In vitro development and optimization of cell-laden injectable bioprinted gelatin methacryloyl (GelMA) microgels mineralized on the nanoscale.

Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98 % viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, showing that mineralization can effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.

In vitro development and optimization of cell-laden injectable bioprinted gelatin methacryloyl (GelMA) microgels mineralized on the nanoscale.

Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98% viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, indicating that mineralization effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.

Opportunities and challenges to engineer 3D models of tumor-adaptive immune interactions.

Augmenting adaptive immunity is a critical goal for developing next-generation cancer therapies. T and B cells infiltrating the tumor dramatically influence cancer progression through complex interactions with the local microenvironment. Cancer cells evade and limit these immune responses by hijacking normal immunologic pathways. Current experimental models using conventional primary cells, cell lines, or animals have limitations for studying cancer-immune interactions directly relevant to human biology and clinical translation. Therefore, engineering methods to emulate such interplay at local and systemic levels are crucial to expedite the development of better therapies and diagnostic tools. In this review, we discuss the challenges, recent advances, and future directions toward engineering the tumor-immune microenvironment (TME), including key elements of adaptive immunity. We first offer an overview of the recent research that has advanced our understanding of the role of the adaptive immune system in the tumor microenvironment. Next, we discuss recent developments in 3D in-vitro models and engineering approaches that have been used to study the interaction of cancer and stromal cells with B and T lymphocytes. We summarize recent advancement in 3D bioengineering and discuss the need for 3D tumor models that better incorporate elements of the complex interplay of adaptive immunity and the tumor microenvironment. Finally, we provide a perspective on current challenges and future directions for modeling cancer-immune interactions aimed at identifying new biological targets for diagnostics and therapeutics.

Antibacterial Nanostructured Surfaces Modulate Protein Adsorption, Inflammatory Responses, and Fibrous Capsule Formation.

The present study interrogates the interaction of highly efficient antibacterial surfaces containing sharp nanostructures with blood proteins and the subsequent immunological consequences, processes that are of key importance for the fate of every implantable biomaterial. Studies with human serum and plasma pointed to significant differences in the composition of the protein corona that formed on control and nanostructured surfaces. Quantitative analysis using liquid chromatography-mass spectrometry demonstrated that the nanostructured surface attracted more vitronectin and less complement proteins compared to the untreated control. In turn, the protein corona composition modulated the adhesion and cytokine expression by immune cells. Monocytes produced lower amounts of pro-inflammatory cytokines and expressed more anti-inflammatory factors on the nanostructured surface. Studies using an in vivo subcutaneous mouse model showed reduced fibrous capsule thickness which could be a consequence of the attenuated inflammatory response. The results from this work suggest that antibacterial surface modification with sharp spike-like nanostructures may not only lead to the reduction of inflammation but also more favorable foreign body response and enhanced healing, processes that are beneficial for most medical devices implanted in patients.

Plasma polymerized bio-interface directs fibronectin adsorption and functionalization to enhance "epithelial barrier structure" formation via FN-ITG ß1-FAK-mTOR signaling cascade.

BACKGROUND: Transepithelial medical devices are increasing utilized in clinical practices. However, the damage of continuous natural epithelial barrier has become a major risk factor for the failure of epithelium-penetrating implants. How to increase the "epithelial barrier structures" (focal adhesions, hemidesmosomes, etc.) becomes one key research aim in overcoming this difficulty. Directly targeting the in situ "epithelial barrier structures" related proteins (such as fibronectin) absorption and functionalization can be a promising way to enhance interface-epithelial integration. METHODS: Herein, we fabricated three plasma polymerized bio-interfaces possessing controllable surface chemistry. Their capacity to adsorb and functionalize fibronectin (FN) from serum protein was compared by Liquid Chromatography-Tandem Mass Spectrometry. The underlying mechanisms were revealed by molecular dynamics simulation. The response of gingival epithelial cells regarding the formation of epithelial barrier structures was tested. RESULTS: Plasma polymerized surfaces successfully directed distinguished protein adsorption profiles from serum protein pool, in which plasma polymerized allylamine (ppAA) surface favored adsorbing adhesion related proteins and could promote FN absorption and functionalization via electrostatic interactions and hydrogen bonds, thus subsequently activating the ITG ß1-FAK-mTOR signaling and promoting gingival epithelial cells adhesion. CONCLUSION: This study offers an effective perspective to overcome the current dilemma of the inferior interface-epithelial integration by in situ protein absorption and functionalization, which may advance the development of functional transepithelial biointerfaces. Tuning the surface chemistry by plasma polymerization can control the adsorption of fibronectin and functionalize it by exposing functional protein domains. The functionalized fibronectin can bind to human gingival epithelial cell membrane integrins to activate epithelial barrier structure related signaling pathway, which eventually enhances the formation of epithelial barrier structure.

Bio-Inspired Nanostructured Ti-6Al-4V Alloy: The Role of Two Alkaline Etchants and the Hydrothermal Processing Duration on Antibacterial Activity.

Inspired by observations that the natural topography observed on cicada and dragonfly wings may be lethal to bacteria, researchers have sought to reproduce these nanostructures on biomaterials with the goal of reducing implant-associated infections. Titanium and its alloys are widely employed biomaterials with excellent properties but are susceptible to bacterial colonisation. Hydrothermal etching is a simple, cost-effective procedure which fabricates nanoscale protrusions of various dimensions upon titanium, depending on the etching parameters used. We investigated the role of etching time and the choice of cation (sodium and potassium) in the alkaline heat treatment on the topographical, physical, and bactericidal properties of the resulting modified titanium surfaces. Optimal etching times were 4 h for sodium hydroxide (NaOH) and 5 h for potassium hydroxide (KOH). NaOH etching for 4 h produced dense, but somewhat ordered, surface nanofeatures with 75 nanospikes per µm2. In comparison, KOH etching for 5 h resulted sparser but nonetheless disordered surface morphology with only 8 spikes per µm2. The NaOH surface was more effective at eliminating Gram-negative pathogens, while the KOH surface was more effective against the Gram-positive strains. These findings may guide further research and development of bactericidal titanium surfaces which are optimised for the predominant pathogens associated with the intended application.

Nanoparticles Surface Chemistry Influence on Protein Corona Composition and Inflammatory Responses.

Nanoparticles are widely used for biomedical applications such as vaccine, drug delivery, diagnostics, and therapeutics. This study aims to reveal the influence of nanoparticle surface functionalization on protein corona formation from blood serum and plasma and the subsequent effects on the innate immune cellular responses. To achieve this goal, the surface chemistry of silica nanoparticles of 20 nm diameter was tailored via plasma polymerization with amine, carboxylic acid, oxazolines, and alkane functionalities. The results of this study show significant surface chemistry-induced differences in protein corona composition, which reflect in the subsequent inflammatory consequences. Nanoparticles rich with carboxylic acid surface functionalities increased the production of pro-inflammatory cytokines in response to higher level of complement proteins and decreased the number of lipoproteins found in their protein coronas. On another hand, amine rich coatings led to increased expressions of anti-inflammatory markers such as arginase. The findings demonstrate the potential to direct physiological responses to nanomaterials via tailoring their surface chemical composition.

Ultra-small gold nanoclusters assembled on plasma polymer-modified zeolites: a multifunctional nanohybrid with anti-haemorrhagic and anti-inflammatory properties.

Hemostatic agents are pivotal for managing clinical and traumatic bleeding during emergency and domestic circumstances. Herein, a novel functional hybrid nanocomposite material consisting of plasma polymer-modified zeolite 13X and ultra-small gold nanoclusters (AuNCs) was fabricated as an efficient hemostatic agent. The surface of zeolite 13X was functionalised with amine groups which served as binding sites for carboxylate terminated AuNCs. Protein corona studies revealed the enhanced adsorption of two proteins, namely, coagulation factors and plasminogen as a result of AuNCs immobilization on the zeolite surface. The immune response studies showed that the hybrid nanocomposites are effective in reducing inflammation, which combined with a greater attachment of vitronectin, may promote wound healing. The hemostatic potential of the nanocomposite could be directly correlated with their immunomodulatory and anti-haemorrhagic properties. Together, the hybrid nanoengineered material developed in this work could provide a new avenue to tackle life-threatening injuries in civilian and other emergencies.

Dermal Pericytes Exhibit Declined Ability to Promote Human Skin Regeneration with Ageing in 3D Organotypic Culture Models.

The well documented decline in the regenerative ability of ageing human skin has been attributed to many factors including genomic instability, telomere shortening, poor nutrient sensing, cellular senescence, and stem cell exhaustion. However, a role for the dermal cellular and molecular microenvironment in skin ageing is just emerging. We previously showed that dermal pericytes co-operate with fibroblasts to improve human skin regeneration in an organotypic skin culture model, and even do so in the absence of fibroblasts. Here, we report that the number of dermal cells, particularly pericytes, declines significantly in human skin of donors aged > 50 years. Notably, aged pericytes promoted epidermal regeneration of neonatal keratinocytes in organotypic cultures and the resulting epithelium exhibited a Ki67+/ΔNp63+ basal layer and terminal differentiation. However, the epithelium lacked several features of homeostasis displaying lower levels of ΔNp63 expression, decreased LAMA5 deposition at the dermo-epidermal junction, and the absence of basement membrane and hemi-desmosome assembly. We conclude that a decline in pericyte incidence and function contribute to an impaired epidermal microenvironment and poor skin regeneration with ageing in the human skin.

Mechanistic Insight in Surface Nanotopography Driven Cellular Migration.

Cellular migration plays a vital role in many physiological processes. To elucidate the role of surface nanotopography on the downstream signaling pathways underlying cell migration, model surfaces having well-defined hill-like surface nanotopography and uniform surface chemistry were designed and implemented using plasma polymerization and covalent attachment of nanoparticles of predetermined size. A scratch wound assay, immunostaining, and gene expression of focal adhesion (FA) proteins were performed to determine the influence of surface nanotopography on cell migration. The results of this study demonstrate that the gap closure between cell monolayers is faster on surfaces having greater nanoscale topography. The phenomenon is predominantly driven by cell migration and was independent from cell proliferation. Qualitative and quantitative assessment of proteins involved in the signaling pathways underlying cell migration showed significant modulation by surface nanotopography. Specifically, focal adhesion sites decreased with the increase in surface nanotopography scale while the expression of FA proteins increased. This implies that nanotopography mediated modulation of cell migration is directly governed by the recruitment of receptor and adapter proteins responsible for cell-surface interaction. The results of this study indicate that biomaterial devices and constructs having rationally designed surface nanotopography and chemistry could be utilized to regulate wound healing and tissue regeneration.

In Vitro Bactericidal Efficacy of Nanostructured Ti6Al4V Surfaces is Bacterial Load Dependent.

The demand for medical implants globally has increased significantly due to an aging population amongst other reasons. Despite the overall increase in the survivorship of Ti6Al4V implants, implant infection rates are increasing due to factors such as diabetes, obesity, and bacterial resistance to antibiotics. Two commonly found bacteria implicated in implant infections are Staphylococcus aureus and Pseudomonas aeruginosa. Based on prior work that showed nanostructured surfaces might have potential in passively killing these bacterial species, we developed a hierarchical, hydrothermally etched, nanostructured titanium surface. To evaluate the antibacterial efficacy of this surface, etched and as-received surfaces were inoculated with S. aureus or P. aeruginosa at concentrations ranging from 102 to 109 colony-forming units per disc. Live/dead staining revealed there was a 60% decrease in viability for S. aureus and greater than a 98% decrease for P. aeruginosa on etched surfaces at the lowest inoculum of 102 CFU/disc, when compared to the control surface. Bactericidal efficiency decreased with increasing bacterial concentrations in a stepwise manner, with decreases in bacterial viability noted for S. aureus above 105 CFU/disc and above 106 CFU/disc for P. aeruginosa. Surprisingly, biofilm depth analysis revealed a decrease in bacterial viability in the 2 µm layer furthest from the nanostructured surface. The nanostructured Ti6Al4V surface developed here holds the potential to reduce the rate of implant infections.

Synergistic Effect of Surface Chemistry and Surface Topography Gradient on Osteogenic/Adipogenic Differentiation of hMSCs.

Much attention has been paid to understanding the individual effects of surface chemistry or topography on cell behavior. However, the synergistic influence of both surface chemistry and surface topography on differentiation of human mesenchymal stem cells (hMSCs) should also be addressed. Here, gold nanoparticles were immobilized in an increasing number density manner to achieve a surface topography gradient; a thin film rich in amine (-NH2) or methyl (-CH3) chemical groups was plasma-polymerized to adjust the surface chemistry of the outermost layer (ppAA and ppOD, respectively). hMSCs were cultured on these model substrates with defined surface chemistry and surface topography gradient. The morphology and focal adhesion (FA) formation of hMSCs were first examined. hMSC differentiation was then co-induced in osteogenic and adipogenic medium, as well as in the presence of extracellular-signal-regulated kinase1/2 (ERK1/2) and RhoA/Rho-associated protein kinase (ROCK) inhibitors. The results show that the introduction of nanotopography could enhance FA formation and osteogenesis but inhibited adipogenesis on both ppAA and ppOD surfaces, indicating that the surface chemistry could regulate hMSC differentiation, in a surface topography-dependent manner. RhoA/ROCK and ERK1/2 signaling pathways may participate in this process. This study demonstrated that surface chemistry and surface topography can jointly affect cell morphology, FA formation, and thus osteogenic/adipogenic differentiation of hMSCs. These findings highlight the importance of the synergistic effect of different material properties on regulation of cell response, which has important implications in designing functional biomaterials.

Synergistic effect of deep ball burnishing and HA coating on surface integrity, corrosion and immune response of biodegradable AZ31B Mg alloys.

The fast degradation and consequent loss of mechanical integrity is a major problem of biodegradable Mg alloy, which limits its clinical viability. This paper presents the influence of a synergistic approach combining deep ball burnishing and hydroxyapatite (HA) coating on biomechanical integrity, degradation and immune response of Mg alloy (AZ31B). The burnishing resulted in smooth surface topography, increased hardness from 0.87 to 1.45 GPa and induced microstructural disturbances with deformation twins/twin bands, which enabled formation of a dense and compact platelet-like crystals HA coating of 110 µm thickness. Compared to the untreated and burnished specimens, the burnished + HA coated surface provided remarkably higher corrosion resistance as indicated by lower corrosion current density and smaller mass loss. HA coating and surface integrity enhancement by burnishing were predominantly responsible for improved corrosion resistance. HA coating on the burnished surface exhibited hydrophilic properties and adequate bonding strength. While the modified surfaces promoted cell growth, the burnished + HA surface outperformed in exhibiting less pro-inflammatory and high anti-inflammatory cytokines, demonstrating that the treated surfaces were not posing any threat to immune cells. The findings indicate that the synergistic surface treatment can be a viable means to enhance corrosion resistance and immune response of Mg alloys implants.

The Influence of Nanoparticle Shape on Protein Corona Formation.

Nanoparticles have become an important utility in many areas of medical treatment such as targeted drug and treatment delivery as well as imaging and diagnostics. These advances require a complete understanding of nanoparticles' fate once placed in the body. Upon exposure to blood, proteins adsorb onto the nanoparticles surface and form a protein corona, which determines the particles' biological fate. This study reports on the protein corona formation from blood serum and plasma on spherical and rod-shaped nanoparticles. These two types of mesoporous silica nanoparticles have identical chemistry, porosity, surface potential, and size in the y-dimension, one being a sphere and the other a rod shape. The results show a significantly larger amount of protein attaching from both plasma and serum on the rod-like particles compared to the spheres. Interrogation of the protein corona by liquid chromatography-mass spectrometry reveals shape-dependent differences in the adsorption of immunoglobulins and albumin proteins from both plasma and serum. This study points to the need for taking nanoparticle shape into consideration because it can have a significant impact on the fate and therapeutic potential of nanoparticles when placed in the body.

It takes two for chronic wounds to heal: dispersing bacterial biofilm and modulating inflammation with dual action plasma coatings.

Chronic wounds are affecting increasingly larger portions of the general population and their treatment has essentially remained unchanged for the past century. This lack of progress is due to the complex problem that chronic wounds are simultaneously infected and inflamed. Both aspects need to be addressed together to achieve a better healing outcome. Hence, we hereby demonstrate that the stable nitroxide radical (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) can be plasma polymerized into smooth coatings (TEMPOpp), as seen via atomic force microscopy, X-ray photoelectron spectroscopy and ellipsometry. Upon contact with water, these coatings leach nitroxides into aqueous supernatant, as measured via EPR. We then exploited the known cell-signalling qualities of TEMPO to change the cellular behaviour of bacteria and human cells that come into contact with the surfaces. Specifically, the TEMPOpp coatings not only suppressed biofilm formation of the opportunistic bacterium Staphylococcus epidermidis but also dispersed already formed biofilm in a dose-dependent manner; a crucial aspect in treating chronic wounds that contain bacterial biofilm. Thus the coatings' microbiological efficacy correlated with their thickness and the thickest coating was the most efficient. Furthermore, this dose-dependent effect was mirrored in significant cytokine reduction of activated THP-1 macrophages for the four cytokines TNF-α, IL-1ß, IL-6 and IP-10. At the same time, the THP-1 cells retained their ability to adhere and colonize the surfaces, as verified via SEM imaging. Thus, summarily, we have exploited the unique qualities of plasma polymerized TEMPO coatings in targeting both infection and inflammation simultaneously; demonstrating a novel alternative to how chronic wounds could be treated in the future.

Biomaterial Surface Hydrophobicity-Mediated Serum Protein Adsorption and Immune Responses.

The nature of the protein corona forming on biomaterial surfaces can affect the performance of implanted devices. This study investigated the role of surface chemistry and wettability on human serum-derived protein corona formation on biomaterial surfaces and the subsequent effects on the cellular innate immune response. Plasma polymerization, a substrate-independent technique, was employed to create nanothin coatings with four specific chemical functionalities and a spectrum of surface charges and wettability. The amount and type of protein adsorbed was strongly influenced by surface chemistry and wettability but did not show any dependence on surface charge. An enhanced adsorption of the dysopsonin albumin was observed on hydrophilic carboxyl surfaces while high opsonin IgG2 adsorption was seen on hydrophobic hydrocarbon surfaces. This in turn led to a distinct immune response from macrophages; hydrophilic surfaces drove greater expression of anti-inflammatory cytokines by macrophages, whilst surface hydrophobicity caused increased production of proinflammatory signaling molecules. These findings map out a unique relationship between surface chemistry, hydrophobicity, protein corona formation, and subsequent cellular innate immune responses; the potential outcomes of these studies may be employed to tailor biomaterial surface modifications, to modulate serum protein adsorption and to achieve the desirable innate immune response to implanted biomaterials and devices.

Plasma deposited poly-oxazoline nanotextured surfaces dictate osteoimmunomodulation towards ameliorative osteogenesis.

Developing "osteoimmune-smart" bone substitute materials have become the forefront of research in bone regeneration. Biocompatible polymer coatings are applied widely to improve the bioactivity of bone substitute materials. In this context, polyoxazolines (Pox) have attracted substantial attention recently due to properties such as biocompatibility, stability, and low biofouling. In view of these useful properties, it is interesting to explore the capacity of Pox as an osteoimmunomodulatory agent to generate a favorable osteoimmune environment for osteogenesis. We applied a technique called plasma polymerization and succeeded in preparing Pox-like coatings (Ppox) and engineered their nanotopography at the nanoscale. We found that Ppox switched macrophages towards M2 extreme, thus inhibiting the release of inflammatory cytokines. The underlying mechanism may be related to the suppression of TLR pathway. The generated osteoimmune environment improved osteogenesis while inhibited osteoclastogenesis. This may be related to the release of osteogenic factors, especially Wnt10b from macrophages. The addition of nanotopography (16 nm, 38 nm, 68 nm) can tune the Ppox-mediated inhibition on inflammation and osteoclastic activities, while no significant effects were observed within the tested nano sizes on the Ppox-mediated osteogenesis. These results collectively suggest that Ppox can be useful as an effective osteoiumunomodulatory agent to endow bone substitute materials with favourable osteoimmunomodulatory property. STATEMENT OF SIGNIFICANCE: In this study, we succeeded in preparing plasma deposited Pox-like nano-coatings (Ppox) via plasma polymerization and found that Ppox nanotopographies are useful osteoimmunomodulatory tools. Their osteoimmunodolatory effects and underlying mechanisms are unveiled. It is the first investigation into the feasibility of applying poly-oxazoline as an osteoimmunomodulatory agent. This expand the application of poly-oxazoline into the forefront in bone regeneration area for the development of advanced "osteoimmune-smart" bone substitute materials.

Magnetic separation technology: Functional group efficiency in the removal of haze-forming proteins from wines.

Magnetic nanoparticles were modified by plasma polymerization, using allylamine, acrylic acid and 2-methyl-2-oxazoline as precursor to produce amine, carboxyl and oxazoline functional group rich surfaces. The nanoparticles were characterized by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and zeta potential measurements. The capacity of nanoparticles carrying different surface properties to remove haze-forming proteins from Sémillon and Sauvignon Blanc unfined wines was examined by high-performance liquid chromatography (HPLC). The protein capture efficiency of the modified surfaces decreases in the following order: COOH > POx > NH2. This study will help optimising the design of this new technology for the selective removal of haze proteins from white wines.

Plasma Polymer Coatings To Direct the Differentiation of Mouse Kidney-Derived Stem Cells into Podocyte and Proximal Tubule-like Cells.

Kidney disease is now recognized as a global health problem and is associated with increased morbidity and mortality, along with high economic costs. To develop new treatments for ameliorating kidney injury and preventing disease progression, there is a need for appropriate renal culture systems for screening novel drugs and investigating the cellular mechanisms underlying renal pathogenesis. There is a need for in vitro culture systems that promote the growth and differentiation of specialized renal cell types. In this work, we have used plasma polymerization technology to generate gradients of chemical functional groups to explore whether specific concentrations of these functional groups can direct the differentiation of mouse kidney-derived stem cells into specialized renal cell types. We found that amine-rich (-NH2) allylamine-based plasma-polymerized coatings could promote differentiation into podocyte-like cells, whereas methyl-rich (CH3) 1,7-octadiene-based coatings promoted differentiation into proximal tubule-like cells (PTC). Importantly, the PT-like cells generated on the substrates expressed the marker megalin and were able to endocytose albumin, indicating that the cells were functional.