TWIST1 interacts with ß/δ-catenins during neural tube development and regulates fate transition in cranial neural crest cells.

Cell fate determination is a necessary and tightly regulated process for producing different cell types and structures during development. Cranial neural crest cells (CNCCs) are unique to vertebrate embryos and emerge from the neural plate borders into multiple cell lineages that differentiate into bone, cartilage, neurons and glial cells. We have previously reported that Irf6 genetically interacts with Twist1 during CNCC-derived tissue formation. Here, we have investigated the mechanistic role of Twist1 and Irf6 at early stages of craniofacial development. Our data indicate that TWIST1 is expressed in endocytic vesicles at the apical surface and interacts with ß/δ-catenins during neural tube closure, and Irf6 is involved in defining neural fold borders by restricting AP2α expression. Twist1 suppresses Irf6 and other epithelial genes in CNCCs during the epithelial-to-mesenchymal transition (EMT) process and cell migration. Conversely, a loss of Twist1 leads to a sustained expression of epithelial and cell adhesion markers in migratory CNCCs. Disruption of TWIST1 phosphorylation in vivo leads to epidermal blebbing, edema, neural tube defects and CNCC-derived structural abnormalities. Altogether, this study describes a previously uncharacterized function of mammalian Twist1 and Irf6 in the neural tube and CNCCs, and provides new target genes for Twist1 that are involved in cytoskeletal remodeling.

Tuning Polymer Hydrophilicity to Regulate Gel Mechanics and Encapsulated Cell Morphology.

Mechanically tunable hydrogels are attractive platforms for 3D cell culture, as hydrogel stiffness plays an important role in cell behavior. Traditionally, hydrogel stiffness has been controlled through altering either the polymer concentration or the stoichiometry between crosslinker reactive groups. Here, an alternative strategy based upon tuning the hydrophilicity of an elastin-like protein (ELP) is presented. ELPs undergo a phase transition that leads to protein aggregation at increasing temperatures. It is hypothesized that increasing this transition temperature through bioconjugation with azide-containing molecules of increasing hydrophilicity will allow direct control of the resulting gel stiffness by making the crosslinking groups more accessible. These azide-modified ELPs are crosslinked into hydrogels with bicyclononyne-modified hyaluronic acid (HA-BCN) using bioorthogonal, click chemistry, resulting in hydrogels with tunable storage moduli (100-1000 Pa). Human mesenchymal stromal cells (hMSCs), human umbilical vein endothelial cells (HUVECs), and human neural progenitor cells (hNPCs) are all observed to alter their cell morphology when encapsulated within hydrogels of varying stiffness. Taken together, the use of protein hydrophilicity as a lever to tune hydrogel mechanical properties is demonstrated. These hydrogels have tunable moduli over a stiffness range relevant to soft tissues, support the viability of encapsulated cells, and modify cell spreading as a consequence of gel stiffness.

Cancer-associated mesothelial cells promote ovarian cancer chemoresistance through paracrine osteopontin signaling.

Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-ß signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer.

Cabozantinib Reverses Renal Cell Carcinoma-mediated Osteoblast Inhibition in Three-dimensional Coculture In Vitro and Reduces Bone Osteolysis In Vivo.

Renal cell carcinoma bone metastases (RCCBM) are typically osteolytic. We previously showed that BIGH3 (beta Ig-h3/TGFBI), secreted by 786-O renal cell carcinoma, plays a role in osteolytic bone lesion in RCCBM through inhibition of osteoblast (OSB) differentiation. To study this interaction, we employed three-dimensional (3D) hydrogels to coculture bone-derived 786-O (Bo-786) renal cell carcinoma cells with MC3T3-E1 pre-OSBs. Culturing pre-OSBs in the 3D hydrogels preserved their ability to differentiate into mature OSB; however, this process was decreased when pre-OSBs were cocultured with Bo-786 cells. Knockdown of BIGH3 in Bo-786 cells recovered OSB differentiation. Furthermore, treatment with bone morphogenetic protein 4, which stimulates OSB differentiation, or cabozantinib (CBZ), which inhibits VEGFR1 and MET tyrosine kinase activities, also increased OSB differentiation in the coculture. CBZ also inhibited pre-osteoclast RAW264.7 cell differentiation. Using RCCBM mouse models, we showed that CBZ inhibited Bo-786 tumor growth in bone. CBZ treatment also increased bone volume and OSB number, and decreased osteoclast number and blood vessel density. When tested in SN12PM6 renal cell carcinoma cells that have been transduced to overexpress BIGH3, CBZ also inhibited SN12PM6 tumor growth in bone. These observations suggest that enhancing OSB differentiation could be one of the therapeutic strategies for treating RCCBM that exhibit OSB inhibition characteristics, and that this 3D coculture system is an effective tool for screening osteoanabolic agents for further in vivo studies.

Perlecan domain I gradients establish stable biomimetic heparin binding growth factor gradients for cell migration in hydrogels.

Growth factor gradients orchestrate many biological processes including organogenesis, wound healing, cancer invasion, and metastasis. Heparin-binding growth factor (HBGF) gradients are established in living systems by proteoglycans including the extracellular matrix heparan sulfate proteoglycan, perlecan/HSPG2. Three potential HBGF-binding glycosaminoglycan attachment sites occur in N-terminal domain I of perlecan's five domains. Our overarching goal was to form stable, biomimetic non-covalently bound HBGF gradients surrounding cells encapsulated in hyaluronate-based hydrogels by first establishing perlecan domain I (PlnD1) gradients. A versatile multichannel gradient maker device (MGMD) was designed and 3D printed, then used to create desired gradients of microparticles in hydrogels. Next, we used the device to covalently incorporate gradients of PEGylated PlnD1 in hydrogels with high-low-high or high-medium-low concentrations across the hydrogel width. Fluorescently-labeled fibroblast growth factor-2 was delivered to hydrogels in phosphate-buffered saline and allowed to electrostatically bind to the covalently pre-incorporated PlnD1, producing stable non-covalent HBGF gradients. To test cell viability after flow through the MGMD, delicate primary human salivary stem/progenitor cells were encapsulated in gradient hydrogels where they showed high viability and continued to grow. Next, to test migratory behavior in response to HBGF gradients, two cell types, preosteoblastic MC3T3-E1 cell line and breast cancer cell line MDA-MB-231 were encapsulated in or adjacent to PlnD1-modified hydrogels. Both cell lines migrated toward HBGFs bound to PlnD1. We conclude that establishing covalently-bound PlnD1 gradients in hydrogels provides a new means to establish physiologically-relevant gradients of HBGFs that are useful for a variety of applications in tissue engineering and cancer biology. STATEMENT OF SIGNIFICANCE: Gradients of heparin binding growth factors (HBGFs) direct cell behavior in living systems. HBGFs bind electrostatically to gradients of HS proteoglycans in the extracellular matrix creating HBGF gradients. We recreated HBGF gradients in physiological hyaluronate-based hydrogels using a 3D-printed multichannel gradient maker device (MGMD) that created gradients of HS proteoglycan-derived perlecan/HSPG2 domain I. We demonstrated the ability of a variety of cells, including primary salivary stem/progenitor cells, pre-osteoblastic cells and an invasive breast cancer cell line, to be co-encapsulated in gradient hydrogels by flowing them together through the MGMD. The versatile device and the ability to create HBGF gradients in hydrogels for a variety of applications is innovative and of broad utility in both cancer biology and tissue engineering applications.

Perlecan/HSPG2: Signaling role of domain IV in chondrocyte clustering with implications for Schwartz-Jampel Syndrome.

Perlecan/heparan sulfate proteoglycan 2 (HSPG2), a large HSPG, is indispensable for the development of musculoskeletal tissues, where it is deposited within the pericellular matrix (PCM) surrounding chondrocytes and disappears nearly completely at the chondro-osseous junction (COJ) of developing long bones. Destruction of perlecan at the COJ converts an avascular cartilage compartment into one that permits blood vessel infiltration and osteogenesis. Mutations in perlecan are associated with chondrodysplasia with widespread musculoskeletal and joint defects. This study elucidated novel signaling roles of perlecan core protein in endochondral bone formation and chondrocyte behavior. Perlecan subdomains were tested for chondrogenic properties in ATDC5 cells, a model for early chondrogenesis. A region within domain IV of perlecan (HSPG2 IV-3) was found to promote rapid prechondrocyte clustering. Introduction of the mutation (R3452Q) associated with the human skeletal disorder Schwartz-Jampel syndrome limited HSPG2 IV-3-induced clustering. HSPG2 IV-3 activity was enhanced when thermally unfolded, likely because of increased exposure of the active motif(s). HSPG2 IV-3-induced clustering was accompanied by the deactivation of key components of the focal adhesion complex, FAK and Src, with increased messenger RNA (mRNA) levels of precartilage condensation markers Sox9 and N-cadherin ( Cdh2), and cartilage PCM components collagen II ( Col2a1) and aggrecan ( Acan). HSPG2 IV-3 reduced signaling through the ERK pathway, where loss of ERK1/2 phosphorylation coincided with reduced FoxM1 protein levels and increased mRNA levels cyclin-dependent kinase inhibitor 1C (Cdkn1c) and activating transcription factor 3 ( Atf3), reducing cell proliferation. These findings point to a critical role for perlecan domain IV in cartilage development through triggering chondrocyte condensation.

Enhancing chondrogenic phenotype for cartilage tissue engineering: monoculture and coculture of articular chondrocytes and mesenchymal stem cells.

Articular cartilage exhibits an inherently low rate of regeneration. Consequently, damage to articular cartilage often requires surgical intervention. However, existing treatments generally result in the formation of fibrocartilage tissue, which is inferior to native articular cartilage. As a result, cartilage engineering strategies seek to repair or replace damaged cartilage with an engineered tissue that restores full functionality to the impaired joint. These strategies often involve the use of chondrocytes, yet in vitro expansion and culture can lead to undesirable changes in chondrocyte phenotype. This review focuses on the use of articular chondrocytes and mesenchymal stem cells (MSCs) in either monoculture or coculture for the enhancement of chondrogenesis. Coculture strategies increasingly outperform their monoculture counterparts with regard to chondrogenesis and present unique opportunities to attain chondrocyte phenotype stability in vitro. Methods to prevent chondrocyte dedifferentiation and promote chondrocyte redifferentiation as well as to promote the chondrogenic differentiation of MSCs while preventing MSC hypertrophy are discussed.

Hair regrowth following a Wnt- and follistatin containing treatment: safety and efficacy in a first-in-man phase 1 clinical trial.

Research has shown the importance of follistatin, Wnt 7a, and wound healing growth factors on the stimulation of bulge cells and inter-follicular stem cells to induce hair growth. We have studied the effects of a bioengineered, non-recombinant, human cell-derived formulation, termed Hair Stimulating Complex (HSC), containing these factors to assess its hair growth activity in male pattern baldness. HSC showed in vitro Wnt activity and contained follistatin, KGF, and VEGF. The clinical study was a double-blind, placebo-controlled, randomized single site trial and was designed to evaluate safety of the HSC product and assess efficacy in stimulating hair growth. All 26 subjects tolerated the single, intradermal injection of HSC procedures well, and no signs of an adverse reaction were reported. Histopathological evaluation of the treatment site biopsies taken at 22 and 52 weeks post-treatment revealed no abnormal morphology, hamartomas, or other pathological responses. Trichoscan image analysis of HSC-treated sites at 12 and 52 weeks showed significant improvements in hair growth over the placebo. At the initial 12-week evaluation period, HSC-treated sites demonstrated an increase in hair shaft thickness (6.3%±2.5% vs. -0.63%±2.1%; P=0.046), thickness density (12.8%±4.5% vs. -0.2%±2.9%; P=0.028), and terminal hair density (20.6±4.9% vs. 4.4±4.9%; P=0.029). At one year, a statistically significant increase in total hair count (P=0.032) continued to be seen. These results demonstrate that a single intradermal administration of HSC improved hair growth in subjects with androgenetic alopecia and is a clinical substantiation of previous preclinical research with Wnts, follistatin, and other growth factors associated with wound healing and regeneration.