Regenerative Medicine Research Articles

Cone-rod homeobox transcriptionally activates TCF7 to promote the proliferation of retinal pigment epithelial and retinoblastoma cells in vitro

AIM: To investigate the proliferation regulatory effect of cone-rod homeobox (CRX) in retinal pigment epithelium (RPE) and retinoblastoma (RB) cells to explore the potential application and side effect (oncogenic potential) of CRX-based gene therapy in RPE-based retinopathies. METHODS: Adult human retinal pigment epithelial (ARPE)-19 and human retinal pigment epithelial (RPE)-1 cells and Y79 RB cell were used in the study. Genetic manipulation was performed by lentivirus-based technology. The cell proliferation was determined by a CellTiter-Glo Reagent. The mRNA and protein levels were determined by quantitative real-time polymerase chain reaction (qPCR) and Western blot assay. The transcriptional activity of the promoter was determined by luciferase reporter gene assay. The bindings between CRX and transcription factor 7 (TCF7) promoter as well as TCF7 and the promoters of TCF7 target genes were examined by chromatin immunoprecipitation (ChIP) assay. The transcription of the TCF7 was determined by a modified nuclear run-on assay. RESULTS: CRX overexpression and knockdown significantly increased (n=3, P<0.05 in all the cells) and decreased (n=3, P<0.01 in all the cells) the proliferation of RPE and RB cells. CRX overexpression and knockdown significantly increased and deceased the mRNA levels of Wnt signaling target genes [including MYC proto-oncogene (MYC), JUN, FOS like 1 (FOSL1), CCND1, cyclin D2 (CCND2), cyclin D3 (CCND3), cellular communication network factor 4 (CCN4), peroxisome proliferator activated receptor delta (PPARD), and matrix metallopeptidase 7 (MMP7)] and the luciferase activity driven by the Wnt signaling transcription factor (TCF7). TCF7 overexpression and knockdown significantly increased and decreased the proliferation of RPE and RB cells and depletion of TCF7 significantly abolished the stimulatory effect of CRX on the proliferation of RPE and RB cells. CRX overexpression and knockdown significantly increased and decreased the mRNA level of TCF7 and the promoter of TCF7 was significantly immunoprecipitated by CRX antibody. CONCLUSION: CRX transcriptionally activates TCF7 to promote the proliferation of RPE and RB cells in vitro. CRX is a potential target for RPE-based regenerative medicine. The potential risk of this strategy, tumorigenic potential, should be considered.

Exploring mesenchymal stem cells homing mechanisms and improvement strategies.

Mesenchymal stem cells (MSCs) are multipotent cells with high self-renewal and multilineage differentiation abilities, playing an important role in tissue healing. Recent advancements in stem cell-based technologies have offered new and promising therapeutic options in regenerative medicine. Upon tissue damage, MSCs are immediately mobilized from the bone marrow and move to the injury site via blood circulation. Notably, allogenically transplanted MSCs can also home to the damaged tissue site. Therefore, MSCs hold great therapeutic potential for curing various diseases. However, one major obstacle to this approach is attracting MSCs specifically to the injury site following systemic administration. In this review, we describe the molecular pathways governing the homing mechanism of MSCs and various strategies for improving this process, including targeted stem cell administration, target tissue modification, in vitro priming, cell surface engineering, genetic modifications, and magnetic guidance. These strategies are crucial for directing MSCs precisely to the injury site and, consequently, enhancing their migration and local tissue repair properties. Specifically, our review provides a guide to improving the therapeutic efficacy of clinical applications of MSCs through optimized in vivo administration and homing capacities.

A novel method to assess photobiomodulation in stimulating regenerative capacity and vascularization in zebrafish.

Photobiomodulation (PBM) therapy is a continuously growing approach to stimulating healing and reducing inflammation and pain. However, its effects in the fields of regenerative medicine and tissue engineering are still under investigation. Studying PBM effects on the regenerative capacity of zebrafish can allow the application of novel clinical approaches where the impact of PBM will be cross-linked with the stem-cell therapeutic approaches. This study was done to establish an in-vivo experimental setup for studying the effects of laser and ultraviolet therapy on zebrafish caudal-fin regeneration and vascularization. Thirty zebrafish were randomly and equally allocated into three groups. The caudal-fins of all zebrafish were amputated under anaesthesia. In the first control group, the caudal-fin was only monitored until fully regenerated. In the second group, the amputated-fin was irradiated with ultraviolet. Finally, in the third group, the amputated-fin was irradiated with laser. Caudal-fin regeneration and vascularization were assessed at days 0, 3, 6, 9, 12, and 15 in all fish. In terms of regeneration, the results indicated that it is possible to discriminate the regenerative effect of laser with the experimental setup as laser therapy showed a statistically significant difference when compared to control-group. It was also found that regenerative stimulation of the group that received ultraviolet therapy showed significant difference when compared to the control group. In terms of vascularization, there was a statistically significant difference in all groups of the study, which may suggest that laser as well as ultraviolet have limited effects in terms of improving vascularization. This study presented a novel, simple and inexpensive method for the assessment of PBM effects on zebrafish. Laser and ultraviolet therapy appeared to act as regenerative stimulators for caudal-fin regeneration of zebrafish. However, laser therapy results were, to some extent, better than ultraviolet therapy. This novel in-vivo design of the experiment led to more rapid and reproducible results than in-vitro experiments.

Poly(norepinephrine)-Mediated Universal Surface Modification for Patterning Human Pluripotent Stem Cell Culture and Differentiation.

Maintaining undifferentiated states of human pluripotent stem cells (hPSCs) is key to accomplishing successful hPSC research. Specific culture conditions, including hPSC-compatible substrates, are required for the hPSC culture. Over the past two decades, substrates supporting hPSC self-renewal have evolved from undefined and xenogeneic protein components to chemically defined and xenogeneic-free materials. However, these synthetic substrates are often costly and complex to use, leading many laboratories to continue using simpler undefined extracellular matrix (ECM) protein mixtures. In this study, we present a method using poly(norepinephrine) (pNE) for surface modification to enhance the immobilization of ECM proteins on various substrates, including polydimethylsiloxane (PDMS) and ultralow attachment (ULA) hydrogels, thereby supporting hPSC culture and maintenance of pluripotency. The pNE-mediated surface modification enables spatial patterning of ECM proteins on nonadhesive ULA surfaces, facilitating tunable macroscopic cell patterning. This approach improves hPSC attachment and growth and allows for cell patterning to study the effects of anisotropic environments on the hPSC fate. Our findings demonstrate the versatility and simplicity of pNE-mediated surface modification for improving hPSC culture and spatially controlled differentiation into endothelial cells and cardiomyocytes on previously nonamenable substrates, providing a valuable tool for tissue engineering and regenerative medicine applications.

Controlling spheroid attachment improves pancreatic beta cell differentiation from human iPS cells.

Regenerative medicine using human induced pluripotent stem cells (hiPSCs) is available for treating type 1 diabetes; however, the efficiency and maturation of hiPSC differentiation into pancreatic beta cells requires improvement. Various protocols, including three-dimensional (3D) culture, have been developed to improve differentiation efficiency and maturation. Several methods for 3D culture have been reported; however, they require costly and complicated equipment, special materials, and complicated operations. To solve these problems, we developed a simple 3D culture method under static conditions using a cyclo-olefin polymer (COP) characterized by high moisture barrier properties, low surface energy, and hydrophobicity. Using this 3D method and our simple and low-cost protocol, we found that differentiation into the definitive endoderm (DE) was better when the spheroids were attached. Therefore, upon the addition of Y-27632, attached spheroids with unique shapes and cavities were formed, and the differentiation efficiency into DE increased. During DE differentiation, the attachment of spheroids to the substrate and their subsequent floating improved differentiation efficiency. We found that the amount of C-peptide in spheroids differentiated using COP dishes was greater than that in rotary culture. Furthermore, INSULIN was highly expressed in areas with low cell density, suggesting that the unique shape of the spheroids made from COP dishes improved differentiation efficiency. Our study suggests that a device-free, simple 3D culture method that controls spheroid attachment improves the efficiency of hiPSC differentiation into pancreatic beta cells.

Automated Manufacturing Processes and Platforms for Large-scale Production of Clinical-grade Mesenchymal Stem/ Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) hold immense potential for regenerative medicine due to their remarkable regenerative and immunomodulatory properties. However, their therapeutic application requires large-scale production under stringent regulatory standards and Good Manufacturing Practice (GMP) guidelines, presenting significant challenges. This review comprehensively evaluates automated manufacturing processes and platforms for the scalable production of clinical-grade MSCs. Various large-scale culture vessels, including multilayer flasks and bioreactors, are analyzed for their efficacy in MSCs expansion. Furthermore, automated MSCs production platforms, such as Quantum® Cell Expansion System, CliniMACS Prodigy®, NANT001/ XL, CellQualia™, Cocoon® Platform, and Xuri™ Cell Expansion System W25 are reviewed and compared as well. We also underscore the importance of optimizing culture media specifically emphasizing the shift from fetal bovine serum to humanized or serum-free alternatives to meet GMP standards. Moreover, advances in alternative cryopreservation methods and controlled-rate freezing systems, that offer promising improvements in MSCs preservation, are discussed as well. In conclusion, advancing automated manufacturing processes and platforms is essential for realizing the full potential of MSCs-based regenerative medicine and accomplishing the increasing demand for cell-based therapies. Collaborative initiatives involving industry, academia, and regulatory bodies are emphasized to accelerate the translation of MSCs-based therapies into clinical practice.

Biocompatible nanocomposite hydroxyapatite-based granules with increased specific surface area and bioresorbability for bone regenerative medicine applications.

Hydroxyapatite (HA) granules are frequently used in orthopedics and maxillofacial surgeries to fill bone defects and stimulate the regeneration process. Optimal HA granules should have high biocompatibility, high microporosity and/or mesoporosity, and high specific surface area (SSA), which are essential for their bioabsorbability, high bioactivity (ability to form apatite layer on their surfaces) and good osseointegration with the host tissue. Commercially available HA granules that are sintered at high temperatures (≥ 900°C) are biocompatible but show low porosity and SSA (2-5 m2/g), reduced bioactivity, poor solubility and thereby, low bioabsorbability. HA granules of high microporosity and SSA can be produced by applying low sintering temperatures (below 900°C). Nevertheless, although HA sintered at low temperatures shows significantly higher SSA (10-60 m2/g) and improved bioabsorbability, it also exhibits high ion reactivity and cytotoxicity under in vitro conditions. The latter is due to the presence of reaction by-products. Thus, the aim of this study was to fabricate novel biomaterials in the form of granules, composed of hydroxyapatite nanopowder sintered at a high temperature (1100°C) and a biopolymer matrix: chitosan/agarose or chitosan/β-1,3-glucan (curdlan). It was hypothesized that appropriately selected ingredients would ensure high biocompatibility and microstructural properties comparable to HA sintered at low temperatures. Synthesized granules were subjected to the evaluation of their biological, microstructural, physicochemical, and mechanical properties. The obtained results showed that the developed nanocomposite granules were characterized by a lack of cytotoxicity towards both mouse preosteoblasts and normal human fetal osteoblasts, and supported cell adhesion to their surface. Moreover, produced biomaterials had the ability to induce precipitation of apatite crystals after immersion in simulated body fluid, which, combined with high biocompatibility, should ensure good osseointegration after implantation. Additionally, nanocomposite granules possessed microstructural parameters similar to HA sintered at a low temperature (porosity approx. 50%, SSA approx. 30m²/g), Young's modulus (5-8 GPa) comparable to cancellous bone, and high fluid absorption capacity. Moreover, the nanocomposites were prone to biodegradation under the influence of enzymatic solution and in an acidic environment. Additionally, it was noted that the hydroxyapatite nanoparticles remaining after the physicochemical dissolution of the biomaterial were easily phagocytosed by mouse macrophages, mouse preosteoblasts, and normal human fetal osteoblasts (in vitro studies). The obtained materials show great potential as bone tissue implantation biomaterials with improved bioresorbability. The obtained materials show great potential as bone tissue implantation biomaterials with improved bioresorbability.

Proteomic analysis of human Wharton's jelly mesenchymal stem/stromal cells and human amniotic epithelial stem cells: a comparison of therapeutic potential.

Perinatal stem cells have prominent applications in cell therapy and regenerative medicine. Among them, human Wharton's jelly mesenchymal stem/stromal cells (hWJMSCs) and human amniotic epithelial stem cells (hAESCs) have been widely used. However, the distinction in the therapeutic potential of hWJMSCs and hAESCs is poorly understood. In this study, we reported the phenotypic differences between these two distinct cell types and provided the first systematic comparison of their therapeutic potential in terms of immunomodulation, extracellular matrix (ECM) remodelling, angiogenesis and antioxidative stress using proteomics. The results revealed that the two cell types presented different protein expression profiles and were both promising candidates for cell therapy. Both types of cells demonstrated angiogenic and antifibrotic potential, whereas hAESCs presented superior immunological tolerance and antioxidant properties, which were supported by a series of relevant in vitro assays. Our study provides clues for the selection of appropriate cell types for diverse indications in cell therapy, which contributes to the advancement of their clinical translation and application.

Bioactive Three-Dimensional Chitosan-Based Scaffolds Modified with Poly(dopamine)/CBD@Pt/Au/PVP Nanoparticles as Potential NGCs Applicable in Nervous Tissue Regeneration—Preparation and Characterization

Tissue engineering of nervous tissue is a promising direction in the treatment of neurological diseases such as spinal cord injuries or neuropathies. Thanks to technological progress and scientific achievements; the use of cells; artificial scaffolds; and growth factors are becoming increasingly common. Despite challenges such as the complex structure of this tissue, regenerative medicine appears as a promising future approach to improve the quality of life of patients with nervous injuries. Until now; most functional biomaterials used for this purpose were based on decellularized extra cellular matrix (ECM) or nanofibrous materials, whereas current clinically verified ones in most cases do not exhibit bioactivity or the possibility for external stimulation. The aim of this research was to develop a new type of bioactive, chitosan-based 3D materials applicable as nerve guide conduits (NGCs) modified with poly(dopamine), Au/Pt coated with PVP nanoparticles, and cannabidiol. The NGCs were prepared under microwave-assisted conditions and their chemical structure was studied using the FT-IR method. Next, this study will discuss novel biomaterials for morphology and swelling abilities as well as susceptibility to biodegradation in the presence of collagenase and lysozyme. Finally, their potential in the field of nervous tissue engineering has been verified via a cytotoxicity study using the 1321N1 human astrocytoma cell line, which confirmed their biocompatibility in direct contact studies.

Turn Hood into Good: Recycling Silicon from Mesoporous Silica Nanoparticles through Magnesium Modification to Lower Toxicity and Promote Tissue Regeneration.

Mesoporous silica nanoparticles (MSNs) have gained wide application as excellent carrier materials; however, their limited degradation in the biological system and potential chronic toxicity pose challenges to their clinical applications. Previous studies have focused on optimizing the elimination performance of MSNs; interestingly, silicon has been well-documented as an essential body component. Therefore, converting MSNs into a form readily utilizable by the organism is a way to turn waste into a valuable resource. However, the recycling and utilization of MSNs are associated with significant hurdles. This study proposes an approach to impede the formation of siloxane, the crucial core in MSNs, by introducing a gradient concentration of Mg2+. The invasion of Mg2+ significantly reduces the stability of Si-O-Si bonds by substituting silicon ions while preserving the functional three-dimensional structure. Recycling the increased release of Mg and Si ions enhances cellular antioxidant capacity, reduces oxidative stress reactions, improves mitochondrial function, and regulates macrophage inflammatory states. The proposed approach to converting MSN materials shows significant advantages for tissue regeneration in the periodontal defect model. This study opens an insight for applying MSNs in clinical applications in regenerative medicine.

Proteomic and functional comparison between human induced and embryonic stem cells.

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.

Innovative Strategies in Regenerative Medicine: Bridging Science and Clinical Practice

Regenerative medicine is a rapidly advancing field to revolutionize healthcare by offering innovative solutions for repairing or replacing damaged tissues and organs. By addressing significant challenges associated with conventional therapies—such as the shortage of donor organs and complications related to immune rejection—regenerative medicine provides a hopeful alternative for patients suffering from chronic diseases and injuries. This review outlines the urgent need for regenerative medicine to tackle prevalent issues like chronic conditions, organ scarcity, and injury recovery through approaches like stem cell therapy and tissue engineering. Key therapies currently available in the market, such as Carticel and Celution, utilize both autologous and allogeneic cells to promote healing and tissue regeneration. Recent breakthroughs showcase the transformative potential of regenerative medicine, with notable successes including stem cell therapies for spinal cord injuries, 3D-printed skin grafts for burn victims, and the development of lab-grown organs. These advancements highlight regenerative medicine's capability to enhance patient outcomes significantly. Looking ahead, the future of regenerative medicine lies in the personalization of therapies, advanced biomaterials, and cutting-edge technologies like 3D bioprinting. These innovations will enable the creation of complex and functional tissues tailored to individual patients. As research continues to progress, regenerative medicine holds the promise of offering long-term, transformative solutions for a wide range of medical conditions..

Immunomodulatory role of Xenopus tropicalis immature Sertoli cells in tadpole muscle regeneration via macrophage response modulation.

Regenerative medicine and transplantation science continuously seek methods to circumvent immune-mediated rejection and promote tissue regeneration. Sertoli cells, with their inherent immunoprotective properties, emerge as pivotal players in this quest. However, whether Sertoli cells can play immunomodulatory role in tadpole tail regeneration and can thus benefit the regeneration process are needed to be discovered. Immature Sertoli cells from Xenopus tropicalis (XtiSCs) were transplanted into X. tropicalis tadpoles, followed by the amputation of the final third of their tails. We assessed the migration of XtiSCs, tail regeneration length, muscle degradation and growth, and macrophage counts across various regions including the entire tail, tail trunk, injection site, and regeneration site. The interactions between XtiSCs and macrophages were examined using a confocal microscope. To deplete macrophages, clodronate liposomes were administered prior to the transplantation of XtiSCs, while the administration of control liposomes acted as a negative control. Student's t-test was used to compare the effects of XtiSCs injection to those of a 2/3PBS injection across groups with no liposomes, control liposomes, and clodronate liposomes. XtiSCs have excellent viability after transplantation to tadpole tail and remarkable homing capabilities to the regeneration site after tail amputation. XtiSCs injection increased macrophage numbers at 3 days post-amputation and 5 days post-amputation in the tail trunk, specifically at the injection site and at the regenerated tail, in a macrophage depleted environment (clodronate-liposome injection). What's more, XtiSCs injection decreased muscle fibers degradation significantly at 1day post-amputation and facilitated new muscle growth significantly at 3 days post-amputation. In addition, whole-mount immunostaining showed that some XtiSCs co-localized with macrophages. And we observed potential mitochondria transport from XtiSCs to macrophages using MitoTracker staining in tadpole tail. Our study delineates the novel role of XtiSCs in facilitating muscle regeneration post tadpole tail amputation, underscoring a unique interaction with macrophages that is crucial for regenerative success. This study not only highlights the therapeutic potential of Sertoli cells in regenerative medicine but also opens avenues for clinical translation, offering insights into immunoregulatory strategies that could enhance tissue regeneration and transplant acceptance.

Divergent Proteomic Profiles and Uptake Mechanisms of Exosomes Derived from Human Dental Pulp Stem Cells, Endothelial Cells, and Fibroblasts.

Effective intercellular communication is crucial for tissue repair and regeneration, with exosomes playing a key role in mediating these processes by transferring proteins, lipids, and nucleic acids between cells. This study explored the mechanisms underlying the uptake of exosomes derived from human dental pulp stem cells (hDPSCs), human umbilical vein endothelial cells (HUVECs), and human fibroblasts (HFBs). Our findings revealed that hDPSCs exhibited the greatest capacity for exosome uptake across all three cell types. Moreover, exosomes originating from hDPSCs were also taken up in the highest amounts by all three cell types. Proteomic analysis uncovered significant differences in protein expression among exosomes from these different cell types, particularly in proteins related to endocytosis. Clathrin-dependent endocytosis emerged as the primary pathway for exosome uptake in hDPSCs and HUVECs, while HFBs appeared to use a different mechanism. Additionally, proteins such as fibronectin and tetraspanins were found to be highly expressed in hDPSC-derived exosomes, suggesting their potential involvement in exosome-cell interactions. This study offers new insights into exosome uptake mechanisms and highlights the potential of exosomes in advancing tissue engineering and regenerative medicine.

Stem Cells: Innovations, Applications, and Future Directions

ABSTRACT Stem cells offer great promise in dentistry by regenerating critical oral tissues. Derived from sources such as dental pulp, periodontal ligament, and dental follicle, these cells can be pluripotent or multipotent and are capable of differentiating into various cell types, including odontoblasts and osteoblasts. This regenerative potential could transform treatments for dental conditions like periodontal disease and pulpitis. Advancing this field requires understanding stem cell differentiation and exploring tissue engineering and biomaterial scaffolds. Despite the potential, evolving legal and ethical issues shape the integration of these innovations. Research in dental stem cells represents a significant advance in regenerative medicine, promising to enhance dental treatments and patient care.

Activated Growth Factor (AGF): An Advanced Modality of Platelet-Rich Plasma as a New Biological Agent for the Treatment of Degenerative and Traumatic Conditions

Platelet-rich plasma (PRP) has emerged as a promising therapeutic modality for various medical applications, particularly in regenerative medicine and wound healing. This is largely attributed to its rich concentration of growth factors (GFs) that play pivotal roles in tissue repair and regeneration. However, the inherent limitations of PRP, such as the variable GF concentrations and short-lived release kinetics, have spurred the development of advanced modalities to enhance its therapeutic efficacy. Activated growth factor (AGF) represents one such advancement, aiming to optimize the release and bioavailability of GFs from platelets. This comprehensive review delves into the biological mechanisms underlying AGF, its preparation methodologies, preclinical and clinical evidence supporting its use, and its potential applications in treating degenerative and traumatic conditions. Furthermore, it explores the advantages of AGF over conventional PRP and discusses future directions for research and clinical translation.

Apoptosis-induced exosomes from human exfoliated deciduous teeth enhance angiogenesis in human umbilical vein endothelial cells.

Exosomes derived from the stem cells of human exfoliated deciduous teeth (SHED) hold promise for tissue regeneration. Apoptotic cells release a variety of extracellular vesicles that affect intercellular communication. This study aimed to investigate the angiogenic effects of SHED-derived exosomes modified via apoptosis induction on human umbilical vein endothelial cells (HUVECs). Apoptosis was induced in SHED via serum starvation for 3 weeks and confirmed by the upregulation of the apoptotic genes, caspase 3 and 9, and via annexin V staining. The apoptotic SHED-derived exosomes were isolated, characterized, and subjected to proteomic analysis. In vitro experiments were performed to assess the effects of apoptotic SHED exosomes on the proliferation, migration, and tube formation of HUVECs. The apoptosis-induced SHED showed increased cell viability and decreased numbers of dead cells compared with those of conventional cultures while retaining their identity as mesenchymal stem cells positive for CD44, CD73, and CD90. The apoptotic SHED-derived exosomes exhibited characteristic features, such as standard size, cup-shaped morphology, and positive staining, for exosomal markers CD9, CD63, and CD81. Proteins associated with apoptosis, programmed cell death, and cellular senescence were downregulated in the apoptotic SHED exosomes, whereas those associated with extracellular matrix organization were upregulated, indicating positive angiogenesis. HUVECs treated with apoptotic SHED exosomes exhibited significantly enhanced proliferation and migration compared with those treated with normal SHED exosomes. The mesh-like structures in the apoptotic SHED exosomes exhibited significantly increased signs of angiogenesis. The findings of this study provide new insights into the potential use of apoptotic SHED-derived exosomes in regenerative medicine.

Blastoid: The future of human development in the laboratory.

Research on early human development is crucial for understanding the origins of life and mechanisms underlying disease onset. However, these studies have significant challenges owing to ethical restrictions and technical limitations. Stem cell technology advancement has led to the development of blastoids to overcome these challenges." Blastoids are three-dimensional structures produced by pluripotent stem cells (PSCs) that resemble the blastocyst stage of human embryos. Research on blastoids can enhance our understanding of early human development and drive innovations in regenerative medicine and disease modeling. This review outlines the background of blastoid development and highlights the limitations of existing organoid research. It presents developments in blastoid research, from previous studies using animal models to the latest developments using human stem cell-derived blastoids in early human development studies. Additionally, this review provides a comparative analysis of the methods used to develop blastoids across various studies, evaluating their potential as ethical alternatives for regenerative medicine, human developmental biology, and embryonic research. It further assesses the ethical and social considerations surrounding blastoid research, the current strategies to address these concerns, and the potential long-term impact on science and medicine. We aimed to provide a comprehensive understanding of the current trends in blastoid research, offer new insights into early human development, and suggest novel directions and approaches for researchers.

Translational implications of a novel combination of iPRF and collagen scaffold for proliferation of gingival mesenchymal stem cells.

"Tissue engineering," is a concept that involves the use of scaffolds, cells, and growth factors/signaling molecules in the field of regenerative medicine. The present study was carried out to evaluate the attachment and depth of penetration of the Gingival Mesenchymal Stem Cells (GMSCs) onto the collagen scaffold preconditioned with Fetal Bovine Serum (FBS), Injectable Platelet Rich Fibrin (i-PRF) and a combination of FBS with i-PRF using histological analysis and environmental scanning electron microscopy (ESEM) respectively. In the present study, commercially available collagen membranes were used as scaffolds and divided into 3 groups where Group I was preconditioned with FBS, Group II was preconditioned with i-PRF, and Group III with a combination of FBS and i-PRF. Scaffolds were then seeded with GMSCs and incubated in a CO2 incubator at 37 0C for 7 days. Both histological and ESEM analysis showed proliferation, attachment, and depth of penetration along with the combination of cell morphological changes in all the three groups. In group I cells showed mild depth of penetration along with a round morphological appearance. In group II the cells showed moderate depth of penetration and rounded morphology. In group III maximum depth of penetration was seen along with the round and spheroidal morphology of cells. The combination of the growth media showed the best effect on cell attachment, depth of penetration, and cell morphology. This is the first report demonstrating the effect of the combination of collagen and i-PRF supports the proliferation of GMSCs. Since FBS alone and i-PRF alone exhibited similar cell proliferation it is recommended that i-PRF could be used in clinical scenarios making it xenofree.

Improvement of osteogenic differentiation potential of placenta-derived mesenchymal stem cells by metformin via AMPK pathway activation.

Placenta-derived human mesenchymal stem cells (PL-MSCs) have gained a lot of attention in the field of regenerative medicine due to their availability and bone-forming capacity. However, the osteogenic differentiation capacity of these cells remains inconsistent and could be improved to achieve greater efficiency. Although metformin, a widely used oral hypoglycemic agent, has been shown to increase bone formation in various cell types, its effect on osteogenic differentiation of PL-MSCs has not yet been investigated. Therefore, the objective of this study was to examine the effect of metformin on the osteogenic differentiation capacity of PL-MSCs and the underlying mechanisms. The PL-MSCs were treated with 0.5 to 640µM metformin and their osteogenic differentiation capacity was examined by an alkaline phosphatase (ALP) activity assay, Alizarin red S staining and expression levels of osteogenic genes. The role of adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling in mediating the effect of metformin on the osteogenic differentiation capacity of PL-MSCs was also investigated by determining levels of phosphorylated AMPK(pAMPK)/AMPK ratio and by using compound C, an AMPK inhibitor. The results showed that 10-160µM metformin significantly increased the viability of PL-MSCs in a dose- and time-dependent manner. Furthermore, 80-320µM metformin also increased ALP activity, matrix mineralization, and expression levels of osteogenic genes, runt-related transcription factor 2 (RUNX2), osterix (OSX), osteocalcin (OCN) and collagen I (COL1), in PL-MSCs. Metformin increases osteogenic differentiation of PL-MSCs, at least in part, through the AMPK signaling pathway, since the administration of compound C inhibited its enhancing effects on ALP activity, matrix mineralization, and osteogenic gene expression of PL-MSCs. This study demonstrated that metformin at concentrations of 80-320μM significantly enhanced osteogenic differentiation of PL-MSCs in a dose- and time-dependent manner, primarily through activation of the AMPK signaling pathway. This finding suggests that metformin could be used with other conventional drugs to induce bone regeneration in various bone diseases. Additionally, this study provides valuable insights for future osteoporosis treatment by highlighting the potential of modulating the AMPK pathway to improve bone regeneration.