A Survey of Transcription Factors in Cell Fate Control.

Transcription factors (TFs) play a cardinal role in the development and maintenance of human physiology by acting as mediators of gene expression and cell state control. Recent advancements have broadened our knowledge on the potency of TFs in governing cell physiology and have deepened our understanding of the mechanisms through which they exert this control. The ability of TFs to program cell fates has gathered significant interest in recent decades, and high-throughput technologies now allow for the systematic discovery of forward programming factors to convert pluripotent stem cells into numerous differentiated cell types. The next generation of these technologies has the potential to improve our understanding and control of cell fates and states and provide advanced therapeutic modalities to address many medical conditions.

Laponite-Based Nanomaterials for Drug Delivery.

Laponite is a clay-based material composed of synthetic disk-shaped crystalline nanoparticles with highly ionic, large surface area. These characteristics enable the intercalation and dissolution of biomolecules in Laponite-based drug delivery systems. Furthermore, Laponite's innate physicochemical properties and architecture enable the development of tunable pH-responsive drug delivery systems. Laponite's coagulation capacity and cation exchangeability determine its exchange capabilities, drug encapsulation efficiency, and release profile. These parameters are exploited to design highly controlled and efficacious drug delivery platforms for sustained drug release. In this review, they provide an overview of how to design efficient delivery of therapeutics by leveraging the properties and specific interactions of various Laponite-polymer composites and drug moieties.

Neuronal Cell-type Engineering by Transcriptional Activation.

Gene activation with the CRISPR-Cas system has great implications in studying gene function, controlling cellular behavior, and modulating disease progression. In this review, we survey recent studies on targeted gene activation and multiplexed screening for inducing neuronal differentiation using CRISPR-Cas transcriptional activation (CRISPRa) and open reading frame (ORF) expression. Critical technical parameters of CRISPRa and ORF-based strategies for neuronal programming are presented and discussed. In addition, recent progress on in vivo applications of CRISPRa to the nervous system are highlighted. Overall, CRISPRa represents a valuable addition to the experimental toolbox for neuronal cell-type programming.

Cell therapy strategies for COVID-19: Current approaches and potential applications.

Coronavirus disease 2019 (COVID-19) continues to burden society worldwide. Despite most patients having a mild course, severe presentations have limited treatment options. COVID-19 manifestations extend beyond the lungs and may affect the cardiovascular, nervous, and other organ systems. Current treatments are nonspecific and do not address potential long-term consequences such as pulmonary fibrosis, demyelination, and ischemic organ damage. Cell therapies offer great potential in treating severe COVID-19 presentations due to their customizability and regenerative function. This review summarizes COVID-19 pathogenesis, respective areas where cell therapies have potential, and the ongoing 89 cell therapy trials in COVID-19 as of 1 January 2021.

A comprehensive library of human transcription factors for cell fate engineering.

Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.

Enabling large-scale genome editing at repetitive elements by reducing DNA nicking.

To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements-ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.

Comparison of visible and UVA phototoxicity in neural culture systems micropatterned with digital projection photolithography.

Photopolymerization provides a favorable method for hydrogel formation due to its simplicity, convenience, and versatility. However, the light exposure required to initiate photopolymerization is known to have a cytotoxic effect on encapsulated cells. Here, a 3D in vitro model of the nervous system microenvironment, micropatterned through the use of digital projection photolithography using a single hydrogel formulation that cross-links similarly under ultraviolet A (UVA, 315-400 nm) and visible light (400-700 nm) exposure, is presented. This setup allowed for the investigation of neuronal responses to different light wavelengths and exposure times during photoencapsulation, while ruling out effects due to the hydrogel formulation or photoinitiators used. Cellular studies-including neurite viability, DNA fragmentation, and neurite outgrowth for both UVA and visible light irradiation, the most common spectra used in biological photomicropatterning applications-were performed to assess the effect of light source on neuronal cultures. These studies indicated that while cell death occurs after exposure to either spectrum, visible light was less phototoxic than UVA, when using comparable levels of irradiation, and interestingly, glial cells were more susceptible to phototoxicity than neuronal cells. Thus, while utilizing visible light for micropatterning and cell encapsulation for nervous system applications is beneficial, it is helpful to keep the light exposure low to ensure optimal neuronal survival and growth. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 134-144, 2019.

A novel gel patch for minimally invasive repair of tympanic membrane perforations.

OBJECTIVE: Evaluate the efficacy of a photocrosslinkable gel patch for repairing tympanic membrane (TM) perforations using a minimally invasive procedure. METHODS: 38 adult male chinchillas underwent bilateral TM perforation via CO2 laser (n = 76 TMs). Eight weeks post-perforation induction, either a gel patch (n = 26) or EpiDisc (n = 12) was applied to the perforation through the ear canal. Perforation margins were not abraded prior to gel patch application in order to make the procedure minimally invasive. During the study, the application process was refined, and 9 of 26 gel-treated TMs received a second gel-patch augmentation. Perforations were observed for 14 weeks post-treatment to determine healing rates, after which animals were euthanized and their TMs and cochlea removed for histological analysis. RESULTS: 38 perforations (50%) persisted for 8 weeks without manipulation. Healing rates stabilized within three weeks post-treatment. Of the gel-treated TMs, 14 TMs healed after one application, 7 TMs healed after a second application, and 5 TMs did not heal, yielding an 81% total healing rate. Six of 12 EpiDisc-treated TMs healed (50%). There was no statistical difference (p = 0.06) in perforation size between gel-treated (25.1 ± 12.5% total TM area) and Epidisc-treated (36.4 ± 22.5). The largest perforation healed with gel patch was 60% total TM area. Histological analysis showed gel-treated TMs to have trilaminar regeneration with substantial lamina propria thickness. Gel-treated TMs had thickness of statistical equivalence to untreated TMs (47.1 ± 29.0 and 54.8 ± 12.1 µm, respectively (p = 0.40)). EpiDisc-treated TMs showed a cell monolayer of substantially less thickness (9.04 ± 6.26 µm, p < 0.05) than gel-treated TMs. No evidence of ototoxicity was present in cochlea from either gel patch or Epidisc treatment. CONCLUSIONS: The gel is promising regarding thickness and trilaminar regenerated tissue, perhaps due to the biomechanical properties of the gel, and further refinements in the material and technique are anticipated to increase ease and efficacy of treatment while minimizing complications.

Methods for fabrication and evaluation of a 3D microengineered model of myelinated peripheral nerve.

OBJECTIVE: The cost and low success rates of the neurological drug development pipeline have diverted the pharmaceutical industry to 'nerve-on-a-chip' systems as preclinical models to streamline drug development. We present a novel micro-engineered 3D hydrogel platform for the culture of myelinated embryonic peripheral neural tissue to serve as an effective in vitro model for electrophysiological and histological analysis that could be adopted for preclinical testing. APPROACH: Dorsal root ganglions (DRG) from 15 d old embryonic rats were cultured in 3D hydrogel platforms. The interaction between Schwann cells (SC) and neurons during axonal development and regeneration affects the direction of growth and the synthesis of myelin sheaths. Induction of myelination was performed with two approaches: the addition of exogenous SC and promoting migration of endogenous SC. MAIN RESULTS: Histological analysis of the preparation utilizing exogenous SC showed aligned, highly fasciculated axonal growth with noticeable myelin sheaths around axons. Separately, electrophysiological testing of the preparation utilizing endogenous SC showed increased amplitude of the compound action potential and nerve conduction velocity in the presence of ascorbic acid (AA). SIGNIFICANCE: This platform has immense potential to be a useful and translatable in vitro testing tool for drug discovery and myelination studies.

Microfluidics-Enabled Multimaterial Maskless Stereolithographic Bioprinting.

A stereolithography-based bioprinting platform for multimaterial fabrication of heterogeneous hydrogel constructs is presented. Dynamic patterning by a digital micromirror device, synchronized by a moving stage and a microfluidic device containing four on/off pneumatic valves, is used to create 3D constructs. The novel microfluidic device is capable of fast switching between different (cell-loaded) hydrogel bioinks, to achieve layer-by-layer multimaterial bioprinting. Compared to conventional stereolithography-based bioprinters, the system provides the unique advantage of multimaterial fabrication capability at high spatial resolution. To demonstrate the multimaterial capacity of this system, a variety of hydrogel constructs are generated, including those based on poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacryloyl (GelMA). The biocompatibility of this system is validated by introducing cell-laden GelMA into the microfluidic device and fabricating cellularized constructs. A pattern of a PEGDA frame and three different concentrations of GelMA, loaded with vascular endothelial growth factor, are further assessed for its neovascularization potential in a rat model. The proposed system provides a robust platform for bioprinting of high-fidelity multimaterial microstructures on demand for applications in tissue engineering, regenerative medicine, and biosensing, which are otherwise not readily achievable at high speed with conventional stereolithographic biofabrication platforms.

Starting a Medical Technology Venture as a Young Academic Innovator or Student Entrepreneur.

Following the footprints of Bill Gates, Steve Jobs and Mark Zuckerberg, there has been a misconception that students are better off quitting their studies to bring to life their ideas, create jobs and monetize their inventions. Having historically transitioned from manpower to mind power, we live in one of the most rapidly changing times in human history. As a result, academic institutions that are supposed to be pioneers and educators of the next generations have started to realize that they need to adapt to a new system, and change their policies to be more flexible towards patent ownership and commercialization. There is an infrastructure being developed towards students starting their own businesses while continuing with their studies. This paper aims to provide an overview of the existing landscape, the exciting rewards as well as risks awaiting a student entrepreneur, the challenges of the present ecosystem, and questions to consider prior to embarking on such a journey. Various entities influencing the start-up environment are considered, specifically for the medical technology sector. These parties include but are not limited to: scientists, clinicians, investors, academic institutions and governments. A special focus will be set on the seemingly unbridgeable gap between founding a company and a scientific career.

Development and characterization of a bioglass/chitosan composite as an injectable bone substitute.

SiO2-CaO-P2O5 based bioglass (BG) systems constitute a group of materials that have wide applications in bone implants. Chitosan (Cn) is a biocompatible and osteoconductive natural polymer that can promote wound healing. In this study, bioactivity of chitosan/bioglass (CnB) composites as minimally invasive bone regenerative materials was assessed both in vitro and in vivo. Injectability tests and scanning electron microscopy (SEM) results demonstrated the formation of uniform injectable paste-like composites using BG particles and Cn. Fourier transform infrared spectroscopy (FTIR) and SEM images confirmed hydroxyapatite deposition in vitro after incubation in simulated body fluid (SBF). Higher BG content in the composite correlated with increased human osteoblast proliferation. An in vivo study in a rat spinal fusion model confirmed that increasing the amount of BG improved osteoconductivity. Manual palpation, radiographic images and pathological assessments proved that the composites promote bone formation. Based on these data, the synthesized composites have a potential application in orthopedic and reconstructive surgeries as a minimally invasive bone substitute.

Rapid Continuous Multimaterial Extrusion Bioprinting.

The development of a multimaterial extrusion bioprinting platform is reported. This platform is capable of depositing multiple coded bioinks in a continuous manner with fast and smooth switching among different reservoirs for rapid fabrication of complex constructs, through digitally controlled extrusion of bioinks from a single printhead consisting of bundled capillaries synergized with programmed movement of the motorized stage.

Graphene-based materials for tissue engineering.

Graphene and its chemical derivatives have been a pivotal new class of nanomaterials and a model system for quantum behavior. The material's excellent electrical conductivity, biocompatibility, surface area and thermal properties are of much interest to the scientific community. Two-dimensional graphene materials have been widely used in various biomedical research areas such as bioelectronics, imaging, drug delivery, and tissue engineering. In this review, we will highlight the recent applications of graphene-based materials in tissue engineering and regenerative medicine. In particular, we will discuss the application of graphene-based materials in cardiac, neural, bone, cartilage, skeletal muscle, and skin/adipose tissue engineering. We will also discuss the potential risk factors of graphene-based materials in tissue engineering. In conclusion, we will outline the opportunities in the usage of graphene-based materials for clinical applications.

Photoreactive interpenetrating network of hyaluronic acid and Puramatrix as a selectively tunable scaffold for neurite growth.

The reconstruction of soft tissue, such as that which is found in the nervous system, is governed by the mechanical cues of the growth microenvironment. The complexity of the nervous system, particularly in cases of nerve repair and reconstruction, necessitates the development of facile high-throughput investigational tools. This study assesses the hypothesis that a mechanically tunable photoreactive interpenetrating network (IPN) of hyaluronic acid and Puramatrix can be manipulated in order to demonstrate that 3-D environmental stiffness influences neurite growth and proliferation. For these studies we employed photocrosslinkable glycidyl methacrylate hyaluronic acid (GMHA) and Puramatrix, a self-assembling peptide scaffold, leading to a structurally adjustable IPN system. Our in vitro model provides us with a simple, reproducible environment to generate different properties in a single specimen. Mechanically manipulated IPN systems with different degrees of methacrylation were fabricated using a dynamic mask projection photolithography apparatus and characterized. To gauge the impact of IPN stiffness on neurite outgrowth, dorsal root ganglia (DRG) explants were cultured in the hydrogels. We found that neurite outgrowth in 3-D was more likely to happen in an environment with a lesser degree of methacrylation, which corresponded to structures that were more compliant and more porous. Overall, tuning the mechanical behavior of our IPN systems led to statistically significant (p<0.05) differences in cellular growth and extension that warrants further investigations.

Effect of alumina on microstructure and compressive strength of a porous silicated hydroxyapatite.

PURPOSE: We investigated the effects of alumina addition on microstructure and compressive strength of a porous silicate substituted hydroxyapatite (Si-HA). METHODS: Hydroxyapatite (HA) was synthesized under precipitation conditions and 10 %Wt. of sol-gel derived CaO.P2O5.SiO2 based bioglass (BG) powder was added to HA. Polyurethane foam was used to form a high porous structure with integral porosity of 70%. Phase analysis was performed using XRD and FTIR and the microstructure was studied using SEM. RESULTS: The results confirmed that the Si-HA was the only formed phase before Al2O3 addition while after addition the presence of silicon-incorporated HA and alumina without any other phases was proved using these analyses. CONCLUSIONS: The porous structures of Si-HA and Al2O3 were synthesized using the replication technique. The compressive strength of porous bioceramics increased with increasing Al2O3 content up to 30 wt% (ANOVA, P<.05).

Light-reactive dextran gels with immobilized guidance cues for directed neurite growth in 3D models.

We present here a novel light-reactive dextran gel for immobilizing guidance cues in neural growth models. The dextran gel is functionalized with glycidyl methacrylate to afford crosslinking abilities, and is combined with polyethylene glycol (PEG) acrylate grafted with thiol groups caged by a UV-light sensitive moiety. The gel is chemically crosslinked within a cell-restrictive PEG micromold with two channels, and then irradiated with UV light to liberate the thiol groups in a spatially defined manner. Maleimide-conjugated NeutrAvidin (NA), neurotrophin-3 (NT-3) and semaphorin 3A (Sema3A) are then bound to the free thiols, resulting in regions of immobilized guidance cues. Dorsal root ganglia explants were cultured in these dual hydrogel constructs, and the neurite response was quantified by comparing the neurite growth in the channel with the immobilized cue to the channel without any protein. We found that immobilized NT-3 elicited a moderate attractive response, while bound Sema3A elicited a strong repulsive response from neurites. This work establishes a model for investigating growth cone responses to immobilized cues in three dimensions.