The re-proliferation of quiescent cancer cells is considered to be the primary contributor to prostate cancer (Pca) recurrence and progression. In this study, we investigated the inhibitory effect of safranal, a monoterpene aldehyde isolated from Crocus sativus (saffron), on the re-proliferation of quiescent Pca cells in vitro and in vivo. The results showed that safranal efficiently blocked the re-activation of quiescent Pca cells by downregulating the G0/G1 cell cycle regulatory proteins CDK2, CDK4, CDK6, and phospho-Rb at Ser807/811 and elevating the levels of cyclin-dependent kinase inhibitors, p21 and p27. Further investigation on the underlying mechanisms revealed that safranal suppressed the mRNA and protein expression levels of Skp2, possibly through the deregulation of the transcriptional activity of two major transcriptional factors, E2F1 and NF-κB subunits. Moreover, safranal inhibited AKT phosphorylation at Ser473 and deregulated both canonical and non-canonical NF-κB signaling pathways. Safranal suppressed the tumor growth of quiescent Pca cell xenografts in vivo. Furthermore, safranal-treated tumor tissues exhibited a reduction in Skp2, E2F1, NF-κB p65, p-IκBα (Ser32), c-MYC, p-Rb (Ser807), CDK4, CDK6, and CDK2 and an elevation of p27 and p21 protein levels. Therefore, our findings demonstrate that safranal suppresses cell cycle re-entry of quiescent Pca cells in vitro and in vivo plausibly by repressing the transcriptional activity of two major transcriptional activators of Skp2, namely, E2F1 and NF-κB, through the downregulation of AKT phosphorylation and NF-κB signaling pathways, respectively.
Non-viral gene delivery holds promises for treating inherited diseases. However, the limited cloning capacity of plasmids may hinder the co-delivery of distinct genes to the transfected cells. Previously, the conjugation of maleimide-functionalized polyurethane grafted with small molecular weight polyethylenimine (PU-PEI600-Mal) using 1,6-hexanedithiol (HDT) could promote the co-delivery and extensive co-expression of two different plasmids in target cells. Herein, we designed HDT-conjugated PU-PEI600-Mal for the simultaneous delivery of CRISPR/Cas9 components to achieve efficient gene correction in the induced pluripotent stem cell (iPSC)-derived model of Fabry cardiomyopathy (FC) harboring GLA IVS4 + 919 G > A mutation. This FC in vitro model recapitulated several clinical FC features, including cardiomyocyte hypertrophy and lysosomal globotriaosylceramide (Gb3) deposition. As evidenced by the expression of two reporter genes, GFP and mCherry, the addition of HDT conjugated two distinct PU-PEI600-Mal/DNA complexes and promoted the co-delivery of sgRNA/Cas9 and homology-directed repair DNA template into target cells to achieve an effective gene correction of IVS4 + 919 G > A mutation. PU-PEI600-Mal/DNA with or without HDT-mediated conjugation consistently showed neither the cytotoxicity nor an adverse effect on cardiac induction of transfected FC-iPSCs. After the gene correction and cardiac induction, disease features, including cardiomyocyte hypertrophy, the mis-regulated gene expressions, and Gb3 deposition, were remarkably rescued in the FC-iPSC-differentiated cardiomyocytes. Collectively, HDT-conjugated PU-PEI600-Mal-mediated dual DNA transfection system can be an ideal approach to improve the concurrent transfection of non-viral-based gene editing system in inherited diseases with specific mutations.
Endothelial cells lining the inner vascular wall form a monolayer that contributes to the selective permeability of endothelial barrier. This selective permeability is mainly regulated by an endothelium-specific adherens junctional protein, known as vascular endothelial-cadherin (VE-cadherin). In endothelial cells, the adherens junction comprises of VE-cadherin and its associated adhesion molecules such as p120, α-catenin, and β-catenin, in which α-catenin links cytoplasmic tails of VE-cadherin to actin cytoskeleton through β-catenin. Proinflammatory stimuli such as lipopolysaccharide (LPS) are capable of attenuating vascular integrity through the disruption of VE-cadherin adhesion in endothelial cells. To date, numerous studies demonstrated the disruption of adherens junction as a result of phosphorylation-mediated VE-cadherin disruption. However, the outcomes from these studies were inconsistent and non-conclusive as different cell fractions were used to examine the effect of LPS on the disruption of VE-cadherin. By using Western Blot, some studies utilized total protein lysate and reported decreased protein expression while some studies reported unchanged expression. Other studies which used membrane and cytosolic fractions of protein extract demonstrated decreased and increased VE-cadherin expression, respectively. Despite the irregularities, the results of immunofluorescence staining are consistent with the formation of intercellular gap. Besides that, the overall underlying disruptive mechanisms of VE-cadherin remain largely unknown. Therefore, this mini review will focus on different experiment approaches in terms of cell fractions used in different human endothelial cell studies, and relate these differences to the results obtained in Western blot and immunofluorescence staining in order to give some insights into the overall differential regulatory mechanisms of LPS-mediated VE-cadherin disruption and address the discrepancy in VE-cadherin expression.
Bone fractures have a high degree of severity. This is usually a result of the physical trauma of diseases that affect bone tissues, such as osteoporosis. Due to its highly vascular nature, the bone is in a constant state of remodeling. Although those of younger ages possess bones with high regenerative potential, the impact of a disrupted vasculature can severely affect the recovery process and cause osteonecrosis. This is commonly seen in the neck of femur, scaphoid, and talus bone. In recent years, mesenchymal stem cell (MSC) therapy has been used to aid in the regeneration of afflicted bone. However, the cut-off in blood supply due to bone fractures can lead to hypoxia-induced changes in engrafted MSCs. Researchers have designed several oxygen-generating biomaterials and yielded varying degrees of success in enhancing tissue salvage and preserving cellular metabolism under ischemia. These can be utilized to further improve stem cell therapy for bone repair. In this review, we touch on the pathophysiology of these bone fractures and review the application of oxygen-generating biomaterials to further enhance MSC-mediated repair of fractures in the three aforementioned parts of the bone.
ATP-induced cell death has emerged as a captivating realm of inquiry with profound ramifications in the context of osteoporosis. This study unveils a paradigm-shifting hypothesis that illuminates the prospective involvement of ATP-induced cellular demise in the etiology of osteoporosis. Initially, we explicate the morphological attributes of ATP-induced cell death and delve into the intricacies of the molecular machinery and regulatory networks governing ATP homeostasis and ATP-induced cell death. Subsequently, our focus pivots towards the multifaceted interplay between ATP-induced cellular demise and pivotal cellular protagonists, such as bone marrow-derived mesenchymal stem cells, osteoblasts, and osteoclasts, accentuating their potential contributions to secondary osteoporosis phenotypes, encompassing diabetic osteoporosis, glucocorticoid-induced osteoporosis, and postmenopausal osteoporosis. Furthermore, we probe the captivating interplay between ATP-induced cellular demise and alternative modalities of cellular demise, encompassing apoptosis, autophagy, and necroptosis. Through an all-encompassing inquiry into the intricate nexus connecting ATP-induced cellular demise and osteoporosis, our primary goal is to deepen our comprehension of the underlying mechanisms propelling this malady and establish a theoretical bedrock to underpin the development of pioneering therapeutic strategies.
Chronic inflammation is a common underlying factor in osteoarthritis (OA) and most age-related degenerative diseases. As conventional therapies help only in partial alleviation of symptoms in OA, stem cell-based therapies and herbal supplements are being widely explored. Thymoquinone (TQ), an active ingredient of Nigella sativa is reported to have immunomodulatory, anti-inflammatory and antioxidant properties. We evaluated the effects of TQ on bone marrow MSCs (BM-MSCs) derived from OA patients and its interrelated pathways in inflammation and age-related degenerative diseases using Ingenuity Pathway Analysis (IPA) as well as possible molecular targets using SwissTargetPrediction. BM-MSCs were derived from OA patients and their stemness properties were characterized by studying the MSCs related CD surface marker expression and differentiation into adipocytes, osteoblasts, and chondrocytes. Treatment with TQ (100 nM-5 μM) demonstrated cell death, especially at higher concentrations. MTT assay demonstrated a significant concentration-dependent decrease in cell viability which ranged from 20.04% to 69.76% with higher doses (300 nM, 1 μM, and 5 μM), especially at 48h and 72h. Additional cell viability testing with CellTiter-Blue also demonstrated a significant concentration-dependent decrease in cell viability which ranged from 27.80 to 73.67% with higher doses (300 nM, 1 μM, 3 μM, and 5 μM). Gene expression analysis following treatment of BM-MSCs with TQ (1 and 3 μM) for 48h showed upregulation of the anti-inflammatory genes IL-4 and IL-10. In contrast, the pro-inflammatory genes namely IFN-γ, TNF-α, COX-2, IL-6, IL-8, IL-16, and IL-12A although were upregulated, compared to the lower concentration of TQ (1 μM) they were all decreased at 3 μM. The pro-apoptotic BAX gene was downregulated while the SURVIVIN gene was upregulated. IPA of the molecular interaction of TQ in inflammation and age-related degenerative diseases identified canonical pathways directly related to synaptogenesis, neuroinflammation, TGF-β, and interleukin signaling. Further screening led to the identification of 36 molecules that are involved in apoptosis, cell cycle regulation, cytokines, chemokines, and growth factors. SwissTargetPrediction of TQ identified potential molecular targets with high probability. TQ exerted anti-inflammatory effects and therefore can be a useful adjuvant along with conventional therapies against inflammation in OA and other age-related degenerative diseases.
Osteoarthritis (OA) is a chronic degenerative joint disorder associated with degradation and decreased production of the extracellular matrix, eventually leading to cartilage destruction. Limited chondrocyte turnover, structural damage, and prevailing inflammatory milieu prevent efficient cartilage repair and restoration of joint function. In the present study, we evaluated the role of secreted cytokines, chemokines, and growth factors present in the culture supernatant obtained from an ex vivo osteochondral model of cartilage differentiation using cartilage pellets (CP), bone marrow stem cells (BM-MSCs), and/or BM-MSCs + CP. Multiplex cytokine analysis showed differential secretion of growth factors (G-CSF, GM-CSF, HGF, EGF, VEGF); chemokines (MCP-1, MIP1α, MIP1β, RANTES, Eotaxin, IP-10), pro-inflammatory cytokines (IL-1β, IL-2, IL-5, IL-6, IL-8, TNFα, IL-12, IL-15, IL-17) and anti-inflammatory cytokines (IL-4, IL-10, and IL-13) in the experimental groups compared to the control. In silico analyses of the role of stem cells and CP in relation to the expression of various molecules, canonical pathways and hierarchical cluster patterns were deduced using the Ingenuity Pathway Analysis (IPA) software (Qiagen, United States). The interactions of the cytokines, chemokines, and growth factors that are involved in the cartilage differentiation showed that stem cells, when used together with CP, bring about a favorable cell signaling that supports cartilage differentiation and additionally helps to attenuate inflammatory cytokines and further downstream disease-associated pro-inflammatory pathways. Hence, the autologous or allogeneic stem cells and local cartilage tissues may be used for efficient cartilage differentiation and the management of OA.
The gut microbiome is implicated in the pathogenesis of polycystic ovary syndrome (PCOS), and prenatal androgen exposure is involved in the development of PCOS in later life. Our previous study of a mouse model of PCOS induced by prenatal dihydrotestosterone (DHT) exposure showed that the reproductive phenotype of PCOS appears from puberty, followed by the appearance of the metabolic phenotype after young adulthood, while changes in the gut microbiota was already apparent before puberty. To determine whether the prenatal or postnatal nurturing environment primarily contributes to these changes that characterize prenatally androgenized (PNA) offspring, we used a cross-fostering model to evaluate the effects of changes in the postnatal early-life environment of PNA offspring on the development of PCOS-like phenotypes and alterations in the gut microbiota in later life. Female PNA offspring fostered by normal dams (exposed to an abnormal prenatal environment only, fostered PNA) exhibited less marked PCOS-like phenotypes than PNA offspring, especially with respect to the metabolic phenotype. The gut microbiota of the fostered PNA offspring was similar to that of controls before adolescence, but differences between the fostered PNA and control groups became apparent after young adulthood. In conclusion, both prenatal androgen exposure and the postnatal early-life environment created by the DHT injection of mothers contribute to the development of PCOS-like phenotypes and the alterations in the gut microbiota that characterize PNA offspring. Thus, both the pre- and postnatal environments represent targets for the prevention of PCOS and the associated alteration in the gut microbiota in later life.
Aging is a complex biological process that occurs in all living organisms. Aging is initiated by the gradual accumulation of biomolecular damage in cells leading to the loss of cellular function and ultimately death. Cellular senescence is one such pathway that leads to aging. The accumulation of nucleic acid damage and genetic alterations that activate permanent cell-cycle arrest triggers the process of senescence. Cellular senescence can result from telomere erosion and ribosomal DNA instability. In this review, we summarize the molecular mechanisms of telomere length homeostasis and ribosomal DNA stability, and describe how these mechanisms are linked to cellular senescence and longevity through lessons learned from budding yeast.
Over the past 2 decades, mesenchymal stem cells (MSCs) have attracted a lot of interest as a unique therapeutic approach for a variety of diseases. MSCs are capable of self-renewal and multilineage differentiation capacity, immunomodulatory, and anti-inflammatory properties allowing it to play a role in regenerative medicine. Furthermore, MSCs are low in tumorigenicity and immune privileged, which permits the use of allogeneic MSCs for therapies that eliminate the need to collect MSCs directly from patients. Induced pluripotent stem cells (iPSCs) can be generated from adult cells through gene reprogramming with ectopic expression of specific pluripotency factors. Advancement in iPS technology avoids the destruction of embryos to make pluripotent cells, making it free of ethical concerns. iPSCs can self-renew and develop into a plethora of specialized cells making it a useful resource for regenerative medicine as they may be created from any human source. MSCs have also been used to treat individuals infected with the SARS-CoV-2 virus. MSCs have undergone more clinical trials than iPSCs due to high tumorigenicity, which can trigger oncogenic transformation. In this review, we discussed the overview of mesenchymal stem cells and induced pluripotent stem cells. We briefly present therapeutic approaches and COVID-19-related diseases using MSCs and iPSCs.
Mesenchymal stem cells (MSC) are highly regarded as a potential treatment for retinal degenerative disorders like retinitis pigmentosa and age-related macular degeneration. However, donor cell heterogeneity and inconsistent protocols for transplantation have led to varied outcomes in clinical trials. We previously showed that genetically-modifying MSCs to express erythropoietin (MSCEPO) improved its regenerative capabilities in vitro. Hence, in this study, we sought to prove its potential in vivo by transplanting MSCsEPO in a rat retinal degeneration model and analyzing its retinal transcriptome using RNA-Seq. Firstly, MSCsEPO were cultured and expanded before being intravitreally transplanted into the sodium iodate-induced model. After the procedure, electroretinography (ERG) was performed bi-weekly for 30 days. Histological analyses were performed after the ERG assessment. The retina was then harvested for RNA extraction. After mRNA-enrichment and library preparation, paired-end RNA-Seq was performed. Salmon and DESeq2 were used to process the output files. The generated dataset was then analyzed using over-representation (ORA), functional enrichment (GSEA), and pathway topology analysis tools (SPIA) to identify enrichment of key pathways in the experimental groups. The results showed that the MSCEPO-treated group had detectable ERG waves (P <0.05), which were indicative of successful phototransduction. The stem cells were also successfully detected by immunohistochemistry 30 days after intravitreal transplantation. An initial over-representation analysis revealed a snapshot of immune-related pathways in all the groups but was mainly overexpressed in the MSC group. A subsequent GSEA and SPIA analysis later revealed enrichment in a large number of biological processes including phototransduction, regeneration, and cell death (P adj <0.05). Based on these pathways, a set of pro-survival gene expressions were extracted and tabulated. This study provided an in-depth transcriptomic analysis on the MSCEPO-treated retinal degeneration model as well as a profile of pro-survival genes that can be used as candidates for further genetic enhancement studies on stem cells.
Mesenchymal stem cells (MSC) have shown promise in restoring the vision of patients in clinical trials. However, this therapeutic effect is not observed in every treated patient and is possibly due to the inefficacies of cell delivery and high cell death following transplantation. Utilizing erythropoietin can significantly enhance the regenerative properties of MSCs and hence improve retinal neuron survivability in oxidative stress. Hence, this study aimed to investigate the efficacy of conditioned medium (CM) obtained from transgenic human erythropoietin-expressing MSCs (MSC EPO ) in protecting human retinal pigment epithelial cells from sodium iodate (NaIO3)-induced cell death. Human MSC and MSC EPO were first cultured to obtain conditioned media (CM). The IC50 of NaIO3 in the ARPE-19 culture was then determined by an MTT assay. After that, the efficacy of both MSC-CM and MSC-CM EPO in ARPE-19 cell survival were compared at 24 and 48 h after NaIO3 treatment with MTT. The treatment effects on mitochondrial membrane potential was then measured by a JC-1 flow cytometric assay. The MTT results indicated a corresponding increase in cell survivability (5-58%) in the ARPE-19 cell cultures. In comparison to MSC-CM, the use of conditioned medium collected from the MSC-CM EPO further enhanced the rate of ARPE-19 survivability at 24 h (P < 0.05) and 48 h (P < 0.05) in the presence of NaIO3. Furthermore, more than 90% were found viable with the JC-1 assay after MSC-CM EPO treatment, showing a positive implication on the mitochondrial dynamics of ARPE-19. The MSC-CM EPO provided an enhanced mitigating effect against NaIO3-induced ARPE-19 cell death over that of MSC-CM alone during the early phase of the treatment, and it may act as a future therapy in treating retinal degenerative diseases.
Extensive clinical efforts have been made to control the severity of dengue diseases; however, the dengue morbidity and mortality have not declined. Dengue virus (DENV) can infect and cause systemic damage in many organs, resulting in organ failure. Here, we present a novel report showing a tailored stem-cell-based therapy that can aid in viral clearance and rescue liver cells from further damage during dengue infection. We administered a combination of hematopoietic stem cells and endothelial progenitor cells in a DENV-infected BALB/c mouse model and found that delivery of this cell cocktail had improved their liver functions, confirmed by hematology, histopathology, and next-generation sequencing. These stem and progenitor cells can differentiate into target cells and repair the damaged tissues. In addition, the regime can regulate endothelial proliferation and permeability, modulate inflammatory reactions, enhance extracellular matrix production and angiogenesis, and secrete an array of growth factors to create an enhanced milieu for cell reparation. No previous study has been published on the treatment of dengue infection using stem cells combination. In conclusion, dengue-induced liver damage was rescued by administration of stem cell therapy, with less apoptosis and improved repair and regeneration in the dengue mouse model.
Osteoarthritis (OA) is a joint degenerative disease that is an exceedingly common problem associated with aging. Aging is the principal risk factor for OA, but damage-related physiopathology of articular chondrocytes probably drives the mechanisms of joint degeneration by a progressive decline in the homeostatic and regenerative capacity of cells. Cellular aging is the manifestation of a complex interplay of cellular and molecular pathways underpinned by transcriptional, translational, and epigenetic mechanisms and niche factors, and unraveling this complexity will improve our understanding of underlying molecular changes that affect the ability of the articular cartilage to maintain or regenerate itself. This insight is imperative for developing new cell and drug therapies for OA disease that will target the specific causes of age-related functional decline. This review explores the key age-related changes within articular chondrocytes and discusses the molecular mechanisms that are commonly perturbed as cartilage ages and degenerates. Current efforts and emerging potential therapies in treating OA that are being employed to halt or decelerate the aging processes are also discussed.
Colorectal cancer (CRC) is one of the most widely diagnosed cancers worldwide. It has been shown that the body-mass index (BMI) of the patients could influence the tumor microenvironment, treatment response, and overall survival rates. Nevertheless, the mechanism on how BMI affects the tumorigenesis process, particularly the tumor microenvironment is still elusive. Herein, we postulate that extracellular vesicles (EVs) from CRC patients and non-CRC volunteers with different BMI could affect immune cells differently, in CD8 T cells particularly. We isolated the EVs from the archived serum of CRC patients with high and low BMI, as well as healthy controls with similar BMI status. The EVs were further characterized via electron microscopy, western blot and dynamic light scattering. Then, functional analysis was performed on CD8 T cells including apoptosis, cell proliferation, gene expression profiling and cytokine release upon co-incubation with the different EVs. Our results suggest that CRC-derived EVs were able to regulate the CD8 T cells. In some assays, low BMI EVs were functionally different than high BMI EVs. This study highlights the possible difference in the regulatory mechanism of cancer patients-derived EVs, especially on CD8 T cells.