Cartilage tissue engineering using decellularized biomatrix hydrogel containing TGF-ß-loaded alginate microspheres in mechanically loaded bioreactor.

Physiochemical tissue inducers and mechanical stimulation are both efficient variables in cartilage tissue fabrication and regeneration. In the presence of biomolecules, decellularized extracellular matrix (ECM) may trigger and enhance stem cell proliferation and differentiation. Here, we investigated the controlled release of transforming growth factor beta (TGF-ß1) as an active mediator of mesenchymal stromal cells (MSCs) in a biocompatible scaffold and mechanical stimulation for cartilage tissue engineering. ECM-derived hydrogel with TGF-ß1-loaded alginate-based microspheres (MSs) was created to promote human MSC chondrogenic development. Ex vivo explants and a complicated multiaxial loading bioreactor replicated the physiological conditions. Hydrogels with/without MSs and TGF-ß1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-ß1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-ß1 added in culture media or those without TGF-ß1. However, constructs with TGF-ß1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-ß1/MSs under loading circumstances formed better cartilage matrix in an ex vivo osteochondral defect than control settings. This study demonstrates that controlled local delivery of TGF-ß1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy.

Good manufacturing practices production of human placental derived mesenchymal stem cells for therapeutic applications: focus on multiple sclerosis.

BACKGROUND: Among neurological diseases, multiple sclerosis (MS) affects mostly young adults and can cause long-term disability. While most medications with approval from regulatory agencies are very effective in treating MS disease, they are unable to repair the tissue damage found in the central nervous system (CNS). Consequently, Cell-based therapy particularly using mesenchymal stem/stromal cells (MSCs), holds promise for neuroprotection and tissue repair in MS treatment. Furthermore, placenta-derived MSCs (PLMSCs) have shown the potential to treat MS due to their abundance, noninvasive isolation from discarded tissues, no ethical problems, anti-inflammatory, and reparative properties. Accordingly, good manufacturing practices (GMPs) plays a crucial part in clinical SCs manufacturing. The purpose of our article is to discuss GMP-grade PLMSC protocols for treating MS as well as other clinical applications. METHODS AND RESULTS: Placental tissue obtained of a healthy donor during the caesarean delivery and PLMSCs isolated by GMP standards. Flow cytometry was used to assess the expression of the CD markers CD34, CD105, CD90, and CD73 in the MSCs and the mesodermal differentiation ability was evaluated. Furthermore, Genetic evaluation of PLMSCs was done by G-banded karyotyping and revealed no chromosomal instability. In spite of the anatomical origin of the starting material, PLMSCs using this method of culture were maternal in origin. CONCLUSIONS: We hope that our protocol for clinical manufacturing of PLMSCs according to GMP standards will assist researchers in isolating MSCs from placental tissue for clinical and pre-clinical applications.

Development of new nanofibrous nerve conduits by PCL-Chitosan-Hyaluronic acid containing Piracetam-Vitamin B12 for sciatic nerve: A rat model.

Peripheral nerve injury is a critical condition that can disrupt nerve functions. Despite the progress in engineering artificial nerve guidance conduits (NGCs), nerve regeneration remains challenging. Here, we developed new nanofibrous NGCs using polycaprolactone (PCL) and chitosan (CH) containing piracetam (PIR)/vitamin B12(VITB12) with an electrospinning method. The lumen of NGCs was coated by hyaluronic acid (HA) to promote regeneration in sciatic nerve injury. The NGCs were characterized via Scanning Electron Microscopy (SEM), Fourier transform infrared (FTIR), tensile, swelling, contact angle, degradation, and drug release tests. Neuronal precursor cell line (PCL12 cell) and rat mesenchymal stem cells derived from bone marrow (MSCs) were seeded on the nanofibrous conduits. After that, the biocompatibility of the NGCs was evaluated by the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, 4',6-diamidino-2-phenylindole (DAPI) staining, and SEM images. The SEM demonstrated that PCL/CH/PIR/VITB12 NGCs had nonaligned, interconnected, smooth fibers. The mechanical properties of these NGCs were similar to rat sciatic nerve. These conduits had an appropriate swelling and degradation rate. The In Vitro studies exhibited favorable biocompatibility of the PCL/CH/PIR/VITB12 NGCs towards PC12 cells and MSCs. The in vitro studies exhibited favorable biocompatibility of the PCL/CH/PIR/VIT B12 NGCs towards MSCs and PC12 cells. To analyze functional efficacy, NGCs were implanted into a 10 mm Wistar rat sciatic nerve gap and bridged the proximal and distal stump of the defect. After three months, the results of sciatic functional index (55.3 ± 1.8), hot plate latency test (5.6 ± 0.5 s), gastrocnemius muscle wet weight-loss (38.57 ± 1.6 %) and histopathological examination using hematoxylin-eosin (H&E) /toluidine blue/ Anti-Neurofilament (NF200) staining demonstrated that the produced conduit recovered motor and sensory functions and had comparable nerve regeneration compared to the autograft that can be as the gold standard to bridge the nerve gaps.

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.

Cytotoxicity of WT1-reactive T cells against Wilms tumor: An implication for antigen-specific adoptive immunotherapy.

Introduction: T cells that recognize WT1 peptides have been shown to efficiently eliminate WT1-expressing tumor cells. This study was designed to investigate the feasibility of isolating WT1-reactive T cells from peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with Wilms tumor, and to assess the cytotoxicity mediated by these cells against Wilms tumor cells (WiTu cells). Methods: WT1-reactive T cells were enriched and isolated by stimulating PBMCs with a WT1 peptide pool and interferon-γ capture-based immunomagnetic separation (IMS). Using the lactate dehydrogenase release assay, the in vitro cytotoxicity of the isolated cells and standard chemotherapy was evaluated on WiTu cells. Results: Higher proportions of WT1-reactive T cells were isolated from patients with Wilms tumor compared to those isolated from HDs. WT1-reactive T cells produced > 50% specific lysis when co-cultured with WT1+ WiTu cells at the highest effector-to-target (E:T) ratio in this study (i.e., 5:1), compared to <23% when co-cultured with WT1- WiTu cells at the same ratio. WT1-reactive T cells showed anti-tumoral activity in a dose-dependent manner and mediated significantly greater cytotoxicity than the non-WT1-reactive fraction of PBMCs on WT1+ WiTu cells. The cytotoxicity of standard chemotherapy was significantly lower than that of WT1-reactive T cells when co-cultured with WT1+ WiTu cells at E:T ratios of 2:1 and 5:1. Conclusion: WT1-reactive T cells can be effectively enriched from the PBMCs of patients with Wilms tumor. Ex vivo generated WT1-reactive T cells might be considered an adoptive immunotherapeutic option for WT1+ Wilms tumors.

Chemo-immune cell therapy by intratumoral injection of adoptive NK cells with capecitabine in gastric cancer xenograft model.

Introduction: Gastric cancer is one of the most commonly known malignancies and is the fifth cancer-related death globally. Whereas natural killer (NK) cells play a critical role in tumor elimination; therefore, adoptive NK cell therapy has become a promising approach in cancer cytotherapy. Hence, this study investigated the chemo-immune cell therapy in MKN-45 derived xenograft gastric cancer model. Methods: Three groups of animals have received the following treatments separately: activated NK cells, capecitabine, the combination of capecitabine and activated NK cells, and one was considered as the control group. Morphometric properties of tumor samples were evaluated at the end of the study. NK cells infiltration was evaluated by immunohistochemistry (IHC) of hCD56. Mitotic count and treatment response was assessed by hematoxylin and eosin (H&E) staining. The proliferation ratio to apoptosis was determined by IHC assessment of Ki67 and caspase 3. Results: The results indicated that the NK cell therapy could effectively decrease the mitotic count in pathology assessment, but the tumor was not completely eradicated. In combination with metronomic chemotherapy (MC) of capecitabine, NK cell therapy demonstrated a significant difference in tumor morphometric properties compared to the control group. The proliferation ratio to apoptosis was also in line with pathology data. Conclusion: Although NK cell therapy could effectively decrease the mitotic count in vivo, the obtained findings indicated lesser potency than MC despite ex vivo activation. In order to enhance NK cell therapy effectiveness, suppressive features of the tumor microenvironment and inhibitory immune checkpoints blockade should be considered.

Cancer immunotherapy via stem cell-derived NK cells.

NK cells are the first sentinels of the immune system that can recognize and eradicate transformed cells. Their activation without a need for additional signaling have attracted great attention on the use of NK cells as a promising option in cancer immunotherapy. However, the large-scale production of NK cells for successful NK cells therapy is a challenge that needs to be tackled. Engineering NK cells to avoid tumor escape and improve their antitumor potency are the other matters of focus that have widely been studied in the recent years. This paper reviews the most recent advances in the stem cell-derived NK cell technology and discusses the potential of the engineered NK cells for clinical applications in cancer immunotherapy.

NK cells are important cells in our body's defense system that can find and destroy tumor cells. These cells are made in bone marrow (in adults) or umbilical cord (in the embryonic period) from a population of cells called stem cells, and then released into the blood and lymph. Stem cells are the early ancestral cells that can differentiate into multiple cell types. Because the number and function of NK cells in a tumor context are reduced, thus we can use these stem cells to make lots of NK cells for treatment purposes. Scientists can also make these cells even better at killing tumors by changing them to have special sensors. In the end, NK cells are like superheroes that fight and kill tumor cells, and using stem cells to make them is a really promising way to help treat malignant diseases.

Assessment of the Efficacy of an LL-37-Encapsulated Keratin Hydrogel for the Treatment of Full-Thickness Wounds.

Wound healing remains a burdensome healthcare problem due to moisture loss and bacterial infection. Advanced hydrogel dressings can help to resolve these issues by assisting and accelerating regenerative processes such as cell migration and angiogenesis because of the similarities between their composition and structure with natural skin. In this study, we aimed to develop a keratin-based hydrogel dressing and investigate the impact of the delivery of LL-37 antimicrobial peptide using this hydrogel in treating full-thickness rat wounds. Therefore, oxidized (keratose) and reduced (kerateine) keratins were utilized to prepare 10% (w/v) hydrogels with different ratios of keratose and kerateine. The mechanical properties of these hydrogels with compressive modulus of 6-32 kPa and tan δ <1 render them suitable for wound healing applications. Also, sustained release of LL-37 from the keratin hydrogel was achieved, which can lead to superior wound healing. In vitro studies confirmed that LL-37 containing 25:75% of keratose/kerateine (L-KO25:KN75) would result in significant fibroblast proliferation (∼85% on day 7), adhesion (∼90 cells/HPF), and migration (73% scratch closure after 12 h and complete closure after 24 h). Also, L-KO25:KN75 is capable of eradicating both Gram-negative and Gram-positive bacteria after 18 h. According to in vivo assessment of L-KO25:KN75, wound closure at day 21 was >98% and microvessel density (>30 vessels/HPF at day 14) was significantly superior in comparison to other treatment groups. The mRNA expression of VEGF and IL-6 was also increased in the L-KO25:KN75-treated group and contributed to proper wound healing. Therefore, the LL-37-containing keratin hydrogel ameliorated wound closure, and also angiogenesis was enhanced as a result of LL-37 delivery. These results suggested that the L-KO25:KN75 hydrogel could be a sustainable substitute for skin tissue regeneration in medical applications.

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.

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.

Li-Doped Bioactive Ceramics: Promising Biomaterials for Tissue Engineering and Regenerative Medicine.

Lithium (Li) is a metal with critical therapeutic properties ranging from the treatment of bipolar depression to antibacterial, anticancer, antiviral and pro-regenerative effects. This element can be incorporated into the structure of various biomaterials through the inclusion of Li chloride/carbonate into polymeric matrices or being doped in bioceramics. The biocompatibility and multifunctionality of Li-doped bioceramics present many opportunities for biomedical researchers and clinicians. Li-doped bioceramics (capable of immunomodulation) have been used extensively for bone and tooth regeneration, and they have great potential for cartilage/nerve regeneration, osteochondral repair, and wound healing. The synergistic effect of Li in combination with other anticancer drugs as well as the anticancer properties of Li underline the rationale that bioceramics doped with Li may be impactful in cancer treatments. The role of Li in autophagy may explain its impact in regenerative, antiviral, and anticancer research. The combination of Li-doped bioceramics with polymers can provide new biomaterials with suitable flexibility, especially as bio-ink used in 3D printing for clinical applications of tissue engineering. Such Li-doped biomaterials have significant clinical potential in the foreseeable future.

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.

The anti-angiogenic effect of atorvastatin loaded exosomes on glioblastoma tumor cells: An in vitro 3D culture model.

Exosomes are endogenous nanoparticles with a lipid bilayer membrane whose natural function as carriers of biological materials has attracted much attention. The ability of exosomes to cross biological barriers, especially the blood-brain barrier, has highlighted them as tools of drug delivery to brain tumors. In a previous study, we isolated and characterized exosomes derived from human endometrial mesenchymal stem cells (hEnMSCs exosomes). In the present study, we used hEnMSCs exosomes as carriers for atorvastatin and investigated its pro-apoptotic and anti-angiogenic effects on U87 glioblastoma spheroids 3D co-cultured with Human Umbilical Vein Endothelial cells (HUVECs). In the study of HUVEC proliferation by using MTT assay, cell treatments with concentrations of 5 and 10 µM of free atorvastatin and atorvastatin-loaded hEnMSCs exosomes (AtoEXOs) showed significant differences in inhibition of proliferation compared to other concentrations. Also, 5 and 10 µM of AtoEXOs inhibited HUVEC migration in both scratch closure and transwell migration assays significantly more than that of free atorvastatin. In addition, in vitro HUVEC capillary tube network formation was inhibited by 5 and 10 µM treatment of AtoEXOs significantly more that of free atorvastatin. Moreover, a significant decrease in VEGF secretion and a significant increase in Bax/Bcl2 expression ratio were observed in U87 spheroids 3D co-cultured with HUVECs, especially for 10 µM AtoEXOs compared to other treated cell groups. Our results showed that hEnMSCs exosomes loaded with atorvastatin not only mimicked the anti-tumor effects of free atorvastatin but also potentiated its anti-tumor effects on glioblastoma cells. The enhanced pro-apoptotic and anti-angiogenic capabilities of atorvastatin loaded in hEnMSCs exosomes offer promising new perspectives for the treatment of glioblastoma.

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.