Histological assessment for investigation of dose-dependent ovarian toxicity of cyclophosphamide in the rat.

Background: Cyclophosphamide (CPA) have significant effects on ovarian follicles which lead to ovarian toxicity and impair the normal female reproductive function. This study aimed to evaluate the dose-dependent effects of CPA on rat follicle numbers. Methods: The experimental groups consisted of rats administered a single intraperitoneal injection of CPA at doses of either 50, 75,150, or 200 mg/kg followed by daily doses of 8 mg/kg for 14 days and control group given no treatment. After the treatment period, the histological evaluation was done. Results: Primordial and primary follicles were affected by all doses of CPA, but differential follicle counts revealed that graaf and preantral follicles were most sensitive to CPA, followed by primary and primordial follicles. The greatest reduction in all type of studied follicles caused by CPA doses of 50 mg/kg. Conclusion: Differential follicle counts revealed that CPA-induced ovarian toxicity is exhibited in structural feature of the ovary, particularly in destruction of graaf and preantral follicles in a dose-dependent manner so that the highest decrease in all type of studied follicles caused by 50 mg/kg of CPA and is suggested as the best concentration for ovotoxicity induction. These findings give insight into ovarian response to structural disruption of folliculogenesis.

Therapeutic potential of endometrial stem cells encapsulated in alginate/gelatin hydrogel to treat of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder in women, often leading to infertility due to anovulation. Recent advances suggest that endometrial stem cells (EnSCs) hold considerable promise for tissue regeneration, which could be pivotal in treating PCOS. To enhance the survival and stabilization of EnSCs within the ovary, the EnSCs were encapsulated in an injectable alginate/gelatin hydrogel (SC-H), which has excellent biocompatibility to support the survival of EnSCs. Polycystic ovary syndrome was induced in female Wistar rats using intraperitoneal injection of letrozole over 21 days. Then the rats were treated with SC, SC-H and clomiphene citrate for one-month post-PCOS induction. The effects of these treatments were evaluated based on changes in body and ovarian weights, inflammatory markers, endocrine profiles, and ovarian histology. The Induction of PCOS led to a significant increase in body and ovarian cyst weight, elevated serum levels of testosterone, luteinizing hormone (LH), and anti-Müllerian hormone (AMH), alongside reduced follicle-stimulating hormone (FSH) and progesterone levels. Histologically, there was a decrease in granulosa cells, immature follicles, and corpus luteum numbers. Treatment with SC and SC-H significantly mitigated these alterations, indicating improved PCOS conditions. Our findings demonstrate that SC and SC-H treatments can effectively ameliorate the symptoms of letrozole-induced PCOS in rats, primarily through their anti-inflammatory effects. This study lays the groundwork for potential clinical applications of EnSCs encapsulated in alginate/gelatin hydrogel as a novel therapeutic strategy for PCOS, highlighting the importance of biomaterials in stem cell-based therapies.

Fabrication and Evaluation of a Soy Protein Isolate/Collagen/Sodium Alginate Multifunctional Bilayered Wound Dressing: Release of Cinnamaldehyde, Artemisia absinthium, and Oxygen.

Chronic wounds, such as diabetic ulcers and pressure sores, pose significant challenges in modern healthcare due to their prolonged healing times and susceptibility to infections. This study aims to engineer a bilayered wound dressing (BLWD) composed of soy protein isolate/collagen with the ability to release Cinnamaldehyde, Artemisia absinthium (AA), and oxygen. Cinnamaldehyde, magnesium peroxide (MgO2), and AA extract were encapsulated. Nanoparticles were evaluated using scanning electron microscopy (SEM), dynamic light scattering, and ZETA potential tests. Swelling, degradation, water vapor penetration, tensile, MTT, SEM, oxygen release, AA extract release, and antibacterial properties were performed. An in vivo study was carried out to assess the final wound dressing under Hematoxiline&Eosin and Masson trichrome staining analysis and compared to a commercial product. According to the results, the synthesized nanoparticles had an average diameter of about 20 nm with a zeta potential in the range of -20 to -30 mV. The layers had uniform and dense surfaces. The maximum swelling and degradation of the dressing was about 130 and 13% respectively. Generally, better mechanical properties were observed in BLWD than in the single-layer case. More than 90% biocompatibility for the wound dressing was reported. The BLWD could inhibit the growth of Gram-positive and Gram-negative microorganisms. Histopathological analysis showed an acceptable wound-healing property. To sum up, the engineered wound dressing can be a good candidate for more clinical trials.

Evaluation of the effect of co-transplantation of collagen-hydroxyapatite bio-scaffold containing nanolycopene and human endometrial mesenchymal stem cell derived exosomes to regenerate bone in rat critical size calvarial defect.

This study aimed to evaluate the effect of nanoparticles based on the PLGA and biomolecule of lycopene (i.e. NLcp) and exosomes loaded on hydroxyapatite/collagen-based scaffolds (HA/Coll), on human endometrial MSCs (hEnMSCs) differentiation into osteoblast cells. To this end, after synthesizing NLcp and isolating hEnMSC-derived exosomes, and studying their characterizations, HA/Coll scaffold with/without NLcp and exosome was fabricated. In following, the rat skull-defect model was created on 54 male Sprague-Dawley rats (12 weeks old) which were classified into 6 groups [control group (4 healthy rats), negative control group: bone defect without grafting (10 rats), and experimental groups including bone defect grafted with HA/Coll scaffold (10 rats), HA/Coll/NLcp scaffold (10 rats), HA/Coll scaffold + exosome (10 rats), and HA/Coll-NLcp scaffold + exosome (10 rats)]. Finally, the grafted membrane along with its surrounding tissues was removed at 90 days after surgery, to assess the amount of defect repair by Hematoxylin and eosin staining. Moreover, immunohistochemical and X-ray Micro-Computed Tomography (Micro-CT) analyses were performed to assess osteocalcin and mean bone volume fraction (BVF). Based on the results, although, the existence of the exosome in the scaffold network can significantly increase mean BVF compared to HA/Coll scaffold and HA/Coll-NLcp scaffold (2.25-fold and 1.5-fold, respectively). However, the combination of NLcp and exosome indicated more effect on mean BVF; so that the HA/Coll-NLcp scaffold + exosome led to a 15.95 % increase in mean BVF than the HA/Coll scaffold + exosome. Hence, synthesized NLcp in this study can act as a suitable bioactive to stimulate the osteogenic, promotion of cell proliferation and its differentiation when used in the polymer scaffold structure or loaded into polymeric carriers containing the exosome.

Injectable multifunctional hydrogel containing Sphingosine 1-phosphate and human acellular amniotic membrane for skin wound healing.

Objectives: The skin serves as the main defense barrier, protecting against injuries, and preventing infection and water loss. Consequently, wound healing and skin regeneration are crucial aspects of wound management. A novel hydrogel scaffold was developed by incorporating carboxymethyl cellulose (CMC) and gelatin (Gel) hydrogels cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) containing Sphingosine 1-phosphate (S1P). This hydrogel is applied topically to treat acute wounds and is covered with a human acellular amniotic membrane (hAAM) as a secondary dressing. Materials and Methods: The scaffold was subjected to in vitro cell viability, red blood cell hemolysis, blood clotting index, and in vivo assays. Real-time PCR was implemented to verify the expression of genes involved in skin wounds. The physical and chemical properties of the scaffolds were also tested using weight loss, swelling ratio, scanning electron microscopy (SEM), Fourier transform infrared (FTIR), and mechanical tensile analysis. Results: The synthetic scaffold is biocompatible as evidenced by the high percentage of 3T3 cell viability (127%) after 72 hr. Additionally, excellent hemocompatibility with a low hemolytic effect (2.26%) was observed. Our in vivo wound healing assay demonstrated that CMC/Gel/S1P/hAAM wound dressing led to faster wound healing in treated rats compared to the control group over 14.Also, the mechanical tests showed that the amniotic membrane and the hAAM had very different Young's modulus and elongation at break values. Conclusion: This study demonstrates the effectiveness of the CMC/Gel/EDC hydrogel with S1P as a wound dressing. Additionally, hAAM exhibits excellent characteristics as a protective layer for the treatment of acute wounds.

3D-bioprinted GelMA/gelatin/amniotic membrane extract (AME) scaffold loaded with keratinocytes, fibroblasts, and endothelial cells for skin tissue engineering.

Gelatin-methacryloyl (GelMA) is a highly adaptable biomaterial extensively utilized in skin regeneration applications. However, it is frequently imperative to enhance its physical and biological qualities by including supplementary substances in its composition. The purpose of this study was to fabricate and characterize a bi-layered GelMA-gelatin scaffold using 3D bioprinting. The upper section of the scaffold was encompassed with keratinocytes to simulate the epidermis, while the lower section included fibroblasts and HUVEC cells to mimic the dermis. A further step involved the addition of amniotic membrane extract (AME) to the scaffold in order to promote angiogenesis. The incorporation of gelatin into GelMA was found to enhance its stability and mechanical qualities. While the Alamar blue test demonstrated that a high concentration of GelMA (20%) resulted in a decrease in cell viability, the live/dead cell staining revealed that incorporation of AME increased the quantity of viable HUVECs. Further, gelatin upregulated the expression of KRT10 in keratinocytes and VIM in fibroblasts. Additionally, the histological staining results demonstrated the formation of well-defined skin layers and the creation of extracellular matrix (ECM) in GelMA/gelatin hydrogels during a 14-day culture period. Our study showed that a 3D-bioprinted composite scaffold comprising GelMA, gelatin, and AME can be used to regenerate skin tissues.

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.

Tissue engineering and stem cell-based therapeutic strategies for premature ovarian insufficiency.

Premature ovarian insufficiency (POI), also known as premature ovarian failure (POF), is a complex endocrine disease that commonly affects women under the age of 40. It is characterized by the cessation of ovarian function before the age of 40, leading to infertility and hormonal imbalances. The currently available treatment options for POI are limited and often ineffective. Tissue engineering and stem cell-based therapeutic strategies have emerged as promising approaches to restore ovarian function and improve the quality of life for women affected by POI. This review aims to provide a comprehensive overview of the types of stem cells and biomaterials used in the treatment of POI, including their biological characteristics and mechanisms of action. It explores various sources of stem cells, including embryonic stem cells, induced pluripotent stem cells, and adult stem cells, and their potential applications in regenerating ovarian tissue. Additionally, this paper discusses the development of biomaterials and scaffolds that mimic the natural ovarian microenvironment and support the growth and maturation of ovarian cells and follicles. Furthermore, the review highlights the challenges and ethical considerations associated with tissue engineering and stem cell-based therapies for POI and proposes potential solutions to address these issues. Overall, this paper aims to provide a comprehensive overview of the current state of research in tissue engineering and stem cell-based therapeutic strategies for POI and offers insights into future directions for improving treatment outcomes in this debilitating condition.

Targeted expression of a designed fusion protein containing BMP2 into the lumen of exosomes.

BACKGROUND: Exosomes are 30-150 nm membrane vesicles, originating from the endocytic pathway. By acting as natural carriers of biomolecules, they can transfer various materials to recipient cells. Therefore, discovering novel strategies for cargo packaging into exosomes is crucial. METHODS: The fusion constructs, consisting of protein of interest (BMP2) along with the targeting motif, linkers, tracking proteins, and enzyme cleavage sites, were computationally designed. Following the homology modeling, the best structure was selected and subjected to molecular dynamics (MD) simulation and docking analyses. The fusion protein gene was expressed in the HEK-293LTV cell line. The high-efficiency transfected and transduced cells were screened and their exosomes were isolated. Finally, cell and exosome lysates were evaluated for expression of the fusion protein. RESULTS: A total of 12 constructs with lengths ranging from 483 to 496 were designed. The top three templates, 1REW, 2H5Q, and 2MOF were screened. MD simulation and docking analyses of the structures revealed their stability and functionality. In the protein expression analyses, three bands at sizes of approximately 60, 25, and 12.5 kDa were observed, consistent with the sizes of the complete fusion protein, dimeric, and monomeric BMP2 protein. The presence of a 12.5 kDa band at exosome lysate analysis might suggest that it was loaded and cleaved inside exosomes. CONCLUSION: In summary, these findings revealed that the proposed idea for cargo sorting within the exosome lumen through incorporating an appropriate cleavage site was effective, thus providing further insight into the potential of exosomes as nano-shuttles bearing therapeutic biomolecules.

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.

A Review on Treatment of Premature Ovarian Insufficiency: Characteristics, Limitations, and Challenges of Stem Cell versus ExosomeTherapy.

Premature ovarian insufficiency (POI) is a complex disorder that can result in varying degrees of infertility. Recently, mesenchymal stem cell (MSC) therapy and its derivatives, such as exosomes, have been introduced as novel strategies for the treatment of POI. This review discusses the features, limitations, and challenges of MSC and exosome therapy in the treatment of POI and provides readers with new insights for comparing and selecting chemical agents, optimizing doses, and other factors involved in study design and treatment strategies. MSC therapy has been shown to improve ovarian function in some animals with POI, but it can also have side effects such as high cost, time-consuming processes, limited lifespan and cell sources, loss of original characteristics during in vitro proliferation, dependence on specific culture environments, potential immune reactions, unknown therapeutic mechanisms, etc. However, exosome therapy is a newer therapy that has not been studied as extensively as MSC therapy, but that it has shown some promise in animal studies. The evidence for the effectiveness of MSC and exosome therapy is still limited, and more research is needed to determine whether these therapies are effective and safe for women with POI. This study presents a new perspective for researchers to advance their research in the fields of cell-based and cell-free therapies.

Tissue engineering studies in male infertility disorder.

Infertility is an important issue among couples worldwide which is caused by a variety of complex diseases. Male infertility is a problem in 7% of all men. In vitro spermatogenesis (IVS) is the experimental approach that has been developed for mimicking seminiferous tubules-like functional structures in vitro. Currently, various researchers are interested in finding and developing a microenvironmental condition or a bioartificial testis applied for fertility restoration via gamete production in vitro. The tissue engineering (TE) has developed new approaches to treat male fertility preservation through development of functional male germ cells. This makes TE a possible future strategy for restoration of male fertility. Although 3D culture systems supply the perception of the effect of cellular interactions in the process of spermatogenesis, formation of a native gradient of autocrine/paracrine factors in 3D culture systems have not been considered. These results collectively suggest that maintaining the microenvironment of testicular cells even in the form of a 3D-culture system is crucial in achieving spermatogenesis ex vivo. It is also possible to engineer the testicular structures using biomaterials to provide a supporting scaffold for somatic and stem cells. The insemination of these cells with GFs is possible for temporally and spatially adjusted release to mimic the microenvironment of the in situ seminiferous epithelium. This review focuses on recent studies and advances in the application of TE strategies to cell-tissue culture on synthetic or natural scaffolds supplemented with growth factors.

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.

Enhanced spinal cord regeneration by gelatin/alginate hydrogel scaffolds containing human endometrial stem cells and curcumin-loaded PLGA nanoparticles in rat.

Spinal cord injury (SCI) is a serious problem with a high prevalence worldwide. The weak capability of the spinal cord for regeneration in association with upregulation of inflammatory factors is two key obstacles against a full SCI repair. Curcumin is a natural substance with anti-inflammatory and neuroprotective effects. Here, we have used a combined strategy using stem cells and hybrid hydrogel scaffolds loaded with curcumin for SCI repair. Curcumin-loaded PLGA nanoparticles were prepared, characterized, and encapsulated into gelatin/alginate hydrogel scaffolds, which were then seeded by human endometrial stem cells (hEnSCs). The resulting construct was studied using in vitro and in vivo experiments on rat models. DLS, SEM, Zeta potential, and FTIR data confirmed the successful addition of curcumin to PLGA nanoparticles. SEM analyses indicated the successful addition of curcumin-loaded nanoparticles into the gelatin/alginate scaffold, as well as the adherence of the seeded EnSCs. Based on the results, the prepared constructs not only allowed the controlled release of curcumin but also could support the survival and growth of hEnSCs. Based on the results of BBB and histological experiments, the highest BBB score was related to the combined strategy, consistent with histological outcomes, in which our hEnSC-seeded gelatin/alginate scaffold containing curcumin-loaded nanoparticles led to improved structures of the white and gray matters in the SCI site, being indicative of the superior nerve fiber regeneration, compared to other studied groups. These results indicate the efficiency of the proposed method for SCI repair and broaden the scope for subsequent studies on spinal cord regeneration.

Differentiation of human endometrial stem cells encapsulated in alginate hydrogel into oocyte-like cells.

Introduction: Human endometrial mesenchymal stem cells (hEnMSCs) are a rich source of mesenchymal stem cells (MSCs) with multi-lineage differentiation potential, making them an intriguing tool in regenerative medicine, particularly for the treatment of reproductive and infertility issues. The specific process of germline cell-derived stem cell differentiation remains unknown, the aim is to study novel ways to achieve an effective differentiation method that produces adequate and functioning human gamete cells. Methods: We adjusted the optimum retinoic acid (RA) concentration for enhancement of germ cell-derived hEnSCs generation in 2D cell culture after 7 days in this study. Subsequently, we developed a suitable oocyte-like cell induction media including RA and bone morphogenetic protein 4 (BMP4), and studied their effects on oocyte-like cell differentiation in 2D and 3D cell culture media utilizing cells encapsulated in alginate hydrogel. Results: Our results from microscopy analysis, real-time PCR, and immunofluorescence tests revealed that 10 µM RA concentration was the optimal dose for inducing germ-like cells after 7 days. We examined the alginate hydrogel structural characteristics and integrity by rheology analysis and SEM microscope. We also demonstrated encapsulated cell viability and adhesion in the manufactured hydrogel. We propose that in 3D cell cultures in alginate hydrogel, an induction medium containing 10 µM RA and 50 ng/mL BMP4 can enhance hEnSC differentiation into oocyte-like cells. Conclusion: The production of oocyte-like cells using 3D alginate hydrogel may be viable in vitro approach for replacing gonad tissues and cells.