Exploring the various effects of Cu doping in hydroxyapatite nanoparticle.

Adding foreign ions to hydroxyapatite (HAp) is a popular approach for improving its properties. This study focuses on the effects of calcium substitution with copper in HAp. Instead of calcium, copper ions were doped into the structure of hydroxyapatite nanoparticles at 1%, 3%, and 5% concentrations. XRD analysis showed that the amount of substituted copper was less than needed to generate a distinct phase, yet its lattice parameters and crystallinity slightly decreased. Further, the results of degradation tests revealed that copper doping in hydroxyapatite doubled calcium ion release in water. The incorporation of copper into the apatite structure also boosted the HAp zeta potential and FBS protein adsorption onto powders. According to antibacterial investigations, a concentration of 200 mg/ml of hydroxyapatite containing 5% copper was sufficient to effectively eradicate E. coli and S. aureus bacteria. Furthermore, copper improved hydroxyapatite biocompatibility. Alkaline phosphatase activity and alizarin red tests showed that copper in hydroxyapatite did not inhibit stem cell differentiation into osteoblasts. Also, the scratch test demonstrated that copper-containing hydroxyapatite extract increased HUVEC cell migration. Overall, our findings demonstrated the utility of incorporating copper into the structure of hydroxyapatite from several perspectives, including the induction of antibacterial characteristics, biocompatibility, and angiogenesis.

Advances in Management of Spinal Cord Injury Using Stem Cell-derived Extracellular Vesicles: A Review Study.

Introduction: Spinal cord injury (SCI) is characterized by serious both motor and sensory disability of the limbs below the injured segment. It is the most traumatic disorder among central nervous system (CNS) conditions which not only leads to psychological and physical harm to patients but also results in a dramatic loss in the life quality. Many efforts have been developed to find a therapeutic approach for SCI; however, an effective treatment has not yet been found. The lack of effective treatment approach and rehabilitation of SCI underscores the need to identify novel approaches. Tissue engineering associated with stem cells has been recently introduced as an effective treatment approaches for traumatic SCI. Although, low survival rates, immune rejection, cell dedifferentiation, and tumorigenicity have been addressed for tissue engineering. Regenerative medicine is an interdisciplinary field developing and applying tissue engineering, stem cell (SC) therapy, and SC-derived extracellular vesicle therapy that aims to provide reliable and safe ways to replace injured tissues and organs. The application of mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) has recently attracted attention to improve central nervous system dysfunction such as SCI, mainly by promoting neurogenesis and angiogenesis. Methods: In this review article the latest information of SCI improvement using stem cell-derived extracellular vesicles published data in the Web of Science, Scopus, Science Direct and Pub Med databases were collected. Results: The data collected show that MSC-EVs, including exosomes, alone or in combination with scaffolds can can regenerate the injured nerve in SCI. Conclusion: This study summarizes the efficacy of MSC-EVs, including exosomes, alone or in combination with scaffolds in the treatment of SCI and then discusses the therapeutic outcomes observed in SCI experimental models following treatment with MSC-EVs alone or loaded on scaffolds in particular collagen-based scaffolds. Highlights: The pathological process of SCI being very complex.A complete effective strategy has yet to be found for treatment of SCI in human.Exosomes derived-stem cells alone have great potential for the treatment of SCI.Various biocompatible scaffolds are good drug carriers for SCI treatment.Various biocompatible scaffolds are good carriers for exosomes. Plain Language Summary: Human with spinal cord injury (SCI) show serious motor and sensory disability of the limbs. Since there is no an effective treatment for SCI, researchers are trying to develop and find a new therapeutic approach for SCI. CNS tissue engineering with various stem cells sources as well as their derived extracellular vesicle has been extensively attracted for providing reliable and safe approach for SCI treatment. Extracellular vesicles are lipid bilayer membrane-enclosed organelles containing various biomolecules involved in a variety of complex intercellular communication systems. They are released from all cell types into their surrounding environment and are important vehicles for paracrine The application of stem cells-derived extracellular vesicles (MSC-EVs) has recently attracted attention to improve central nervous system dysfunction such as SCI, mainly by promoting neurogenesis and angiogenesis.

Cell-free therapy based on extracellular vesicles: a promising therapeutic strategy for peripheral nerve injury.

Peripheral nerve injury (PNI) is one of the public health concerns that can result in a loss of sensory or motor function in the areas in which injured and non-injured nerves come together. Up until now, there has been no optimized therapy for complete nerve regeneration after PNI. Exosome-based therapies are an emerging and effective therapeutic strategy for promoting nerve regeneration and functional recovery. Exosomes, as natural extracellular vesicles, contain bioactive molecules for intracellular communications and nervous tissue function, which could overcome the challenges of cell-based therapies. Furthermore, the bioactivity and ability of exosomes to deliver various types of agents, such as proteins and microRNA, have made exosomes a potential approach for neurotherapeutics. However, the type of cell origin, dosage, and targeted delivery of exosomes still pose challenges for the clinical translation of exosome therapeutics. In this review, we have focused on Schwann cell and mesenchymal stem cell (MSC)-derived exosomes in nerve tissue regeneration. Also, we expressed the current understanding of MSC-derived exosomes related to nerve regeneration and provided insights for developing a cell-free MSC therapeutic strategy for nerve injury.

Evaluation of the effect of mesenchymal stem cells injection in the nucleus accumbens on the morphine reinstatement behavior in a conditioned place preference model in Wistar rat: Expression changes of NMDA receptor subunits and NT-3.

Mesenchymal stem cells (MSCs) have been recently shown to improve functional recovery in animal models of CNS disorders and are currently being examined in clinical studies for sclerosis, stroke, and CNS lesions. The activation of endogenous CNS protection and repair mechanisms is unclear. MSC-based approaches are considered a new potential target for neurodegenerative disorders. This study was designed to discover the effect of MSCs injection in the nucleus accumbens (NAc) on the reinstatement of behavior in morphine-induced conditioned place preference (CPP) in male rats. The CPP was induced via intra-peritoneal (i.p.) morphine injection (5 mg/kg) for three consecutive days. After being tested for CPP induction, animals received MSCs or culture medium (DMEM F-12) in their NAc using stereotaxic surgery. Following extinction, a priming dose of morphine (2 mg/kg) was administered to induce reinstatement. Expression of GluN1, GluN2A, and GluN2B subunits of the NMDA receptor and the NT-3 gene in the NAc was assessed on the last day of extinction and following CPP reinstatement. The results showed that local injection of MSCs attenuated reinstatement after receiving a priming dose of morphine, and also shortened the period of CPP extinction. The mRNA expression of the NT-3 gene in the group receiving MSCs was increased compared to control animals, as was observed for GluN1 and GluN2B, but not GluN2A. It is concluded that intra-NAc injection of MSCs may facilitate morphine extinction and alleviate reinstatement behavior which may be via expression changes in NMDA receptor subunits and NT-3 gene.

The Effect of Collagen and Fibrin Hydrogels Encapsulated with Adipose Tissue Mesenchymal Stem Cell-Derived Exosomes for Treatment of Spinal Cord Injury in a Rat Model.

Background: Mesenchymal stem cell (MSC) derived exosomes (MSC-DE) have been demonstrated to be potential candidates for the treatment of rat spinal cord injury (SCI). Objective: The effect of AD-MSC and AD-MSC-DE encapsulated into collagen and fibrin hydrogels on the treatment of SCI in a rat animal model was investigated for introducing a new effective SCI treatment method. Materials and Methods: The AD-MSC-DE was isolated using ultra-centrifugation at 100,000×g for 120 min and characterized by different methods. Fibrin and collagen hydrogels were synthesized and then mixed with AD-MSC-DE suspension. the characterized AD-MSC-DE were encapsulated into collagen and fibrin hydrogels. eighteen adult male Wister rats were randomly classified into 3 equal groups (n=6): the control group (SCI rat without treatment), SCI rat treated with either AD-MSC-DE encapsulated in collagen hydrogel or encapsulated in fibrin hydrogel groups. the treatment approaches were evaluated using clinical, histological, and molecular assays. Results: The AD-MSC-DE encapsulated into fibrin and collagen groups showed better clinical function than the control group. The AD-MSC-DE encapsulated into fibrin and collagen also improved SCI-induced polio and leuko-myelomalacia and leads to higher expression of NF protein than the control group. In the AD-MSC-DE encapsulated into collagen and fibrin leads to up-regulation the mean levels of NEFL (23.82 and 24.33, respectively), eNOS (24.31 and 24.53, respectively), and CK19 mRNAs (24.23 and 23.98, respectively) compared to the control group. Conclusion: The AD-MSC-DE encapsulated within ECM-based hydrogel scaffolds such as collagen and fibrin can regenerate the injured nerve in SCI rats and reduce spinal cord lesion-induced central neuropathic pain.

Novel therapeutic approach to slow down the inflammatory cascade in acute/subacute spinal cord injury: Early immune therapy with lipopolysaccharide enhanced neuroprotective effect of combinational therapy of granulocyte colony-stimulating factor and bone-marrow mesenchymal stem cell in spinal cord injury.

Bone-marrow mesenchymal stem cells (BM-MSCs) have not yet proven any significant therapeutic efficacy in spinal cord injury (SCI) clinical trials, due to the hostile microenvironment of the injured spinal cord at the acute phase. This study aims to modulate the inflammatory milieu by lipopolysaccharide (LPS) and granulocyte colony-stimulating factor (G-CSF) to improve the BM-MSCs therapy. For this purpose, we determined the optimum injection time and sub-toxic dosage of LPS following a T10 contusion injury. Medium-dose LPS administration may result in a local anti-inflammatory beneficial role. This regulatory role is associated with an increase in NF-200-positive cells, significant tissue sparing, and improvement in functional recovery compared to the SCI control group. The second aim was to examine the potential ability of LPS and LPS + G-CSF combination therapy to modulate the lesion site before BM-MSC (1 × 105 cells) intra-spinal injection. Our results demonstrated combination therapy increased potency to enhance the anti-inflammatory response (IL-10 and Arg-1) and decrease inflammatory markers (TNF-α and CD86) and caspase-3 compared to BM-MSC monotherapy. Histological analysis revealed that combination groups displayed better structural remodeling than BM-MSC monotherapy. In addition, Basso-Beattie-Bresnahan (BBB) scores show an increase in motor recovery in all treatment groups. Moreover, drug therapy shows faster recovery than BM-MSC monotherapy. Our results suggest that a sub-toxic dose of LPS provides neuroprotection to SCI and can promote the beneficial effect of BM-MSC in SCI. These findings suggest that a combination of LPS or LPS + G-CSF prior BM-MSC transplantation is a promising approach for optimizing BM-MSC-based strategies to treat SCI. However, because of the lack of some methodological limitations to examine the survival rate and ultimate fate of transplanted BM-MSCs followed by LPS administration in this study, further research needs to be done in this area. The presence of only one-time point for evaluating the inflammatory response (1 week) after SCI can be considered as one of the limitations of this study. We believed that the inclusion of additional time points would provide more information about the effect of our combination therapy on the microglia/macrophage polarization dynamic at the injured spinal cord.

Reduced inflammation following human endometrial stromal/stem cell injection into male Wistar rats with cisplatin-induced acute kidney injury.

Introduction: Inflammation is one of the most important mechanisms involved in cisplatin-induced acute kidney injury (AKI). Mesenchymal stromal/stem cells (MSCs) exhibit anti-inflammatory and immunomodulatory abilities. Human endometrial stromal/stem cells (hEnSCs) exhibit similar properties to MSCs. These cells secrete immunoregulators, so we investigated the inflammatory aspect of hEnSCs in the treatment of cisplatin-induced AKI in Wistar rats. Methods: Each group consisted of 6 male Wistar rats. Groups were as follows: sham, model (5 mg/kg cisplatin, IP), and treatment (1 million hEnSCs, IV, 3 hours after cisplatin). Renal function, histopathology, proliferation rate, infiltration of CD3+ T cell, and expression of Il-10 and cystatin c (Cst3) were assessed on day 5. DiI-labeled cells were tracked in kidney and liver on days 4 and 14. Results: HEnSC transplantation improved cisplatin-induced injuries such as renal dysfunction and tissue damage. The highest levels of pathologic scores and hyaline cast formation were observed in the model group while hEnSCs transplantation resulted in their reduction (154.00 ± 14.95, 8.00 ± 1.41 vs. 119.40 ± 5.43, 2.50 ± 1.05). The percentage of Ki-67 positive cells in the treatment group increased while cisplatin decreased proliferation (39.91 ± 5.33 vs. 23.91 ± 3.57 in glomeruli and 39.07 ± 2.95 vs. 16.61 ± 3.25 in tubules). The expression of Cst3 and Il-10 was higher in the model and treatment groups, respectively. DiI-labeled cells were observed in the renal tubules and liver lobes on days 4 and 14. Conclusion: HEnSCs may ameliorate cisplatin-induced AKI through anti-inflammatory and immunomodulatory effects and/or through paracrine effects.

Encapsulation of rat bone marrow-derived mesenchymal stem cells (rBMMSCs) in collagen type I containing platelet-rich plasma for osteoarthritis treatment in rat model.

Osteoarthritis (OA) is the most common form of degenerative joint disease, affecting more than 25% of the adults despite its prevalence in the elderly population. Most of the current therapeutic modalities aim at symptomatic treatment which lingers the disease progression. In recent years, regenerative medicine such as stem cell transplantation and tissue engineering has been suggested as a potential curative intervention for OA. The objective of this current study was to assess the safety and efficacy of an injectable tissue-engineered construct composed of rat bone marrow mesenchymal stem cells (rBMMSCs), platelet-rich plasma (PRP), and collagen type I in rat model of OA. To produce collagen type I, PRP and rBMMSCs, male Wistar rats were ethically euthanized. After isolation, culture, expansion and characterization of rBMMSCs, tissue-engineered construct was formed by a combination of appropriate amount of collagen type I, PRP and rBMMSCs. In vitro studies were conducted to evaluate the effect of PRP on chondrogenic differentiation capacity of encapsulated cells. In the following, the tissue-engineered construct was injected in knee joints of rat models of OA (24 rats in 4 groups: OA, OA + MSC, OA + collagen + MSC + PRP, OA + MSC + collagen). After 6 weeks, the animals were euthanized and knee joint histopathology examinations of knee joint samples were performed to evaluate the effect of each treatment on OA. Tissue-engineered construct was successfully manufactured and in vitro assays demonstrated the relevant chondrogenic genes and proteins expression were higher in PRP group than that of others. Histopathological findings of the knee joint samples showed favorable regenerative effect of rBMMSCs + PRP + collagen group compared to others. We introduced an injectable tissue-engineered product composed of rBMMSCs + PRP + collagen with potential regenerative effect on cartilage that has been damaged by OA.

Angiogenesis and Its Targeting in Glioblastoma with Focus on Clinical Approaches.

Angiogenesis is a characteristic of glioblastoma (GBM), the most fatal and therapeutic-resistant brain tumor. Highly expressed angiogenic cytokines and proliferated microvascular system made anti-angiogenesis treatments a thoroughly plausible approach for GBM treatment. Many trials have proved to be not only as a safe but also as an effective approach in GBM retardation in a certain time window as seen in radiographic response rates; however, they have failed to implement significant improvements in clinical manifestation whether alone or in combination with radio/chemotherapy. Bevasizumab, an anti-vascular endothelial growth factor-A (VEGF-A) antibody, is the only agent that exerts meaningful clinical influence by improving progression-free survival (PFS) and partially alleviate clinical symptoms, nevertheless, it could not prolong the overall survival (OS) in patients with GBM. The data generated from phase II trials clearly revealed a correlation between elevated reperfusion, subsequent to vascular normalization induction, and improved clinical outcomes which explicitly indicates anti-angiogenesis treatments are beneficial. In order to prolong these initial benefits observed in a certain period of time after anti-angiogenesis targeting, some aspects of the therapy should be tackled: recognition of other bypass angiogenesis pathways activated following antiangiogenesis therapy, identification of probable pathways that induce insensitivity to shortage of blood supply, and classifying the patients by mapping their GBM-related gene profile as biomarkers to predict their responsiveness to therapy. Herein, the molecular basis of brain vasculature development in normal and tumoral conditions is briefly discussed and it is explained how "vascular normalization" concept opened a window to a better comprehension of some adverse effects observed in anti-angiogenesis therapy in clinical condition. Then, the most targeted angiogenesis pathways focused on ligand/receptor interactions in GBM clinical trials are reviewed. Lastly, different targeting strategies applied in anti-angiogenesis treatment are discussed.

3-Dimensional Model to Study Apoptosis Induction of Activated Natural Killer Cells Conditioned Medium Using Patient-Derived Colorectal Cancer Organoids.

Natural killer (NK) cells are innate lymphocytes that can kill tumor cells via different pathways, including the secretion of cytotoxic granules in immunological synapses and the binding of apoptosis-inducing ligands with cognate death receptors on tumor cells. These ligands are also soluble in NK cells conditioned medium (NK-CM). However, novel preclinical in vitro models are required for solid tumors such as colorectal cancer (CRC) to investigate apoptosis induction of activated NK-CM in a tissue-like structure. In the present study, we established a patient-derived CRC organoid culture system as a new tool for CRC research in the last decade. Tumor organoids were stained with hematoxylin and eosin (H&E) and compared with the original tumor taken from the patient. Goblet cell differentiation and mucus secretion were evaluated using periodic acid-Schiff and alcian blue histochemical staining. Moreover, tumor organoids were stained for CDX2 and Ki67 markers with immunohistochemistry (IHC) to investigate gastrointestinal origin and proliferation. Histopathological evaluations indicated tumor organoids represent patient tumor characteristics. Primary NK cells were isolated and characterized using CD56 marker expression and the lack of the CD3 marker. Flow cytometry results showed the purity of isolated CD3-and CD56 + NK cells about 93%. After further ex vivo expansion, IL-2-activated NK-CM was collected. Secretions of IFN-γ and TNF-α were measured to characterize activated NK-CM. Cytokines levels were significantly elevated in comparison to the control group. Soluble forms of apoptosis-inducing ligands, including TNF-related apoptosis-inducing ligand (TRAIL) and FasL, were detected by western blot assay. Colon cancer organoids were treated by IL-2-activated NK-CM. Apoptosis was assessed by Annexin V-FITC/PI staining and quantified by flow cytometry. In conclusion, despite the activated NK-CM containing apoptosis-inducing ligands, these ligands' soluble forms failed to induce apoptosis in patient-derived colon cancer organoids. Nevertheless, we report a reliable in vitro assessment platform in a personalized setting.

Atorvastatin Inhibits Viability and Migration of MCF7 Breast Cancer Cells.

OBJECTIVE: Atorvastatin is commonly used as a lipid lowering drug. The emerging interest in  statins as anticancer agents is based on their pleiotropic effects on cancer cells. Among the statins, atorvastatin, and in cancers, breast malignancies have received less attention in preclinical investigations. In order to enhance the efficacy of cancer treatment,  adjuvant, less expensive therapeutic strategies have been recently noticed. In this case, we investigated the in-vitro effect of atorvastatin on viability and migration of MCF7 breast cancer cell line. METHODS: We tested the cytotoxicity of atorvastatin on breast cancer cells survival by MTT assay. Annexin-V / PI staining and then flow cytometry of cancer cells in addition to quantitative real-time PCR tests quantified the apoptosis and necrosis of cancer cells. We figured out the impact of atorvastatin on cancer cell migration capability through scratch-wound healing assay and transwell migration examination. Inverted light microscope and fluorescent imaging displayed the morphological changes following treatment of MCF7 cells with atorvastatin. RESULT: We resulted that atorvastatin can trigger MCF7 cancer cells to undergo necrosis and caspase-dependent apoptosis based on the viable/dead cell number, mitotic cell cycle, gene expression, and morphological assays. The results were dose- and time-dependent and the half- maximal inhibitory concentration of atorvastatin for cancer cells' viability inhibition was 9.1 µM/L(nM/mL). Moreover, the migration of MCF7 cells were inhibited in the treated group as we figured out in two- and three-dimensional migration methods. CONCLUSION: In-vitro inspection of drug-cancer cell interactions paves the way  for future in-vivo research studies. These in-vitro results revealed that atorvastatin has anti-viability and anti-migration effects on breast cancer cells.

Fabrication and Characterization of a Three-Dimensional Fibrin Gel Model to Evaluate Anti-Proliferative Effects of Astragalus hamosus Plant Extract on Breast Cancer Cells.

BACKGROUND: Breast Cancer (BC) is a malignancy with high mortality among women. Recently, scaffold-based three-dimensional (3D) models have been developed for anti-cancer drug research. The present study aimed to investigate the anti-proliferative effects of Astragalus hamosus (A. hamosus) in 3D fibrin gel against MCF-7 cell line. We have also evaluated anti-proliferative effect of A. hamosus differences between 3D and 2D cultures. METHODS: The fibrin gel formulation was first optimized by testing the structural and mechanical properties. Then the cytotoxic effect of A. hamosus extract was assessed on MCF-7 cells by MTT assay. Cell apoptosis was evaluated using TUNEL method and flow cytometry. Cell cycle and proliferation were analyzed by flow cytometry. Apoptosis-related gene expression such as Bcl-2, caspase-3, -8 and -9 were quantified by real time-PCR. RESULTS: TUNEL staining showed a significant damage accompanied with cell apoptosis. Flow cytometry analysis revealed that apoptosis increased after treatment with A. hamosus extract in 3D culture model compared to 2D culture. The A. hamosus extract arrested cell cycle in the S and G2/M phases in 3D model while in the 2D culture G0/G1 phase was affected. Treatment with A. hamosus extract led to upregulation of the caspase-3, -8 and -9 genes and downregulation of the Ki-67 in the 3D-culture compared with the 2D culture. CONCLUSION: These results indicated that A. hamosus extract could be used as a therapeutic candidate for BC due to its anti-proliferative effects. Furthermore, 3D fibrin gel could be better than 2D-cultured cells in simulating important tumor characteristics in vivo, namely, anti-proliferative and anti-apoptotic features.

Synergistic inhibitory effect of human umbilical cord matrix mesenchymal stem cells-conditioned medium and atorvastatin on MCF7 cancer cells viability and migration.

Recent studies have demonstrated inhibitory effects of mesenchymal stem cells on breast tumors. Likewise, the emerging interest in statins as anticancer agents is based on their pleiotropic effects. In the present study, we investigated whether atorvastatin and umbilical cord matrix derived mesenchymal stem cells-conditioned medium affect the MCF7 cancer cells viability and interactions. We measured the viability of MCF7 cancer cells by MTT assay, flow cytometry, and quantitative real-time PCR. Two-dimensional culture and hanging drop aggregation assay illustrated the morphological changes. We traced the MCF7 migration via scratch-wound healing test and trans-well assay. The results showed the inhibition of cancer cell viability in all treated groups compared to the control group. The effect of atorvastatin and conditioned medium combination was significantly more than each substance separately. The morphological changes indicated apoptosis in treated cells. The annexin V/PI flow cytometry especially in the combination-treated group displayed decreasing in DNA synthesis and cell cycle arrest in G1 and G2/M phases. As well, the mRNA expressions of caspases 3, 8, 9, and Bcl-2 genes were along with extrinsic and intrinsic apoptosis pathways. Conditioned medium disrupted the connections between cancer cells, so the spheroids in three-dimensional configuration lost their order and dispersed. The migration of treated cells across the wound area and trans-well diminished, particularly by the conditioned medium and atorvastatin combination. There fore, the synergistic anti-proliferative and anti-motility effect of atorvastatin along with human umbilical cord mesenchymal stem cells-derived conditioned medium on MCF7 breast cancer cells have been proved. The results might lead the development of novel adjuvant anticancer therapeutics based on targeting or modifying the extracellular matrix to increase chemotherapy results or to prevent metastatic colonization. Schematic representation of "Synergistic Inhibitory Effect of Human Umbilical Cord Matrix Mesenchymal Stem Cells-Conditioned Medium and Atorvastatin on MCF7 Cancer Cells Viablity and Migration" by: Dr. Reyhaneh Abolghasemi, Dr. Somayeh Ebrahimi-barough, Proffesor. Jafar Ai.

The effects of Sorafenib and Natural killer cell co-injection in combinational treatment of hepatocellular carcinoma; an in vivo approach.

BACKGROUND: Natural killer cells (NKC) and Sorafenib (Sor) are two important agents for the treatment of hepatocellular carcinoma (HCC). Over the past decade, the interaction of Sor and NKC against HCC has been widely challenging. This study aimed to assess the efficacy of NKC & Sor for the treatment of HCC in vivo. METHODS: Subcutaneous xenograft models of HCC were established in nude mice. For safety assessment of treatment, the kidney and liver functions were analyzed. Paraffin embedded tumor sections were histopathologically studied and immunohistochemistry (IHC) tests were done to evaluate the angiogenesis (CD34) and proliferation (Ki67) indexes. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to identify the tumor cells undergoing apoptosis. The serum levels of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were measured by enzyme-linked immunosorbent assay (ELISA) and expression levels of major inflammatory cytokines and cytoplasmic granules in xenograft HCC were quantified using real-time PCR. RESULTS: NKC & Sor significantly inhibited necrosis and apoptosis in tumor cells and increased angiogenesis and proliferation of HCC compared to the monotherapy of NKC or Sor alone. The serum levels of TNF-α, IFN-γ as well as the expression levels of TNF-α, IFN-γ, interleukins (ILs)-1, 6, 10, granzyme-B and perforin in the xenograft HCC tissues of the treated mice with NKC & Sor were significantly lower than those of treated with NKC or Sor alone. CONCLUSION: Therapy with the specific dosage of NKC & Sor could not inhibit the HCC xenograft growth rate through a synergistic effect in a mouse model of HCC.

Development and Clinical Application of Tumor-derived Exosomes in Patients with Cancer.

A tumor is an abnormal growth of cells within a tissue that can lead to death due to late diagnosis, poor prognosis, drug resistance, and finally enhanced metastasis formation. Exosomes are nanovesicles that have been derived from all the different cell types. These vesicles can transfer various molecules, including the distinct form of nucleic acids (mRNA, miRNA, and circRNA) and proteins. Tumor-derived exosomes (TEXs) have exceptionally important roles through multiple molecular and cellular pathways like progression, tumorigenesis, drug resistance, and as well as metastasis. TEXs are detectable in all body fluids such as serum and urine, a convenient and non-invasive way to access these nano-sized vesicles. TEXs lead to the symptom expression of genetic aberrations in the tumor cell population, making them an accurate and sensitive biomarker for the diagnosis and prognosis of tumors. On the other hand, TEXs contain major histocompatibility complexes (MHCs) and play important dual roles in regulating tumor immune responses: they can mediate both immune activation and suppression through tumor-associated immunity. Despite numerous scientific studies, there are still many technical barriers to distinguish TEXs from non-tumor-derived exosomes. Even so, removing exosomes leading to a wide difference in outcomes inside a patient's body. Hence, controversial pieces of evidence have demonstrated the vital role of TEXs as hopeful biomarkers for the early detection of cancers, evaluation of therapeutic effects, and monitoring of the patient.

Improving motor neuron-like cell differentiation of hEnSCs by the combination of epothilone B loaded PCL microspheres in optimized 3D collagen hydrogel.

Spinal cord regeneration is limited due to various obstacles and complex pathophysiological events after injury. Combination therapy is one approach that recently garnered attention for spinal cord injury (SCI) recovery. A composite of three-dimensional (3D) collagen hydrogel containing epothilone B (EpoB)-loaded polycaprolactone (PCL) microspheres (2.5 ng/mg, 10 ng/mg, and 40 ng/mg EpoB/PCL) were fabricated and optimized to improve motor neuron (MN) differentiation efficacy of human endometrial stem cells (hEnSCs). The microspheres were characterized using liquid chromatography-mass/mass spectrometry (LC-mas/mas) to assess the drug release and scanning electron microscope (SEM) for morphological assessment. hEnSCs were isolated, then characterized by flow cytometry, and seeded on the optimized 3D composite. Based on cell morphology and proliferation, cross-linked collagen hydrogels with and without 2.5 ng/mg EpoB loaded PCL microspheres were selected as the optimized formulations to compare the effect of EpoB release on MN differentiation. After differentiation, the expression of MN markers was estimated by real-time PCR and immunofluorescence (IF). The collagen hydrogel containing the EpoB group had the highest HB9 and ISL-1 expression and the longest neurite elongation. Providing a 3D permissive environment with EpoB, significantly improves MN-like cell differentiation and maturation of hEnSCs and is a promising approach to replace lost neurons after SCI.

Human Endometrial Stromal/Stem Cells Inhibit Apoptosis in Cisplatin-Induced Acute Kidney Injury in Male Wistar Rats.

OBJECTIVE: Acute kidney injury (AKI) is referred to as sudden decline in the function of kidney. Human endometrial stromal/stem cells (hEnSCs) are mesenchymal stem cell (MSC)-like cells, which are suitable candidates for regenerative medicine purposes, yet the effect of hEnSCs on cisplatin-induced AKI has not been studied; therefore, the present study was conducted to investigate this gap in the literature. MATERIALS AND METHODS: In this experimental study, hEnSCs were obtained from endometrial biopsy using collagenase I and were then cultured in DMEM/F12 medium. A total of 48 male Wistar rats (150-200 g) were classified into four groups: intact -receiving no treatment, model -receiving 5 mg/kg of body weight cisplatin, as well as phosphate-buffered saline (PBS) and cell -receiving either PBS or hEnSCs for three hours after cisplatin injection, respectively. Biochemical parameters, pathologic scores, apoptosis assay, Bcl-2 and Tnf-α expression were evaluated on day 5. RESULTS: On day 5 post-transplantation we observed that HEnSCs injection has led to a decrease in both blood urea nitrogen (BUN) and serum creatinine (SCr), compared to the model and PBS groups (0.82 ± 0.03 vs. 1.42 ± 0.06, 1.09 ± 0.05 mg/dl and 61.53 ± 3.07 vs. 116.60 ± 2.12, 112.00 ± 1.35 mg/dl, respectively). The highest levels of pathologic scores were observed in model and PBS groups, while hEnSCs transplantation resulted in a decrease in pathologic scores (149.10 ± 7.03, 141.50 ± 4.68 vs. 118 ± 2.16). HEnSCs significantly decreased the percentage of TUNELpositive cells in the cell group compared with model and PBS groups (20.37 ±. 3.37 vs. 33.67 ± 1.79, 31.53 ± 1.05 in glomeruli and 15.10 ± 1.47 vs. 42.33 ± 1.72, 39.23 ± 1.61 in tubules). In addition, HEnSCs resulted in upregulation of Bcl-2 and downregulation of Tnf-α in the cisplatin-induced AKI. CONCLUSION: Our results showed that injection of hEnSCs may improve AKI through lowering the amount of apoptosis in renal cells.

Adaptive NK Cell Therapy Modulated by Anti-PD-1 Antibody in Gastric Cancer Model.

Recently, adaptive NK cell therapy has become a promising treatment but has limited efficacy as a monotherapy. The identification of immune checkpoint inhibitor (ICI) molecules has opened a new horizon of immunotherapy. Herein, we aimed to demonstrate the cytotoxic effects of a polytherapy consisting of ex vivo expanded IL-2-activated NK cells combined with human anti-PD-1 antibody as an important checkpoint molecule in a xenograft gastric cancer mouse model. EBV-LCL cell is used as a feeder to promote NK cell proliferation with a purity of 93.4%. Mice (NOG, female, 6-8 weeks old) with xenograft gastric tumors were treated with PBS, ex vivo IL-2-activated NK cells, IL-2-activated NK cell along with human anti-PD-1 (Nivolumab), and IL-2-activated pretreated NK cells with anti-PD-1 antibody. The cytotoxicity of ex vivo expanded NK cells against MKN-45 cells was assessed by a lactate dehydrogenase (LDH) assay. Tumor volume was evaluated for morphometric properties, and tumor-infiltrating NK cells were assessed by immunohistochemistry (IHC) and quantified by flow cytometry. Pathologic responses were considered by H and E staining. Ex vivo LDH evaluation showed the cytotoxic potential of treated NK cells against gastric cancer cell line. We indicated that the adoptive transfer of ex vivo IL-2-activated NK cells combined with anti-PD-1 resulted in tumor growth inhibition in a xenograft gastric cancer model. Mitotic count was significantly decreased (*p < 0.05), and the tumor was associated with improved infiltration of NK cells in the NK-anti-PD-1 pretreated group (*p < 0.05). In conclusion, the combination approach of activated NK cells and anti-PD-1 therapy results in tumor growth inhibition, accompanied by tumor immune cell infiltration in the gastric tumor model.

Pesticides and Parkinson's disease: Current and future perspective.

Inappropriate use of pesticides has globally exposed mankind to a number of health hazards. Still their production is rising at the rate of 11 % annually and, has already exceeded more than 5 million tons in 2000 (FAO 2017). Plenty of available data reveals that pesticides exposures through agricultural use and food-preservative residue consumption may lead to neurodegenerative disorders like Parkinson's and Alzheimer's diseases. Parkinson's disease (PD) is a progressive motor impairment and a neurodegenerative disorder, considered as the leading source of motor disability. Pesticides strongly inhibit mitochondrial Complex-I, causing mitochondrial dysfunction and death of dopaminergic neurons in the substantia nigra (SN), thus leading to pathophysiologic implications of PD. Current medical treatment strategies, including pharmacotherapeutics and supportive therapies can only provide symptomatic relief. While complementary and alternative medicines including traditional medicine or acupuncture are considered as beneficial ways of treatment with significant clinical effect. Medically non-responding cases can be treated by surgical means, 'Deep Brain Stimulation'. Cell therapy is also an emerging and promising technology for disease modeling and drug development in PD. Their main aim is to replace and/or support the lost and dying dopaminergic neurons in the SN. Recently I/II clinical phase trial (Japan) have used dopaminergic progenitors generated from induced pluripotent stem (iPS) cells which can unveil a successful cell therapy to treat PD symptoms efficiently. This review focuses on PD caused by pesticides use, current treatment modalities, and ongoing research updates. Since PD is not a cell-autonomous disease rather caused by multiple factors, a combinatorial therapeutic approach may address not only the motor-related symptoms but also non-motor cognitive-behavioral issues.

Standard Operating Procedure for the Good Manufacturing Practice-Compliant Production of Human Endometrial Stem Cells for Multiple Sclerosis.

Multiple sclerosis (MS) is the most common cause of neurological diseases. Although, there are some effective medications with regulatory approval for treating MS, they are only partially effective and cannot promote repairing of tissue damage directly which occurs in the central nervous system. Therefore, there is an essential need to develop novel therapeutic approaches for neuroprotection or repairing damaged tissue in MS. Accordingly, cell-based therapies as a novel therapeutic strategy have opened a new horizon in treatment of MS. Each setting in cell therapy has potential benefits. Human endometrial stem cells as an invaluable source for cell therapy have introduced treatment for MS. In this respect, good manufacturing practice (GMP) has a pivotal role in clinical production of stem cells. This chapter tries to describe the protocol of GMP-grade endometrial stem cells for treatment of MS.