inhibition, markedly weakened the therapeutic effect of ADSC-sEVs on LPS-induced PMVECs barrier damage and acute lung injury. In addition, autophagy inhibition, prohibited the expression of five specific miRNAs in ADSC-sEVs -under LPS-induced inflammatory conditions. Conclusions: Our results indicate that ADSC-sEVs protect against LPS-induced pulmonary microvascular barrier damage and acute lung injury. Autophagy is a positive mediator of sEVs function, at least in part through controlling the expression of bioactive miRNAs in sEVs.