Soy Protein Nanofiber Scaffolds for Uniform Maturation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells, called induced retinal pigment epithelium (iRPE), is being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration. The success of RPE implantation is linked to the use of biomimetic scaffolds that simulate Bruch's membrane and promote RPE maturation and integration as a functional tissue. Due to difficulties associated with animal protein-derived scaffolds, including sterility and pro-inflammatory responses, current practices favor the use of synthetic polymers, such as polycaprolactone (PCL), for generating nanofibrous scaffolds. In this study, we tested the hypothesis that plant protein-derived fibrous scaffolds can provide favorable conditions permissive for the maturation of RPE tissue sheets in vitro. Our natural, soy protein-derived nanofibrous scaffolds exhibited a J-shaped stress-strain curve that more closely resembled the mechanical properties of native tissues than PCL with significantly higher hydrophilicity of the natural scaffolds, favoring in vivo implantation. We then demonstrate that iRPE sheets growing on these soy protein scaffolds are equivalent to iRPE monolayers cultured on synthetic PCL nanofibrous scaffolds. Immunohistochemistry demonstrated RPE-like morphology and functionality with appropriate localization of RPE markers RPE65, PMEL17, Ezrin, and ZO1 and with anticipated histotypic polarization of vascular endothelial growth factor and pigment epithelium-derived growth factor as indicated by enzyme-linked immunosorbent assay. Scanning electron microscopy revealed dense microvilli on the cell surface and homogeneous tight junctional contacts between the cells. Finally, comparative transcriptome analysis in conjunction with principal component analysis demonstrated that iRPE on nanofibrous scaffolds, either natural or synthetic, matured more consistently than on nonfibrous substrates. Taken together, our studies suggest that the maturation of cultured iRPE sheets for subsequent clinical applications might benefit from the use of nanofibrous scaffolds generated from natural proteins. Impact statement Induced retinal pigment epithelium (iRPE) from patient-derived induced pluripotent stem cells (iPSCs) may yield powerful treatments of retinal diseases, including age-related macular degeneration. Recent studies, including early human clinical trials, demonstrate the importance of selecting appropriate biomaterial scaffolds to support tissue-engineered iRPE sheets during implantation. Electrospun scaffolds show particular promise due to their similarity to the structure of the native Bruch's membrane. In this study, we describe the use of electroprocessed nanofibrous soy protein scaffolds to generate polarized sheets of human iPSC-derived iRPE sheets. Our evaluation, including RNA-seq transcriptomics, indicates that these scaffolds are viable alternatives to scaffolds electrospun from synthetic polymers.

Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal.

PURPOSE: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. METHODS: We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. RESULTS: We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrate that the addition of 9-cis retinal, instead of the widely used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. CONCLUSION: Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.

Production of high purity alveolar-like cells from iPSCs through depletion of uncommitted cells after AFE induction.

Protocols to differentiate induced pluripotent stem cells (iPSCs) into specialized cells are continually evolving. iPSCs can be differentiated to alveolar cells with protocols that focus on development, specifically by inducing differentiation into definitive endoderm (DE), anterior foregut endoderm (AFE) and then lung bud progenitor intermediaries. However, current protocols result in a relatively low yield of the desired alveolar cells. The aim of this study was to evaluate whether depleting uncommitted cells after AFE induction would have a beneficial effect on alveolar cell yield. iPSCs were differentiated on Matrigel-coated plates for 25days. At each stage, phenotype was assessed using flow cytometry, immunofluorescence and qRT-PCR. Additionally, samples were dissociated in trypsin following AFE induction to improve the purity of the cells for the subsequent lung differentiation phase. Finally, the efficacy of dissociating the samples was confirmed comparing the expression of markers indicative of pluripotency and apoptosis. The ability to differentiate iPSCs to DE was 96% and to AFE was 97% utilizing our current protocol. After depletion of uncommitted cells and 12 days in culture, the purity of lung bud progenitors was 99%. Finally, the percentage of alveolar types I and II at the end of differentiation was 74% as compared to 31% in control cultures that had not been depleted of uncommitted cells after AFE induction. In conclusion, depletion of uncommitted cells after AFE induction, improves terminal differentiation of alveolar cells from 31% to 74%.

Activatable interpolymer complex-superparamagnetic iron oxide nanoparticles as magnetic resonance contrast agents sensitive to oxidative stress.

Magnetic resonance contrast agents that can be activated in response to specific triggers hold potential as molecular biosensors that may be of great utility in non-invasive disease diagnosis. We developed an activatable agent based on superparamagnetic iron oxide nanoparticles (SPIOs) that is sensitive to oxidative stress, a factor in the pathophysiology of numerous diseases. SPIOs were coated with poly(ethylene glycol) (PEG) and complexed with poly(gallol), a synthetic tannin. Hydrogen bonding between PEG and poly(gallol) creates a complexed layer around the SPIO that decreases the interaction of solute water with the SPIO, attenuating its magnetic resonance relaxivity. The complexed interpolymer nanoparticle is in an OFF state (decreased T2 contrast), where the contrast agent has a low T2 relaxivity of 7±2mM-1s-1. In the presence of superoxides, the poly(gallol) is oxidized and the polymers decomplex, allowing solute water to again interact with the SPIO, representing an ON state (increased T2 contrast) with a T2 relaxivity of 70±10mM-1s-1. These contrast agents show promise as effective sensors for diseases characterized in part by oxidative stress such as atherosclerosis, diabetes, and cancer.

Mesenchymal stem cell response to TGF-ß1 in both 2D and 3D environments.

Smooth muscle cells (SMC) are critical in stabilizing developing vascular networks, and transforming growth factor ß1 (TGF-ß1) has been shown to promote SMC differentiation from stem cells. Previously, our lab has developed a chemically modified fibrin-based hydrogel that induces endothelial cell (EC) phenotype and network formation from human mesenchymal stem cells (hMSCs) without exogenous cytokines. Additionally, we have shown that this hydrogel system is capable of releasing growth factors in a controlled manner. In the present work, the effects of TGF-ß1 on hMSCs in both monolayer and fibrin-based gel culture systems were demonstrated. The objective was to enhance SMC properties through TGF-ß1 signaling for vessel stability while maintaining EC gene expression and morphology. Proliferation was decreased with higher TGF-ß1 concentration in both monolayer and 3D gel cultures. EC genes were predominantly downregulated in the presence of TGF-ß1 in monolayer cultures, while SMC genes were generally upregulated. In fibrin-based gels, several SMC genes were significantly upregulated at high concentrations of TGF-ß1. Even at elevated TGF-ß1 concentrations, no significant differences were seen in EC genes for hMSCs in gels compared to controls. Network formation and growth occurred in PEGylated fibrin gels loaded with TGF-ß1 and were not significantly different from gels without loaded growth factor. Additionally, production of smooth muscle α-actin (SMA) was significantly increased in gels loaded with TGF-ß1. These results demonstrate a simultaneous response of hMSCs to both the 3D biomatrix and cytokine signaling cues.

Controlled release of IGF-I from a biodegradable matrix improves functional recovery of skeletal muscle from ischemia/reperfusion.

Ischemia/reperfusion (I/R) injury is a considerable insult to skeletal muscle, often resulting in prolonged functional deficits. The purpose of the current study was to evaluate the controlled release of the pro-regenerative growth factor, insulin-like growth factor-I (IGF-I), from a biodegradable polyethylene glycol (PEG)ylated fibrin gel matrix and the subsequent recovery of skeletal muscle from I/R. To accomplish this, the hind limbs of male Sprague-Dawley rats were subjected to 2-h tourniquet-induced I/R then treated with saline, bolus IGF-I (bIGF), PEGylated fibrin gel (PEG-Fib), or IGF-I conjugated PEGylated fibrin gel (PEG-Fib-IGF). Functional and histological evaluations were performed following 14 days of reperfusion, and muscles from 4-day reperfusion animals were analyzed by Western blotting and histological assessments. There was no difference in functional recovery between saline, bIGF, or PEG-Fib groups. However, PEG-Fib-IGF treatment resulted in significant improvement of muscle function and structure, as observed histologically. Activation of the PI3K/Akt pathway was significantly elevated in PEG-Fib-IGF muscles, compared to PEG-Fib treatment, at 4 days of reperfusion, suggesting involvement of the pathway PI3K/Akt as a mediator of the improved function. Surprisingly, myoblast activity was not evident as a result of PEG-Fib-IGF treatment. Taken together, these data give evidence for a protective role for the delivered IGF. These results indicate that PEG-Fib-IGF is a viable therapeutic technique in the treatment of skeletal muscle I/R injury.

Fibronectin-hyaluronic acid composite hydrogels for three-dimensional endothelial cell culture.

Biomaterials that actively promote both wound healing and angiogenesis are of critical importance for many biomedical applications, including tissue engineering. In particular, hyaluronic acid (HA) is an important player that has multiple roles throughout the angiogenic process in the body. Previously, our laboratory has developed photocrosslinkable HA-based scaffolds that promote angiogenesis when implanted in vivo. This paper reports the incorporation of a photocrosslinkable fibronectin (FN) conjugate into three-dimensional (3-D) HA hydrogel networks to enhance endothelial cell adhesion and angiogenesis. The results demonstrate significantly better retention of FN that was photocrosslinked within HA hydrogels compared to FN that was physically adsorbed within HA hydrogels. Increased viability of endothelial cells cultured in 3-D HA hydrogels with photoimmobilized FN, compared to adsorbed FN, was also observed. Endothelial cells were cultured within hydrogels for up to 6 days, a period over which cell proliferation, migration and an angiogenic phenotype were influenced by varying the concentration of incorporated FN. The results demonstrate the potential of these composite hydrogels as biomaterial scaffolds capable of promoting wound healing and angiogenesis.

Multimodal release of transforming growth factor-ß1 and the BB isoform of platelet derived growth factor from PEGylated fibrin gels.

We designed a growth factor release system to potentially stabilize neovascularization in the treatment of ischemic tissue. In this study, the release of PDGF-BB and TGF-ß1 was controlled with distinct kinetics from injectable PEGylated fibrin gels. Growth factors can be loaded into PEGylated fibrin gels via 3 mechanisms: entrapment, conjugation through a homobifunctional amine reactive PEG linker, and physical affinity with the fibrin matrix. PDGF-BB was entrapped during thrombin-mediated crosslinking leading to a diffusion-controlled release over 2days. TGF-ß1 was both conjugated through the PEG linker and bound to the matrix via physical affinity, delaying the release rate of TGF-ß1 up to 10days. Further, the release rate was highly correlated to gel degradation rate indicating that TGF-ß1 release is degradation-controlled. Therefore, by modulating the molar ratio of PEG to fibrinogen, we were able to control the release rate of TGF-ß1 without altering the release kinetics of PDGF-BB. The bioactivity of loaded TGF-ß1 was maintained upon release as evidenced by the inhibition of cell proliferation. This system could be expanded to incorporate growth factors loaded via 3 schemes with differing release rates from an injectable system allowing for a high degree of flexibility in other combinational drug delivery and tissue engineering systems.

Vascular differentiation of bone marrow stem cells is directed by a tunable three-dimensional matrix.

Microenvironmental cues are critical in regulating cell behavior and fate. The roles that matrix mechanical signals play in regulating cell behavior have recently been elucidated. An artificial matrix that can maintain the appropriate characteristics for transplanted stem cells is therefore needed to achieve a desired cell phenotype. The objective of this study was to develop a three-dimensional (3-D) matrix with tunable physical and mechanical properties and investigate their effects on mesenchymal stem cell (MSC) differentiation towards vascular cell types. In this study we developed an extracellular microenvironment by modifying fibrinogen with various polyethylene glycol (PEG) derivatives. We hypothesized that adjusting the type of PEG derivative to modify the resultant physical and mechanical characteristics of fibrin would allow us to create a tunable system for use in culture or in vivo in conjunction with a regenerative medicine strategy. Human MSC (hMSC) were entrapped into PEGylated fibrin matrices at a density of 50,000 cells ml(-1). Cell phenotypes were confirmed by immunofluorescent staining as well as the use of oligonucleotide arrays. Vascular phenotypes were correlated with measured mechanical properties and fiber diameters of the PEGylated fibrin matrices. Blocking studies were performed to identify mechanistic factors controlling MSC differentiation through selected blocking of matrix degradation or cell contraction. Cell-matrix interactions were also examined in vivo. Our results demonstrate that transdifferentiation of MSC towards an endothelial cell phenotype is profoundly affected by the 3-D matrix microenvironment. Our work provides a predictive road map for the creation of fibrin-based matrices that support robust endothelial cell gene expression and tubulogenesis.