Novel muscle-derived extracellular matrix hydrogel promotes angiogenesis and neurogenesis in volumetric muscle loss.

Volumetric muscle loss (VML) represents a clinical challenge due to the limited regenerative capacity of skeletal muscle. Most often, it results in scar tissue formation and loss of function, which cannot be prevented by current therapies. Decellularized extracellular matrix (DEM) has emerged as a native biomaterial for the enhancement of tissue repair. Here, we report the generation and characterization of hydrogels derived from DEM prepared from WT or thrombospondin (TSP)-2 null muscle tissue. TSP2-null hydrogels, when compared to WT, displayed altered architecture, protein composition, and biomechanical properties and allowed enhanced invasion of C2C12 myocytes and chord formation by endothelial cells. They also displayed enhanced cell invasion, innervation, and angiogenesis following subcutaneous implantation. To evaluate their regenerative capacity, WT or TSP2 null hydrogels were used to treat VML injury to tibialis anterior muscles and the latter induced greater recruitment of repair cells, innervation, and blood vessel formation and reduced inflammation. Taken together, these observations indicate that TSP2-null hydrogels enhance angiogenesis and promote muscle repair in a VML model.

Efficacy of a modified twin block appliance compared with the traditional twin block appliance in children with hyperdivergent mandibular retrognathia: protocol for a single-centre, single-blind, randomised controlled trial.

INTRODUCTION: Compensatory mouth breathing, caused by nasopharyngeal obstructive diseases, is the main cause of hyperdivergent mandibular retrognathia in children. Such deformities require effective growth guidance before pubertal growth peaks. The traditional mandibular advancement device, twin block (TB), can guide the forward development of the mandible. However, the side effect of increasing the vertical dimension of the lower facial third, worsens the facial profile of children with divergent growth trends. To solve this problem, a modified TB (LLTB) appliance was designed to control the vertical dimension by intruding incisors and inhibiting the elongation of posterior teeth during the advancement of the mandible, which could avoid the side effects of traditional appliances and effectively guide the growth of the mandible in a normal direction. METHODS AND ANALYSIS: The study was designed as a single-centre, single-blind, randomised, parallel controlled trial. We aim to enrol 60 children aged 9-14 years with hyperdivergent skeletal class II malocclusion, using a 1:1 allocation ratio. The participants were will be randomly assigned to receive either the TB or LLTB treatment. The primary outcome will be a change in the angle of the mandibular plane relative to the anterior cranial base. The secondary outcomes will include changes in the sagittal maxillomandibular relation, occlusal plane, facial height, morphology of the mandible and upper airway width. Safety endpoints will also be evaluated. ETHICS AND DISSEMINATION: Ethical approval was obtained from the ethics committee of Shanghai Stomatological Hospital. Both participants and their guardians will be fully informed of the study and sign an informed consent form before participating in the trial. The results will be publicly available in peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: ChiCTR2000035882.

High power density output and durability of microbial fuel cells enabled by dispersed cobalt nanoparticles on nitrogen-doped carbon as the cathode electrocatalyst.

To endow microbial fuel cells (MFCs) with low cost, long-term stability and high-power output, a novel cobalt-based cathode electrocatalyst (Nano-Co@NC) is synthesized from a polygonal metal-organic framework ZIF-67. After calcining the resultant ZIF-67, the as-synthesized Nano-Co@NC is characteristic of cobalt nanoparticles (Nano-Co) embedded in nitrogen-doped carbon (NC) that inherits the morphology of ZIF-67 with a large surface area. The Nano-Co particles that are highly dispersed and firmly fixed on NC not only ensure electrocatalytic activity of Nano-Co@NC toward the oxygen reduction reaction on the cathode, but also inhibit the growth of non-electrogenic bacteria on the anode. Consequently, the MFC using Nano-Co@NC as the cathode electrocatalyst demonstrates excellent performance, delivering a comparable initial power density and exhibiting far better durability than that using Pt/C (20 wt%) as the cathode electrocatalyst. The low cost and the excellent performance of Nano-Co@NC make it promising for MFCs to be used in practice.

A facile synthesis of FeS/Fe3C nanoparticles highly dispersed on in situ grown N-doped CNTs as cathode electrocatalysts for microbial fuel cells.

A novel composite of iron sulfide, iron carbide and nitrogen carbides (Nano-FeS/Fe3C@NCNTs) as a cathode electrocatalyst for microbial fuel cells (MFCs) is synthesized by a one-pot solid state reaction, which yields a unique configuration of FeS/Fe3C nanoparticles highly dispersed on in situ grown nitrogen-doped carbon nanotubes (NCNTs). The highly dispersed FeS/Fe3C nanoparticles possess large active sites, while the NCNTs provide an electronically conductive network. Consequently, the resultant Nano-FeS/Fe3C@NCNTs exhibit excellent electrocatalytic activity towards the oxygen reduction reaction (ORR), with a half-wave potential close to that of Pt/C (about 0.88 V vs. RHE), and enable MFCs to deliver a power density of 1.28 W m-2 after two weeks' operation, which is higher than that of MFCs with Pt/C as the cathode electrocatalyst (1.02 W m-2). Theoretical calculations and experimental data demonstrate that there is a synergistic effect between Fe3C and FeS in Nano-FeS/Fe3C@NCNTs. Fe3C presents a strong attraction and electron-donating tendency to oxygen molecules, serving as the main active component, while FeS reduces charge transfer resistance by transferring electrons to Fe3C, synergistically improving the kinetics of the ORR and power density of MFCs.

Injectable TG-linked recombinant human collagen hydrogel loaded with bFGF for rat cranial defect repair.

The basic fibroblast growth factor (bFGF) plays a significant role in promoting the process of bone repair, but bFGF cannot keep its biological activity stable under normal physiological conditions. Therefore, the development of better biomaterials to carry bFGF remains a challenge for bone repair and regeneration. Here we designed a novel recombinant human collagen (rhCol), which could be cross-linked by transglutaminase (TG) and loaded bFGF to prepare rhCol/bFGF hydrogels. The rhCol hydrogel possessed a porous structure and good mechanical properties. The assays, including cell proliferation, migration, and adhesion assay, were performed to evaluate the biocompatibility of rhCol/bFGF and the results demonstrated that the rhCol/bFGF promoted cell proliferation, migration and adhesion. The rhCol/bFGF hydrogel degraded and released bFGF controllably, enhancing utilization rate of bFGF and allowing osteoinductive activity. The results of RT-qPCR and immunofluorescence staining also proved that rhCol/bFGF promoted expression of bone-related proteins. The rhCol/bFGF hydrogels were applied in the cranial defect in rats and the results confirmed that it accelerates bone defect repair. In conclusion, rhCol/bFGF hydrogel has excellent biomechanical properties and can continuously release bFGF to promote bone regeneration, suggesting that rhCol/bFGF hydrogel is a potential scaffold in clinic application.

A triple-diazonium reagent for virus crosslinking and the synthesis of an azo-linked molecular cage.

The first bench-stable triple-diazonium reagent (TDA-1) was rationally designed and synthesized for coupling and crosslinking. The three reactive sites of TDA-1 can react with phenol-containing molecules as well as plant viruses in aqueous buffers efficiently. In addition, a new-type azo-linked cage was constructed by the direct reaction of TDA-1 with a triple-phenol molecule and was characterized by X-ray crystallography.

Phylogenetic and Taxonomic Analyses of Three New Wood-Inhabiting Fungi of Xylodon (Basidiomycota) in a Forest Ecological System.

Wood-inhabiting fungi are a cosmopolitan group and show a rich diversity, growing in the vegetation of boreal, temperate, subtropical, and tropical regions. Xylodon grandineus, X. punctus, and X. wenshanensis spp. nov. were found in the Yunnan-Guizhou Plateau, China, suggested here to be new fungal species in light of their morphology and phylogeny. Xylodon grandineus is characterized by a grandinioid hymenophore and ellipsoid basidiospores; X. punctus has a membranous hymenophore, a smooth hymenial surface with a speckled distribution, and absent cystidia; X. wenshanensis has a grandinioid hymenophore with a cream to slightly buff hymenial surface and cystidia of two types. Sequences of the ITS and nLSU rRNA markers of the studied samples were generated, and phylogenetic analyses were performed using the maximum likelihood, maximum parsimony, and Bayesian inference methods. After a series of phylogenetic studies, the ITS+nLSU analysis of the order Hymenochaetales indicated that, at the generic level, six genera (i.e., Fasciodontia, Hastodontia, Hyphodontia, Lyomyces, Kneiffiella, and Xylodon) should be accepted to accommodate the members of Hyphodontia sensu lato. According to a further analysis of the ITS dataset, X. grandineus was retrieved as a sister to X. nesporii; X. punctus formed a monophyletic lineage and then grouped with X. filicinus, X. hastifer, X. hyphodontinus, and X. tropicus; and X. wenshanensis was a sister to X. xinpingensis.

Tailoring conductive inverse opal films with anisotropic elliptical porous patterns for nerve cell orientation.

BACKGROUND: The nervous system is critical to the operation of various organs and systems, while novel methods with designable neural induction remain to exploit. RESULTS: Here, we present a conductive inverse opal film with anisotropic elliptical porous patterns for nerve orientation induction. The films are fabricated based on polystyrene (PS) inverse opal scaffolds with periodical elliptical porous structure and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) mixed polyacrylamide (PAAm) polymers fillers. It is demonstrated that the anisotropic elliptical surface topography allows the nerve cells to be induced into orientation connected with the stretching direction. Because of the anisotropic features of the film which can be stretched into different directions, nerve cells can be induced to grow in one or two directions, forming a neural network and promoting the connection of nerve cells. It is worth mentioning that the PEDOT:PSS-doped PAAm hydrogels endow the film with conductive properties, which makes the composite films be a suitable candidate for neurites growth and differentiation. CONCLUSIONS: All these features of the conductive and anisotropic inverse opal films imply their great prospects in biomedical applications.

Electroconductive and Anisotropic Structural Color Hydrogels for Visual Heart-on-a-Chip Construction.

Heart-on-a-chip plays an important role in revealing the biological mechanism and developing new drugs for cardiomyopathy. Tremendous efforts have been devoted to developing heart-on-a-chip systems featuring simplified fabrication, accurate imitation and microphysiological visuality. In this paper, the authors present a novel electroconductive and anisotropic structural color hydrogel by simply polymerizing non-close-packed colloidal arrays on super aligned carbon nanotube sheets (SACNTs) for visualized and accurate heart-on-a-chip construction. The generated anisotropic hydrogel consists of a colloidal array-locked hydrogel layer with brilliant structural color on one surface and a conductive methacrylated gelatin (GelMA)/SACNTs film on the other surface. It is demonstrated that the anisotropic morphology of the SACNTs could effectively induce the alignment of cardiomyocytes, and the conductivity of SACNTs could contribute to the synchronous beating of cardiomyocytes. Such consistent beating rhythm caused the deformation of the hydrogel substrates and dynamic shifts in structural color and reflection spectra of the whole hybrid hydrogels. More attractively, with the integration of such cardiomyocyte-driven living structural color hydrogels and microfluidics, a visualized heart-on-a-chip system with more consistent beating frequency has been established for dynamic cardiomyocyte sensing and drug screening. The results indicate that the electroconductive and anisotropic structural color hydrogels are potential for various biomedical applications.

Conductive Nerve Guidance Conduits Based on Morpho Butterfly Wings for Peripheral Nerve Repair.

Peripheral nerve injury (PNI), causing loss of sensory and motor function, is a complex and challenging disease in the clinic due to the restricted regeneration capacity. Nerve guidance conduits (NGCs) have become a promising substitute for peripheral nerve regeneration, but their efficacy is often limited. Here, inspired by the physiological structures of peripheral nerves, we present a conductive topological scaffold for nerve repair by modifying Morpho butterfly wing with reduced graphene oxide (rGO) nanosheets and methacrylated gelatin (GelMA) hydrogel encapsulated brain-derived neurotrophic factor (BDNF). Benefiting from the biocompatibility of GelMA hydrogel, the conductivity of rGO and parallel nanoridge structures of wing scales, PC12 cells, and neural stem cells grown on the modified wing have an increased neurite length with guided cellular orientation. In addition, the NGCs are successfully obtained by manually rolling up the scaffolds and exhibited great performance in repairing 10 mm sciatic nerve defects in rats, and we believe that the NGCs can be applied in reparing longer nerve defects in the future by further optimization. We also demonstrate the feasibility of electrically conductive NGCs based on the rGO/BDNF/GelMA-integrated Morpho butterfly wing as functional nerve regeneration conduits, which may have potential value for application in repairing peripheral nerve injuries.

Advances in the development and optimization strategies of the hemostatic biomaterials.

Most injuries are accompanied by acute bleeding. Hemostasis is necessary to relieve pain and reduce mortality in these accidents. In recent years, the traditional hemostatic materials, including inorganic, protein-based, polysaccharide-based and synthetic materials have been widely used in the clinic. The most prominent of these are biodegradable collagen sponges (Helistat®, United States), gelatin sponges (Ethicon®, SURGIFOAM®, United States), chitosan (AllaQuixTM, ChitoSAMTM, United States), cellulose (Tabotamp®, SURGICEL®, United States), and the newly investigated extracellular matrix gels, etc. Although these materials have excellent hemostatic properties, they also have their advantages and disadvantages. In this review, the performance characteristics, hemostatic effects, applications and hemostatic mechanisms of various biomaterials mentioned above are presented, followed by several strategies to improve hemostasis, including modification of single materials, blending of multiple materials, design of self-assembled peptides and their hybrid materials. Finally, the exploration of more novel hemostatic biomaterials and relative coagulation mechanisms will be essential for future research on hemostatic methods.

Novel tissue-engineered skin equivalent from recombinant human collagen hydrogel and fibroblasts facilitated full-thickness skin defect repair in a mouse model.

Tissue-engineered skin equivalent (TESE) is an optimized alternative for the treatment of skin defects. Designing and fabricating biomaterials with desired properties to load cells is critical for the approach. In this study, we aim to develop a novel TESE with recombinant human collagen (rHC) hydrogel and fibroblasts to improve full-thickness skin defect repair. First, the bioactive effect of rHC on fibroblast proliferation, migration and phenotype was assayed. The results showed that rHC had good biocompatibility and could stimulate fibroblasts migration and secrete various growth factors. Then, rHC was cross-linked with transglutaminase (TG) to prepare rHC hydrogel. Rheometer tests indicated that 10% rHC/TG hydrogel could reach a oscillate stress of 251 Pa and remained stable. Fibroblasts were seeded into rHC/TG hydrogel to prepare TESE. Confocal microscope and scanning electronic microscope observation showed that seeded fibroblasts survived well in the hydrogel. Finally, the therapeutic effect of the newly prepared TESE was tested in a mouse full-thickness skin defect model. The results demonstrated that TESE could significantly improve skin defect repair in vivo. Conclusively, TESE prepared from rHC and fibroblasts in this study exhibits great potential for clinical application in the future.

Functional Identification of the Xanthomonas oryzae pv. oryzae Type I-C CRISPR-Cas System and Its Potential in Gene Editing Application.

The type I clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is one of five adaptive immune systems and exists widely in bacteria and archaea. In this study, we showed that Xanthomonas oryzae pv. oryzae (Xoo) possesses a functional CRISPR system by engineering constructs mimicking its CRISPR cassette. CRISPR array analysis showed that the TTC at the 5'-end of the target sequence is a functional protospacer-adjacent motif (PAM) of CRISPR. Guide RNA (gRNA) deletion analysis identified a minimum of 27-bp spacer that was required to ensure successful self-target killing in PXO99A strain. Mutants with deletion of individual Cas genes were constructed to analyze the effects of Cas proteins on mature CRISPR RNA (crRNA), processing intermediates and DNA interference. Results showed that depleting each of the three genes, cas5d, csd1, and csd2 inactivated the pre-crRNA processing, whereas inactivation of cas3 impaired in processing pre-crRNA. Furthermore, the Xoo CRISPR/Cas system was functional in Pseudomonas syringae pv. tomato. Collectively, our results would contribute to the functional study of CRISPR/Cas system of Xoo, and also provide a new vision on the use of bacterial endogenous systems as a convenient tool for gene editing.

Topographically Conductive Butterfly Wing Substrates for Directed Spiral Ganglion Neuron Growth.

Spiral ganglion neuron (SGN) degeneration can lead to severe hearing loss, and the directional regeneration of SGNs has shown great potential for improving the efficacy of auditory therapy. Here, a novel 3D conductive microstructure with surface topologies is presented by integrating superaligned carbon-nanotube sheets (SA-CNTs) onto Morpho Menelaus butterfly wings for SGN culture. The parallel groove-like topological structures of M. Menelaus wings induce the cultured cells to grow along the direction of its ridges. The excellent conductivity of SA-CNTs significantly improves the efficiency of cellular information conduction. When integrating the SA-CNTs with M. Menelaus wings, the SA-CNTs are aligned in parallel with the M. Menelaus ridges, which further strengthens the consistency of the surface topography in the composite substrate. The SA-CNTs integrated onto butterfly wings provide powerful physical signals and regulate the behavior of SGNs, including cell survival, adhesion, neurite outgrowth, and synapse formation. These features indicate the possibility of directed regeneration after auditory nerve injury.

Elastin-like polypeptide modified silk fibroin porous scaffold promotes osteochondral repair.

Silk fibroin (SF) is considered biocompatible and biodegradable for osteochondral repair. However, it lacks a bioactive domain for cell adhesion, proliferation and differentiation, limiting its therapeutic efficacy. To revamp SF as a biomimicking and bioactive microenvironment to regulate cell behaviours, we engineered an elastin-like polypeptide (ELP, Val-Pro-Gly-Xaa-Gly) to modify SF fibers via simple and green dehydrothermal (DHT) treatment. Our results demonstrated that the ELP successfully bound to SF, and the scaffold was reinforced by the fusion of the silk fiber intersections with ELP (S-ELP-DHT) via the DHT treatment. Both bone mesenchymal stem cells (BMSCs) and chondrocytes exhibited improved spreading and proliferation on the S-ELP-DHT scaffolds. The ex vivo and in vivo experiments further demonstrated enhanced mature bone and cartilage tissue formation using the S-ELP-DHT scaffolds compared to the naked SF scaffolds. These results indicated that a recombinant ELP-modified silk scaffold can mimic three-dimensional (3D) cell microenvironment, and improve bone and cartilage regeneration. We envision that our scaffolds have huge clinical potential for osteochondral repair.

Hierarchical Hydrogels with Ordered Micro-Nano Structures for Cancer-on-a-Chip Construction.

In the drug therapy of tumor, efficient and stable drug screening platforms are required since the drug efficacy varies individually. Here, inspired by the microstructures of hepatic lobules, in which hepatocytes obtain nutrients from both capillary vessel and the central vein, we present a novel hierarchical hydrogel system with ordered micro-nano structure for liver cancer-on-a-chip construction and drug screening. The hierarchical hydrogel system was fabricated by using pregel to fill and replicate self-assembled colloidal crystal arrays and microcolumn array template. Due to the synergistic effect of its interconnected micro-nano structures, the resultant system could not only precisely control the size of cell spheroids but also realize adequate nutrient supply of cell spheroids. We have demonstrated that by integrating the hierarchical hydrogel system into a multichannel concentration gradients microfluidic chip, a functional liver cancer-on-a-chip could be constructed for high-throughput drug screening with good repeatability and high accuracy. These results indicated that the hierarchical hydrogel system and its derived liver cancer-on-a-chip are ideal platforms for drug screening and have great application potential in the field of personalized medicine.

Fabricating a novel HLC-hBMP2 fusion protein for the treatment of bone defects.

Treating serious bone trauma with an osteo-inductive agent such as bone morphogenetic proteins (BMPs) has been considered as an optimized option when delivered via a collagen sponge (CS). Previous works have shown that the BMP concentration and release rate from approved CS carriers is difficult to control with precision. Here we presented the fabrication of a recombinant fusion protein from recombinant human-like collagen (HLC) and human BMP-2 (hBMP2). The fusion protein preserved the characteristic of HLC allowing the recombinant protein to be expressed in Yeast (such as Pichia pastoris GS115) and purified rapidly and easily with mass production after methanol induction. It also kept the stable properties of HLC and hBMP2 in the body fluid environment with good biocompatibility and no cytotoxicity. Moreover, the recombinant fusion protein fabricated a vertical through-hole structure with improved mechanical properties, and thus facilitated migration of bone marrow mesenchymal stem cells (MSCs) into the fusion materials. Furthermore, the fusion protein degraded and released hBMP-2 in vivo allowing osteoinductive activity and the enhancement of utilization rate and the precise control of the hBMP2 release. This fusion protein when applied to cranial defects in rats was osteoinductively active and improved bone repairing enhancing the repairing rate 3.5- fold and 4.2- fold when compared to the HLC alone and the control, respectively. There were no visible inflammatory reactions, infections or extrusions around the implantation sites observed. Our data strongly suggests that this novel recombinant fusion protein could be more beneficial in the treatment of bone defects than the simple superposition of the hBMP2/collagen sponge.

Exploring the potential of the recombinant human collagens for biomedical and clinical applications: a short review.

Natural animal collagen and its recombinant collagen are favourable replacements in human tissue engineering due to their remarkable biomedical property. However, this exploitation is largely restricted due to the potential of immunogenicity and virus contamination. Exploring new ways to produce human collagen is fundamental to its biomedical and clinical application. All human fibrillar collagen molecules have three polypeptide chains constructed from a repeating Gly-Xaa-Yaa triplet, where Xaa and Yaa represent one random amino acid. Using cDNA techniques to modify several repeat sequences of the cDNA fragment, a novel human collagen, named recombinant human-like collagen (rHLC), with low immunogenicity and little risk from hidden virus can be engineered and notably tailored to specific applications. Human-like collagen (HLC) was initially used as a coating to modify the tissue engineering scaffold, and then used as the scaffold after cross-link agents were added to increase its mechanical strength. Due to its good biocompatibility, low immunogenicity, stabilised property, and the ability of mass production, HLC has been widely used in skin injury treatments, vascular scaffolds engineering, cartilage, bone defect repair, skincare, haemostatic sponge, and drug delivery, including coating with medical nanoparticles. In this review, we symmetrically reviewed the development, recent advances in design and application of HLC, and other recombinant human collagen-based biomedicine potentials. At the end, future improvements are also discussed.

Bioinspired conductive cellulose liquid-crystal hydrogels as multifunctional electrical skins.

Bionic electronic skin (E-skin) that could convert external physical or mechanical stimuli into output signals has a wide range of applications including wearable devices, artificial prostheses, software robots, etc. Here, we present a chameleon-inspired multifunctional E-skin based on hydroxypropyl cellulose (HPC), Poly(Acrylamide-co-Acrylic acid) (PACA), and carbon nanotubes (CNTs) composited liquid-crystal hydrogel. We found that the HPC could still form cholesteric liquid-crystal photonic structures with the CNTs additive for enhancing their color saturation and PACA polymerization for locating their assembled periodic structures. As the composite hydrogel containing HPC elements and the PACA scaffold responds to different stimuli, such as temperature variations, mechanical pressure, and tension, it could correspondingly change its volume or internal nanostructure and report these as visible color switches. In addition, due to the additive of CNTs, the composite hydrogel could also output these stimuli as electrical resistance signals. Thus, the hydrogel E-skins had the ability of quantitatively feeding back external stimuli through electrical resistance as well as visually mapping the stimulating sites by color variation. This dual-signal sensing provides the ability of visible-user interaction as well as antiinterference, endowing the multifunctional E-skin with great application prospects.

Intrinsic Color Sensing System Allows for Real-Time Observable Functional Changes on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Stem-cell based in vitro differentiation for disease modeling offers great value to explore the molecular and functional underpinnings driving many types of cardiomyopathy and congenital heart diseases. Nevertheless, one major caveat in the application of in vitro differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) involves the immature phenotype of the CMs. Most of the existing methods need complex apparatus and require laborious procedures in order to monitor the cardiac differentiation/maturation process and often result in cell death. Here we developed an intrinsic color sensing system utilizing a microgroove structural color methacrylated gelatin film, which allows us to monitor the cardiac differentiation process of hiPSC-derived cardiac progenitor cells in real time. Subsequently this system can be employed as an assay system to live monitor induced functional changes on hiPSC-CMs stemming from drug treatment, the effects of which are simply revealed through color diversity. Our research shows that early intervention of cardiac differentiation through simple physical cues can enhance cardiac differentiation and maturation to some extent. Our system also simplifies the previous complex experimental processes for evaluating the physiological effects of successful differentiation and drug treatment and lays a solid foundation for future transformational applications.