Photo-crosslinked chitosan-gelatin xerogel-like coating onto "cold" plasma functionalized poly(lactic acid) film as cell culture support.

This paper reports on biofunctionalisation of a poly(lactic acid) (PLA) film by surface activation through cold plasma treatment followed by coating with a chitosan-gelatin xerogel. The UV cross-linking of the xerogel precursor was simultaneously performed with the fixation onto the PLA support. This has a strong effect on surface properties, in terms of wettability, surface free energy, morphology and micromechanical features. The hydrophilic - hydrophobic character of the surface, determined by contact angle measurements, was tuned along the process, passing from moderate hydrophobic PLA to enhanced hydrophilic plasma activated surface, which favors coating adhesion, then to moderate hydrophobic chitosan-gelatin coating. The coating has a Lewis amphoteric surface, with a porous xerogel-like morphology, as revealed by scanning electron microscopy images. By riboflavin mediated UV cross-linking the chitosan-gelatin coating becomes high adhesive and with a more pronounced plasticity, as shown by AFM force-distance spectroscopy. Thus prepared surface-coated PLA supports were successfully tested for growth of dermal fibroblasts, which are known for their induction potential of chondrogenic cells, which is very important in cartilage tissue engineering.

BSA Binding and Aggregate Formation of a Synthetic Amino Acid with Potential for Promoting Fibroblast Proliferation: An In Silico, CD Spectroscopic, DLS, and Cellular Study.

This study presents the chemical synthesis, purification, and characterization of a novel non-natural synthetic amino acid. The compound was synthesized in solution, purified, and characterized using NMR spectroscopy, polarimetry, and melting point determination. Dynamic Light Scattering (DLS) analysis demonstrated its ability to form aggregates with an average size of 391 nm, extending to the low micrometric size range. Furthermore, cellular biological assays revealed its ability to enhance fibroblast cell growth, highlighting its potential for tissue regenerative applications. Circular dichroism (CD) spectroscopy showed the ability of the synthetic amino acid to bind serum albumins (using bovine serum albumin (BSA) as a model), and CD deconvolution provided insights into the changes in the secondary structures of BSA upon interaction with the amino acid ligand. Additionally, molecular docking using HDOCK software elucidated the most likely binding mode of the ligand inside the BSA structure. We also performed in silico oligomerization of the synthetic compound in order to obtain a model of aggregate to investigate computationally. In more detail, the dimer formation achieved by molecular self-docking showed two distinct poses, corresponding to the lowest and comparable energies, with one pose exhibiting a quasi-coplanar arrangement characterized by a close alignment of two aromatic rings from the synthetic amino acids within the dimer, suggesting the presence of π-π stacking interactions. In contrast, the second pose displayed a non-coplanar configuration, with the aromatic rings oriented in a staggered arrangement, indicating distinct modes of interaction. Both poses were further utilized in the self-docking procedure. Notably, iterative molecular docking of amino acid structures resulted in the formation of higher-order aggregates, with a model of a 512-mer aggregate obtained through self-docking procedures. This model of aggregate presented a cavity capable of hosting therapeutic cargoes and biomolecules, rendering it a potential scaffold for cell adhesion and growth in tissue regenerative applications. Overall, our findings highlight the potential of this synthetic amino acid for tissue regenerative therapeutics and provide valuable insights into its molecular interactions and aggregation behavior.

A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors.

Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein-Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors.

Investigations of cardiac fibrosis rheology by in vitro cardiac tissue modeling with 3D cellular spheroids.

Cardiac fibrosis refers to the abnormal accumulation of extracellular matrix within the cardiac muscle, leading to increased stiffness and impaired heart function. From a rheological standpoint, knowledge about myocardial behavior is still lacking, partially due to a lack of appropriate techniques to investigate the rheology of in vitro cardiac tissue models. 3D multicellular cardiac spheroids are powerful and versatile platforms for modeling healthy and fibrotic cardiac tissue in vitro and studying how their mechanical properties are modulated. In this study, cardiac spheroids were created by co-culturing neonatal rat ventricular cardiomyocytes and fibroblasts in definite ratios using the hanging-drop method. The rheological characterization of such models was performed by Atomic Force Microscopy-based stress-relaxation measurements on the whole spheroid. After strain application, a viscoelastic bi-exponential relaxation was observed, characterized by a fast relaxation time (τ1) followed by a slower one (τ2). In particular, spheroids with higher fibroblasts density showed reduction for both relaxation times comparing to control, with a more pronounced decrement of τ1 with respect to τ2. Such response was found compatible with the increased production of extracellular matrix within these spheroids, which recapitulates the main feature of the fibrosis pathophysiology. These results demonstrate how the rheological characteristics of cardiac tissue vary as a function of cellular composition and extracellular matrix, confirming the suitability of such system as an in vitro preclinical model of cardiac fibrosis.

Bone marrow mesenchymal stem cell-derived exosomes shuttle microRNAs to endometrial stromal fibroblasts that promote tissue proliferation /regeneration/ and inhibit differentiation.

BACKGROUND: Human bone marrow-derived stem cells (hBMDSCs) are well characterized mediators of tissue repair and regeneration. An increasing body of evidence indicates that these cells exert their therapeutic effects largely through their paracrine actions rather than clonal expansion and differentiation. Here we studied the role of microRNAs (miRNAs) present in extracellular vesicles (EVs) from hBMDSCs in tissue regeneration and cell differentiation targeting endometrial stromal fibroblasts (eSF). METHODS: Extracellular vesicles (EVs) are isolated from hBMDSCs, characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) techniques. Extracted total RNA from EVs was subjected to RNA seq analysis. Transfection and decidualization studies were carried out in endometrial stromal fibroblasts (eSF). Gene expression was analyzed by qRTPCR. Unpaired t-test with Welch's correction was used for data analysis between two groups. RESULTS: We identified several microRNAs (miRNAs) that were highly expressed, including miR-21-5p, miR-100-5p, miR-143-3p and let7. MiR-21 is associated with several signaling pathways involved in tissue regeneration, quiescence, cellular senescence, and fibrosis. Both miR-100-5p and miR-143-3p promoted cell proliferation. MiR-100-5p specifically promoted regenerative processes by upregulating TGF-ß3, VEGFA, MMP7, and HGF. MiR-100-5p blocked differentiation or decidualization as evidenced by morphologic changes and downregulation of decidualization mediators including HOXA10, IGFBP1, PRL, PR-B, and PR. CONCLUSION: EVs delivered to tissues by hBMDSCs contain specific miRNAs that prevent terminal differentiation and drive repair and regeneration. Delivery of microRNAs is a novel treatment paradigm with the potential to replace BMDSCs in cell-free regenerative therapies.

The Long Non-Coding RNA Gene AC027288.3 Plays a Role in Human Endometrial Stromal Fibroblast Decidualization.

During the secretory phase of the menstrual cycle, endometrial fibroblast cells begin to change into large epithelial-like cells called decidual cells in a process called decidualization. This differentiation continues more broadly in the endometrium and forms the decidual tissue during early pregnancy. The cells undergoing decidualization as well as the resulting decidual cells, support successful implantation and placentation during early pregnancy. This study was carried out to identify new potentially important long non-coding RNA (lncRNA) genes that may play a role in human endometrial stromal fibroblast cells (hESF) undergoing decidualization in vitro, and several were found. The expression of nine was further characterized. One of these, AC027288.3, showed a dramatic increase in the expression of hESF cells undergoing decidualization. When AC027288.3 expression was targeted, the ability of the cells to undergo decidualization as determined by the expression of decidualization marker protein-coding genes was significantly altered. The most affected markers of decidualization whose expression was significantly reduced were FOXO1, FZD4, and INHBA. Therefore, AC027288.3 may be a major upstream regulator of the WNT-FOXO1 pathway and activin-SMAD3 pathways previously shown as critical for hESF decidualization. Finally, we explored possible regulators of AC027288.3 expression during human ESF decidualization. Expression was regulated by cAMP and progesterone. Our results suggest that AC027288.3 plays a role in hESF decidualization and identifies several other lncRNA genes that may also play a role.

Exploring the cellular antioxidant mechanism against cytotoxic silver nanoparticles: a Raman spectroscopic analysis.

Silver nanoparticles (AgNPs) hold great promise for several different applications, from colorimetric sensors to antimicrobial agents. Despite their widespread incorporation in consumer products, limited understanding of the detrimental effects and cellular antioxidant responses associated with AgNPs at sublethal concentrations persists, raising concerns for human and ecological well-being. To address this gap, we synthesized AgNPs of varying sizes and evaluated their cytotoxicity against human dermal fibroblasts (HDF). Our study revealed that toxicity of AgNPs is a time- and size-dependent process, even at low exposure levels. AgNPs exhibited low short-term cytotoxicity but high long-term impact, particularly for the smallest NPs tested. Raman microspectroscopy was employed for in-time investigations of intracellular molecular variations during the first 24 h of exposure to AgNPs of 35 nm. Subtle protein and lipid degradations were detected, but no discernible damage to the DNA was observed. Signals associated with antioxidant proteins, such as superoxide dismutase (SOD), catalase (CAT) and metallothioneins (MTs), increased over time, reflecting the heightened production of these defense agents. Fluorescence microscopy further confirmed the efficacy of overexpressed antioxidant proteins in mitigating ROS formation during short-term exposure to AgNPs. This work provides valuable insights into the molecular changes and remedial strategies within the cellular environment, utilizing Raman microspectroscopy as an advanced analytical technique. These findings offer a novel perspective on the cytotoxicity mechanism of AgNPs, contributing to the development of safer materials and advice on regulatory guidelines for their biomedical applications.

Chromatin profiling and state predictions reveal insights into epigenetic regulation during early porcine development.

BACKGROUND: Given their physiological similarities to humans, pigs are increasingly used as model organisms in human-oriented biomedical studies. Additionally, their value to animal agriculture across the globe has led to the development of numerous studies to investigate how to improve livestock welfare and production efficiency. As such, pigs are uniquely poised as compelling models that can yield findings with potential implications in both human and animal contexts. Despite this, many gaps remain in our knowledge about the foundational mechanisms that govern gene expression in swine across different developmental stages, particularly in early development. To address some of these gaps, we profiled the histone marks H3K4me3, H3K27ac, and H3K27me3 and the SWI/SNF central ATPase BRG1 in two porcine cell lines representing discrete early developmental time points and used the resulting information to construct predicted chromatin state maps for these cells. We combined this approach with analysis of publicly available RNA-seq data to examine the relationship between epigenetic status and gene expression in these cell types. RESULTS: In porcine fetal fibroblast (PFF) and trophectoderm cells (PTr2), we saw expected patterns of enrichment for each of the profiled epigenetic features relative to specific genomic regions. H3K4me3 was primarily enriched at and around global gene promoters, H3K27ac was enriched in promoter and intergenic regions, H3K27me3 had broad stretches of enrichment across the genome and narrower enrichment patterns in and around the promoter regions of some genes, and BRG1 primarily had detectable enrichment at and around promoter regions and in intergenic stretches, with many instances of H3K27ac co-enrichment. We used this information to perform genome-wide chromatin state predictions for 10 different states using ChromHMM. Using the predicted chromatin state maps, we identified a subset of genomic regions marked by broad H3K4me3 enrichment, and annotation of these regions revealed that they were highly associated with essential developmental processes and consisted largely of expressed genes. We then compared the identities of the genes marked by these regions to genes identified as cell-type-specific using transcriptome data and saw that a subset of broad H3K4me3-marked genes was also specifically expressed in either PFF or PTr2 cells. CONCLUSIONS: These findings enhance our understanding of the epigenetic landscape present in early swine development and provide insight into how variabilities in chromatin state are linked to cell identity. Furthermore, this data captures foundational epigenetic details in two valuable porcine cell lines and contributes to the growing body of knowledge surrounding the epigenetic landscape in this species.

Transdifferentiation of fibroblasts into muscle cells to constitute cultured meat with tunable intramuscular fat deposition.

Current studies on cultured meat mainly focus on the muscle tissue reconstruction in vitro, but lack the formation of intramuscular fat, which is a crucial factor in determining taste, texture, and nutritional contents. Therefore, incorporating fat into cultured meat is of superior value. In this study, we employed the myogenic/lipogenic transdifferentiation of chicken fibroblasts in 3D to produce muscle mass and deposit fat into the same cells without the co-culture or mixture of different cells or fat substances. The immortalized chicken embryonic fibroblasts were implanted into the hydrogel scaffold, and the cell proliferation and myogenic transdifferentiation were conducted in 3D to produce the whole-cut meat mimics. Compared to 2D, cells grown in 3D matrix showed elevated myogenesis and collagen production. We further induced fat deposition in the transdifferentiated muscle cells and the triglyceride content could be manipulated to match and exceed the levels of chicken meat. The gene expression analysis indicated that both lineage-specific and multifunctional signalings could contribute to the generation of muscle/fat matrix. Overall, we were able to precisely modulate muscle, fat, and extracellular matrix contents according to balanced or specialized meat preferences. These findings provide new avenues for customized cultured meat production with desired intramuscular fat contents that can be tailored to meet the diverse demands of consumers.

High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos.

Lysine crotonylation (Kcr) is a recently discovered histone acylation modification that is closely associated with gene expression, cell proliferation, and the maintenance of stem cell pluripotency and indicates the transcriptional activity of genes and the regulation of various biological processes. During cell culture, the introduction of exogenous croconic acid disodium salt (Nacr) has been shown to modulate intracellular Kcr levels. Although research on Kcr has increased, its role in cell growth and proliferation and its potential regulatory mechanisms remain unclear compared to those of histone methylation and acetylation. Our investigation demonstrated that the addition of 5 mM Nacr to cultured bovine fibroblasts increased the expression of genes associated with Kcr modification, ultimately promoting cell growth and stimulating cell proliferation. Somatic cell nuclear transfer of donor cells cultured in 5 mM Nacr resulted in 38.1% blastocyst development, which was significantly greater than that in the control group (25.2%). This research is important for elucidating the crotonylation modification mechanism in fibroblast proliferation to promote the efficacy of somatic cell nuclear transfer.

Hemocompatibility and Preangiogenic Attributes of SiBxCyNzOm Coatings for Biomedical Applications.

In current clinical practices related to orthopedics, dental, and cardiovascular surgeries, a number of biomaterial coatings, such as hydroxyapatite (HAp), diamond-like carbon (DLC), have been used in combination with metallic substrates (stainless steel, Ti6Al4V alloy, etc.). Although SiBCN coatings are widely explored in material science for diverse applications, their potential remains largely unexplored for biomedical applications. With this motivation, the present work reports the development of SiBxCyNzOm coatings on a Ti6Al4V substrate, employing a reactive radiofrequency (RF) magnetron sputtering technique. Three different coating compositions (Si0.27B0.10C0.31N0.07O0.24, Si0.23B0.06C0.21N0.22O0.27, and Si0.20B0.05C0.19N0.20O0.35) were obtained using a Si2BC2N target and varying nitrogen flow rates. The hydrophilic properties of the as-synthesized coatings were rationalized in terms of an increase in the number of oxygen-containing functional groups (OH and NO) on the surface, as probed using XPS and FTIR analyses. Furthermore, the cellular monoculture of SVEC4-10 endothelial cells and L929 fibroblasts established good cytocompatibility. More importantly, the coculture system of SVEC4-10 and L929, in the absence of growth factors, demonstrated clear cellular phenotypical changes, with extensive sprouting leading to tube-like morphologies on the coating surfaces, when stimulated using a customized cell stimulator (StimuCell) with 1.15 V/cm direct current (DC) electric field strength for 1 h. In addition, the hemocompatibility assessment using human blood samples revealed clinically acceptable hemolysis, less erythrocyte adhesion, shorter plasma recalcification, and reduced risk for thrombosis on the SiBxCyNzOm coatings, when compared to uncoated Ti6Al4V. Taken together, the present study unambiguously establishes excellent cytocompatibility, hemocompatibility, and defines the preangiogenic properties of SiBxCyNzOm bioceramic coatings for potential biomedical applications.

Direct conversion of cardiac fibroblasts into endothelial-like cells using Sox17 and Erg.

Endothelial cells are a heterogeneous population with various organ-specific and conserved functions that are critical to organ development, function, and regeneration. Here we report a Sox17-Erg direct reprogramming approach that uses cardiac fibroblasts to create differentiated endothelial cells that demonstrate endothelial-like molecular and physiological functions in vitro and in vivo. Injection of these induced endothelial cells into myocardial infarct sites after injury results in improved vascular perfusion of the scar region. Furthermore, we use genomic analyses to illustrate that Sox17-Erg reprogramming instructs cardiac fibroblasts toward an arterial-like identity. This results in a more efficient direct conversion of fibroblasts into endothelial-like cells when compared to traditional Etv2-based reprogramming. Overall, this Sox17-Erg direct reprogramming strategy offers a robust tool to generate endothelial cells both in vitro and in vivo, and has the potential to be used in repairing injured tissue.

Schiff base crosslinked hyaluronic acid hydrogels with tunable and cell instructive time-dependent mechanical properties.

The dynamic interplay between cells and their native extracellular matrix (ECM) influences cellular behavior, imposing a challenge in biomaterial design. Dynamic covalent hydrogels are viscoelastic and show self-healing ability, making them a potential scaffold for recapitulating native ECM properties. We aimed to implement kinetically and thermodynamically distinct crosslinkers to prepare self-healing dynamic hydrogels to explore the arising properties and their effects on cellular behavior. To do so, aldehyde-substituted hyaluronic acid (HA) was synthesized to generate imine, hydrazone, and oxime crosslinked dynamic covalent hydrogels. Differences in equilibrium constants of these bonds yielded distinct properties including stiffness, stress relaxation, and self-healing ability. The effects of degree of substitution (DS), polymer concentration, crosslinker to aldehyde ratio, and crosslinker functionality on hydrogel properties were evaluated. The self-healing ability of hydrogels was investigated on samples of the same and different crosslinkers and DS to obtain hydrogels with gradient properties. Subsequently, human dermal fibroblasts were cultured in 2D and 3D to assess the cellular response considering the dynamic properties of the hydrogels. Moreover, assessing cell spreading and morphology on hydrogels having similar modulus but different stress relaxation rates showed the effects of matrix viscoelasticity with higher cell spreading in slower relaxing hydrogels.

The local mechanosensitive response of primary cardiac fibroblasts is influenced by the microenvironment mechanics.

Cardiac fibroblasts (CFs) are essential for preserving myocardial integrity and function. They can detect variations in cardiac tissue stiffness using various cellular mechanosensors, including the Ca2+ permeable mechanosensitive channel Piezo1. Nevertheless, how CFs adapt the mechanosensitive response to stiffness changes remains unclear. In this work we adopted a multimodal approach, combining the local mechanical stimulation (from 10 pN to 350 nN) with variations of culture substrate stiffness. We found that primary rat CFs cultured on stiff (GPa) substrates showed a broad Piezo1 distribution in the cell with particular accumulation at the mitochondria membrane. CFs displayed a force-dependent behavior in both calcium uptake and channel activation probability, showing a threshold at 300 nN, which involves both cytosolic and mitochondrial Ca2+ mobilization. This trend decreases as the myofibroblast phenotype within the cell population increases, following a possible Piezo1 accumulation at focal adhesion sites. In contrast, the inhibition of fibroblasts to myofibroblasts transition with soft substrates (kPa) considerably reduces both mechanically- and chemically-induced Piezo1 activation and expression. Our findings shed light on how Piezo1 function and expression are regulated by the substrate stiffness and highlight its involvement in the environment-mediated modulation of CFs mechanosensitivity.

A chitosan-coated PCL/nano-hydroxyapatite aerogel integrated with a nanofiber membrane for providing antibacterial activity and guiding bone regeneration.

A guided bone regeneration (GBR) membrane can act as a barrier to prevent the invasion and interference from foreign soft tissues, promoting infiltration and proliferation of osteoblasts in the bone defect area. Herein, a composite scaffold with dual functions of osteogenesis and antibacterial effects was prepared for GBR. A polycaprolactone (PCL)/nano-hydroxyapatite (n-HA) aerogel produced by electrospinning and freeze-drying techniques was fabricated as the loose layer of the scaffold, while a PCL nanofiber membrane was used as the dense layer. Chitosan (CS) solution served as a middle layer to provide mechanical support and antibacterial effects between the two layers. Morphological results showed that the loose layer had a porous structure with n-HA successfully dispersed in the aerogels, while the dense layer possessed a sufficiently dense structure. In vitro antibacterial experiments illustrated that the CS solution in the middle layer stabilized the scaffold structure and endowed the scaffold with good antibacterial properties. The cytocompatibility results indicated that both fibroblasts and osteoblasts exhibited superior cell activity on the dense and loose layers, respectively. In particular, the dense layer made of nanofibers could work as a barrier layer to inhibit the infiltration of fibroblasts into the loose layer. In vitro osteogenesis analysis suggested that the PCL/n-HA aerogel could enhance the bone induction ability of bone mesenchymal stem cells, which was confirmed by the increased expression of the alkaline phosphatase activity. The loose structure facilitated the infiltration and migration of bone mesenchymal stem cells for better osteogenesis. In summary, such a composite scaffold exhibited excellent osteogenic and antibacterial properties as well as the barrier effect, thus holding promising potential for use as GBR materials.

The effects of exosomes originating from different cell sources on the differentiation of bone marrow mesenchymal stem cells into schwann cells.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can differentiate into Schwann cells (SCs) during peripheral nerve injury; in our previous research, we showed that SC-derived exosomes (SC-exos) played a direct induction role while fibroblast-derived exosomes (Fb-exos) had no obvious induction role. The induction role of neural stem cell (NSC)-derived exosomes (NSC-exos) has also been widely confirmed. However, no studies have compared the induction effects of these three types of cells at the same time. Therefore, by investigating the effect of these three cell-derived exosomes upon the induction of BMSCs to differentiate into SCs, this study explored the role of different exosomes in promoting the differentiation of stem cells into SCs cells, and conducted a comparison between the two groups by RNA sequencing to further narrow the range of target genes and related gene pathways in order to study their related mechanisms. MATERIALS AND METHODS: We extracted exosomes from SCs, fibroblasts (Fb) and neural stem cells (NSC) and then investigated the ability of these exosomes to induce differentiation into BMSCs under different culture conditions. The expression levels of key proteins and gene markers were detected in induced cells by fluorescence immunoassays, western blotting and polymerase chain reaction (PCR); then, we statistically compared the relative induction effects under different conditions. Finally, we analyzed the three types of exosomes by RNA-seq to predict target genes and related gene pathways. RESULTS: BMSCs were cultured by three media: conventional (no induction), pre-induction or pre-induction + original induction medium (ODM) with exosomes of the same cell origin under different culture conditions. When adding the three different types of exosomes separately, the overall induction of BMSCs to differentiate into SCs was significantly increased (P < 0.05). The induction ability was ranked as follows: pre-induction + ODM + exosome group > pre-induction + exosome group > non-induction + exosome group. Using exosomes from different cell sources under the same culture conditions, we observed the following trends under the three culture conditions: RSC96-exos group ≥ NSC-exos group > Fb-exos group. The overall ability to induce BMSCs into SCs was significantly greater in the RSC96-exos group and the NSC-exos group. Although there was no significant difference in induction efficiency when comparing these two groups, the overall induction ability of the RSC96-exos group was slightly higher than that of the NSC-exos group. By combining the differentiation induction results with the RNA-seq data, the three types of exosomes were divided into three comparative groups: RSC vs. NSC, RSC vs. Fb and NSC vs. Fb. We identified 203 differentially expressed mRNA target genes in these three groups. Two differentially expressed genes were upregulated simultaneously, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). We did not identify any co-upregulated target genes for the miRNAs, but did identify one target gene of the lncRNAs, namely ENSRNOG00000065005. Analysis identified 90 GO terms related to nerves and axons in the mRNAs; in addition, KEGG enrichment and GASA analysis identified 13 common differential expression pathways in the three groups. CONCLUSIONS: Our analysis found that pre-induction + ODM + RSC96/NSC-exos culture conditions were most conducive with regards to induction and differentiation. RSC96-exos and NSC-exos exhibited significantly greater differentiation efficiency of BMSCs into SCs. Although there was no statistical difference, the data indicated a trend for RSC96-exos to be advantageous We identified 203 differentially expressed mRNAs between the three groups and two differentially expressed target mRNAs were upregulated, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). 90 GO terms were related to nerves and axons. Finally, we identified 13 common differentially expressed pathways across our three types of exosomes. It is hoped that the efficiency of BMSCs induction differentiation into SCs can be improved, bringing hope to patients and more options for clinical treatment.

Establishment of Transgene-Free Porcine Induced Pluripotent Stem Cells.

Although protocols to generate authentic transgene-free mouse and human induced pluripotent stem cells (iPSCs) are now well established, standard methods for reprogramming porcine somatic cells still suffer from low efficiency and transgene retention. The Basic Protocol describes reprogramming procedures to establish transgene-free porcine iPSCs (PiPSCs) from porcine fibroblasts. This method uses episomal plasmids encoding POU5F1, SOX2, NANOG, KLF4, SV40LT, c-MYC, LIN28A, and microRNA-302/367, combined with an optimized medium, to establish PiPSC lines. Support protocols describe the establishment and characterization of clonal PiPSC lines, as well as the preparation of feeder cells and EBNA1 mRNA. This optimized, step-by-step approach tailored to this species enables the efficient derivation of PiPSCs in ∼4 weeks. The establishment of transgene-free PiPSCs provides a new and valuable model for studies of larger mammalian species' development, disease, and regenerative biology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Reprogramming of porcine fibroblasts with episomal plasmids Support Protocol 1: Preparation of mouse embryonic fibroblasts for feeder layer Support Protocol 2: Preparation of in vitro-transcribed EBNA1 mRNA Support Protocol 3: Establishment of clonal porcine induced pluripotent stem cell (PiPSC) lines Support Protocol 4: PiPSC characterization: Genomic DNA PCR and RT-PCR Support Protocol 5: PiPSC characterization: Immunostaining.

The effect of continuous long-term illumination with visible light in different spectral ranges on mammalian cells.

One of the biggest challenges in tissue engineering and regenerative medicine is to ensure oxygen supply of cells in the (temporary) absence of vasculature. With the vision to exploit photosynthetic oxygen production by microalgae, co-cultivated in close vicinity to oxygen-consuming mammalian cells, we are searching for culture conditions that are compatible for both sides. Herein, we investigated the impact of long-term illumination on mammalian cells which is essential to enable photosynthesis by microalgae: four different cell types-primary human fibroblasts, dental pulp stem cells, and osteoblasts as well as the murine beta-cell line INS-1-were continuously exposed to warm white light, red or blue light over seven days. We observed that illumination with red light has no adverse effects on viability, metabolic activity and growth of the cells whereas exposure to white light has deleterious effects that can be attributed to its blue light portion. Quantification of intracellular glutathione did not reveal a clear correlation of this effect with an enhanced production of reactive oxygen species. Finally, our data indicate that the cytotoxic effect of short-wavelength light is predominantly a direct effect of cell illumination; photo-induced changes in the cell culture media play only a minor role.

PURION® processed human amnion chorion membrane allografts retain material and biological properties supportive of soft tissue repair.

The reparative properties of amniotic membrane allografts are well-suited for a broad spectrum of specialties. Further enhancement of their utility can be achieved by designing to the needs of each application through the development of novel processing techniques and tissue configurations. As such, this study evaluated the material characteristics and biological properties of two PURION® processed amniotic membrane products, a lyophilized human amnion, intermediate layer, and chorion membrane (LHACM) and a dehydrated human amnion, chorion membrane (DHACM). LHACM is thicker; therefore, its handling properties are ideal for deep, soft tissue deficits; whereas DHACM is more similar to a film-like overlay and may be used for shallow defects or surgical on-lays. Characterization of the similarities and differences between LHACM and DHACM was conducted through a series of in vitro and in vivo studies relevant to the healing cascade. Compositional analysis was performed through histological staining along with assessment of barrier membrane properties through equilibrium dialysis. In vitro cellular response was assessed in fibroblasts and endothelial cells using cell proliferation, migration, and metabolic assays. The in vivo cellular response was assessed in an athymic nude mouse subcutaneous implantation model. The results indicated the PURION® process preserved the native membrane structure, nonviable cells and collagen distributed in the individual layers of both products. Although, LHACM is thicker than DHACM, a similar composition of growth factors, cytokines, chemokines and proteases is retained and consequently elicit comparable in vitro and in vivo cellular responses. In culture, both treatments behaved as potent mitogens, chemoattractants and stimulants, which translated to the promotion of cellular infiltration, neocollagen deposition and angiogenesis in a murine model. PURION® processed LHACM and DHACM differ in physical properties but possess similar in vitro and in vivo activities highlighting the impact of processing method on the versatility of clinical use of amniotic membrane allografts.

Mechanical stimulation of induced pluripotent stem derived cardiac fibroblasts.

Cardiac fibrosis contributes to the development of heart failure, and is the response of cardiac fibroblasts (CFs) to pressure or volume overload. Limiting factors in CFs research are the poor availability of human cells and the tendency of CFs to transdifferentiate into myofibroblasts when cultured in vitro. The possibility to generate CFs from induced pluripotent stem cells (iPSC), providing a nearly unlimited cell source, opens new possibilities. However, the behaviour of iPSC-CFs under mechanical stimulation has not been studied yet. Our study aimed to assess the behaviour of iPSC-CFs under mechanical stretch and pro-fibrotic conditions. First, we confirm that iPSC-CFs are comparable to primary CFs at gene, protein and functional level. Furthermore, iPSC-derived CFs adopt a pro-fibrotic response to transforming growth factor beta (TGF-ß). In addition, mechanical stretch inhibits TGF-ß-induced fibroblast activation in iPSC-CFs. Thus, the responsiveness to cytokines and mechanical stimulation of iPSC-CFs demonstrates they possess key characteristics of primary CFs and may be useful for disease modelling.