Multifunctional Thermoresponsive Microcarriers for High-Throughput Cell Culture and Enzyme-Free Cell Harvesting.

An effective treatment of human diseases using regenerative medicine and cell therapy approaches requires a large number of cells. Cultivation of cells on microcarriers is a promising approach due to the high surface-to-volume ratios that these microcarriers offer. Here, multifunctional temperature-responsive microcarriers (cytoGel) made of an interpenetrating hydrogel network composed of poly(N-isopropylacrylamide) (PNIPAM), poly(ethylene glycol) diacrylate (PEGDA), and gelatin methacryloyl (GelMA) are developed. A flow-focusing microfluidic chip is used to produce microcarriers with diameters in the range of 100-300 µm and uniform size distribution (polydispersity index of ≈0.08). The mechanical properties and cells adhesion properties of cytoGel are adjusted by changing the composition hydrogel composition. Notably, GelMA regulates the temperature response and enhances microcarrier stiffness. Human-derived glioma cells (U87) are grown on cytoGel in static and dynamic culture conditions with cell viabilities greater than 90%. Enzyme-free cell detachment is achieved at room temperature with up to 70% detachment efficiency. Controlled release of bioactive molecules from cytoGel is accomplished for over a week to showcase the potential use of microcarriers for localized delivery of growth factors to cell surfaces. These microcarriers hold great promise for the efficient expansion of cells for the industrial-scale culture of therapeutic cells.

The Hippo Pathway Targets Rae1 to Regulate Mitosis and Organ Size and to Feed Back to Regulate Upstream Components Merlin, Hippo, and Warts.

Hippo signaling acts as a master regulatory pathway controlling growth, proliferation, and apoptosis and also ensures that variations in proliferation do not alter organ size. How the pathway coordinates restricting proliferation with organ size control remains a major unanswered question. Here we identify Rae1 as a highly-conserved target of the Hippo Pathway integrating proliferation and organ size. Genetic and biochemical studies in Drosophila cells and tissues and in mammalian cells indicate that Hippo signaling promotes Rae1 degradation downstream of Warts/Lats. In proliferating cells, Rae1 loss restricts cyclin B levels and organ size while Rae1 over-expression increases cyclin B levels and organ size, similar to Hippo Pathway over-activation or loss-of-function, respectively. Importantly, Rae1 regulation by the Hippo Pathway is crucial for its regulation of cyclin B and organ size; reducing Rae1 blocks cyclin B accumulation and suppresses overgrowth caused by Hippo Pathway loss. Surprisingly, in addition to suppressing overgrowth, reducing Rae1 also compromises survival of epithelial tissue overgrowing due to loss of Hippo signaling leading to a tissue "synthetic lethality" phenotype. Excitingly, Rae1 plays a highly conserved role to reduce the levels and activity of the Yki/YAP oncogene. Rae1 increases activation of the core kinases Hippo and Warts and plays a post-transcriptional role to increase the protein levels of the Merlin, Hippo, and Warts components of the pathway; therefore, in addition to Rae1 coordinating organ size regulation with proliferative control, we propose that Rae1 also acts in a feedback circuit to regulate pathway homeostasis.

An Engineered Infected Epidermis Model for In Vitro Study of the Skin's Pro-Inflammatory Response.

Wound infection is a major clinical challenge that can significantly delay the healing process, can create pain, and requires prolonged hospital stays. Pre-clinical research to evaluate new drugs normally involves animals. However, ethical concerns, cost, and the challenges associated with interspecies variation remain major obstacles. Tissue engineering enables the development of in vitro human skin models for drug testing. However, existing engineered skin models are representative of healthy human skin and its normal functions. This paper presents a functional infected epidermis model that consists of a multilayer epidermis structure formed at an air-liquid interface on a hydrogel matrix and a three-dimensionally (3D) printed vascular-like network. The function of the engineered epidermis is evaluated by the expression of the terminal differentiation marker, filaggrin, and the barrier function of the epidermis model using the electrical resistance and permeability across the epidermal layer. The results showed that the multilayer structure enhances the electrical resistance by 40% and decreased the drug permeation by 16.9% in the epidermis model compared to the monolayer cell culture on gelatin. We infect the model with Escherichia coli to study the inflammatory response of keratinocytes by measuring the expression level of pro-inflammatory cytokines (interleukin 1 beta and tumor necrosis factor alpha). After 24 h of exposure to Escherichia coli, the level of IL-1ß and TNF-α in control samples were 125 ± 78 and 920 ± 187 pg/mL respectively, while in infected samples, they were 1429 ± 101 and 2155.5 ± 279 pg/mL respectively. However, in ciprofloxacin-treated samples the levels of IL-1ß and TNF-α without significant difference with respect to the control reached to 246 ± 87 and 1141.5 ± 97 pg/mL respectively. The robust fabrication procedure and functionality of this model suggest that the model has great potential for modeling wound infections and drug testing.

Hyaluronic Acid (HA)-Based Silk Fibroin/Zinc Oxide Core-Shell Electrospun Dressing for Burn Wound Management.

Burn injuries represent a major life-threatening event that impacts the quality of life of patients, and places enormous demands on the global healthcare systems. This study introduces the fabrication and characterization of a novel wound dressing made of core-shell hyaluronic acid-silk fibroin/zinc oxide (ZO) nanofibers for treatment of burn injuries. The core-shell configuration enables loading ZO-an antibacterial agent-in the core of nanofibers, which in return improves the sustained release of the drug and maintains its bioactivity. Successful formation of core-shell nanofibers and loading of zinc oxide are confirmed by transmission electron microscopy, Fourier-transform infrared spectroscopy, and energy dispersive X-ray. The antibacterial activity of the dressings are examined against Escherichia coli and Staphylococcus aureus and it is shown that addition of ZO improves the antibacterial property of the dressing in a dose-dependent fashion. However, in vitro cytotoxicity studies show that high concentration of ZO (>3 wt%) is toxic to the cells. In vivo studies indicate that the wound dressings loaded with ZO (3 wt%) substantially improves the wound healing procedure and significantly reduces the inflammatory response at the wound site. Overall, the dressing introduced herein holds great promise for the management of burn injuries.

Double minute amplification of mutant PDGF receptor α in a mouse glioma model.

In primary brain tumors, oncogenes are frequently amplified and maintained on extrachromosomal DNA as double minutes (DM), but the underlying mechanisms remain poorly understood. We have generated a mouse model of malignant glioma based on knock-in of a mutant PDGF receptor α (PDGFRα) that is expressed in oligodendrocyte precursor cells (OPCs) after activation by a Cre recombinase. In the tumor suppressor INK4/Arf(-/-) background, mutant animals frequently developed brain tumors resembling anaplastic human gliomas (WHO grade III). Besides brain tumors, most animals also developed aggressive fibrosarcomas, likely triggered by Cre activation of mutant PDGFRα in fibroblastic cell lineages. Importantly, in the brain tumors and cell lines derived from brain tumor tissues, we identified a high prevalence of DM Pdgfra gene amplification, suggesting its occurrence as an early mutational event contributing to the malignant transformation of OPCs. Amplicons extended beyond the Pdgfra locus and included in some cases neighboring genes Kit and Kdr. Our genetically defined mouse brain tumor model therefore supports OPC as a cell of origin for malignant glioma and offers an example of a defined temporal sequence of mutational events, thus providing an entry point for a mechanistic understanding of DM gene amplification and its functionality in gliomagenesis.

Alcohol interacts with genetic alteration of the Hippo tumor suppressor pathway to modulate tissue growth in Drosophila.

Alcohol-mediated cancers represent more than 3.5% of cancer-related deaths, yet how alcohol promotes cancer is a major open question. Using Drosophila, we identified novel interactions between dietary ethanol and loss of tumor suppressor components of the Hippo Pathway. The Hippo Pathway suppresses tumors in flies and mammals by inactivating transcriptional co-activator Yorkie, and the spectrum of cancers associated with impaired Hippo signaling overlaps strikingly with those associated with alcohol. Therefore, our findings may implicate loss of Hippo Pathway tumor suppression in alcohol-mediated cancers. Ethanol enhanced overgrowth from loss of the expanded, hippo, or warts tumor suppressors but, surprisingly, not from over-expressing the yorkie oncogene. We propose that in parallel to Yorkie-dependent overgrowth, impairing Hippo signaling in the presence of alcohol may promote overgrowth via additional alcohol-relevant targets. We also identified interactions between alcohol and Hippo Pathway over-activation. We propose that exceeding certain thresholds of alcohol exposure activates Hippo signaling to maintain proper growth control and prevent alcohol-mediated mis-patterning and tissue overgrowth.

Rabex-5 ubiquitin ligase activity restricts Ras signaling to establish pathway homeostasis in Drosophila.

The Ras signaling pathway allows cells to translate external cues into diverse biological responses. Depending on context and the threshold reached, Ras signaling can promote growth, proliferation, differentiation, or cell survival. Failure to maintain precise control of Ras can have adverse physiological consequences. Indeed, excess Ras signaling disrupts developmental patterning and causes developmental disorders [1, 2], and in mature tissues, it can lead to cancer [3-5]. We identify Rabex-5 as a new component of Ras signaling crucial for achieving proper pathway outputs in multiple contexts in vivo. We show that Drosophila Rabex-5 restricts Ras signaling to establish organism size, wing vein pattern, and eye versus antennal fate. Rabex-5 has both Rab5 guanine nucleotide exchange factor (GEF) activity that regulates endocytic trafficking [6] and ubiquitin ligase activity [7, 8]. Surprisingly, overexpression studies demonstrate that Rabex-5 ubiquitin ligase activity, not its Rab5 GEF activity, is required to restrict wing vein specification and to suppress the eye phenotypes of oncogenic Ras expression. Furthermore, genetic interaction experiments indicate that Rabex-5 acts at the step of Ras, and tissue culture studies show that Rabex-5 promotes Ras ubiquitination. Together, these findings reveal a new mechanism for attenuating Ras signaling in vivo and suggest an important role for Rabex-5-mediated Ras ubiquitination in pathway homeostasis.