The effect of serum origin on cytokines induced killer cell expansion and function.

BACKGROUND: Cytokine-induced killer (CIK) cells have shown promising results in adoptive immunotherapy. However, serum may play a determining role in the large-scale expansion of these cells for clinical applications. According to Good Manufacturing Practice (GMP) guidelines to reduce the use of animal products in cell-based therapies; therefore, this study sought to investigate the impact of serum origin and the reduced serum concentration on the pattern of cell expansion and function. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from a healthy donor were expanded based on the CIK cell expansion protocol. The cell culture medium was supplemented with three types of sera comprising fetal bovine serum (FBS), human serum (HS), or human-derived platelet lysate (hPL) at different concentrations (10%, 5%, and 2.5%). The proliferation kinetics for each group were investigated for 30 days of cell culture. RESULTS: Cell proliferation in 10% concentration of all sera (hPL, FBS, HS) was higher than their lower concentrations. Moreover, hPL was significantly associated with higher expansion rates than FBS and HS in all three concentrations. Furthermore, cells cultured in hPL showed higher viability, cytotoxicity effect, and CIK CD markers expression. CONCLUSION: hPL at a concentration of 10% showed the best effect on CIK cell proliferation and function.

Optimizing interleukin-2 concentration, seeding density and bead-to-cell ratio of T-cell expansion for adoptive immunotherapy.

BACKGROUND: The successful ex vivo expansion of T-cells in great numbers is the cornerstone of adoptive cell therapy. We aimed to achieve the most optimal T-cell expansion condition by comparing the expansion of T-cells at various seeding densities, IL-2 concentrations, and bead-to-cell ratios. we first expanded the peripheral blood mononuclear cells (PBMCs) of a healthy donor at a range of 20 to 500 IU/mL IL-2 concentrations, 125 × 103 to 1.5 × 106 cell/mL, and 1:10 to 10:1 B:C (Bead-to-cell) ratios and compared the results. We then expanded the PBMC of three healthy donors using the optimized conditions and examined the growth kinetics. On day 28, CD3, CD4, and CD8 expression of the cell populations were analyzed by flow cytometry. RESULTS: T-cells of the first donor showed greater expansion results in IL-2 concentrations higher than 50 IU/mL compared to 20 IU/mL (P = 0.02). A seeding density of 250 × 103 cell/mL was superior to higher or lower densities in expanding T-cells (P = 0.025). Also, we witnessed a direct correlation between the B:C ratio and T-cell expansion, in which, in 5:1 and 10:1 B:C ratios T-cell significantly expanded more than lower B:C ratios. The results of PBMC expansions of three healthy donors were similar in growth kinetics. In the optimized condition, 96-98% of the lymphocyte population expressed CD3. While the majority of these cells expressed CD8, the mean expression of CD4 in the donors was 19.3, 16.5, and 20.4%. CONCLUSIONS: Our methodology demonstrates an optimized culture condition for the production of large quantities of polyclonal T-cells, which could be useful for future clinical and research studies.

An outlook on antigen-specific adoptive immunotherapy for viral infections with a focus on COVID-19.

Although not a standard-of-care yet, adoptive immunotherapeutic approaches have gradually earned a place within the list of antiviral therapies for some of fatal and hard-to-treat viral diseases. To maintain robust antiviral immunity and to effectively target the viral particles and virally-infected cells, immune cells capable of recognizing the viral antigens are required. While conventional vaccination can induce these cells in vivo; another option is to prime and generate antigen-specific immune cells ex vivo. This approach has been successfully trialed for virulent opportunistic viral infections after bone marrow transplantation. Amid the crisis of SARS-CoV2 pandemic, which has been followed by the success of certain early-authorized vaccines; some institutions and companies have explored the effects of viral-specific adoptive cell transfers (ACTs) in trials, as alternative treatments. Aimed at outlining a perspective on antigen-specific adoptive immunotherapy for viral infections, this review article specifically provides an appraisal of ACT-based studies/trials on SARS-CoV2 infection.

Effects of human placenta-derived mesenchymal stem cells with NK4 gene expression on glioblastoma multiforme cell lines.

Poor prognosis and low survival are commonly seen in patients with glioblastoma multiforme (GBM). Due to the specific nature of solid tumors such as GBM, delivery of therapeutic agents to the tumor sites is difficult. So, one of the major challenges in the treatment of these tumors is a selection of appropriate method for drug delivery. Mesenchymal stem cells (MSCs) have a unique characteristic in migration toward the tumor tissue. In this regard, the present study examined the antitumor effects of manipulating human placenta-derived mesenchymal stem cells (PDMSCs) with NK4 expression (PDMSC-NK4) on GBM cells. After separation and characterization of PDMSCs, these cells were transduced with NK4 which was known as the antagonist of hepatocyte growth factor (HGF). The results indicated that engineered PDMSCs preferably migrate into GBM cells by transwell coculture system. In addition, the proliferation of the GBM cells significantly reduced after coculture with these cells. In fact, manipulated PDMSCs inhibited growth of tumor cells by induction of apoptosis. Our findings suggested that besides having antitumor effects, PDMSCs can also be applied as an ideal cellular vehicle to target the glioblastoma multiforme.

MSLN (Mesothelin), ANTXR1 (TEM8), and MUC3A are the potent antigenic targets for CAR T cell therapy of gastric adenocarcinoma.

Gastric adenocarcinoma is usually diagnosed in late stages, necessitating the use of different therapeutic modalities. Currently, antibody-based therapies have also been approved through with limited clinical efficacy. Reinforcing antibody-based immunotherapy by using chimeric antigen receptor (CAR) T cells may enhance the approach. However, the cells can cause severe on-target and off-tumor toxicities owing to their higher sensitivity to low-level antigen expressions. To address the need for safe and reliable targets, we made a bioinformatics pipeline by which we screened overexpressed genes in the disease for off-tumor sites in many normal tissues. Our inspection showed that MSLN (Mesothelin), ANTXR1 (TEM8), and MUC3A are the probable targets of CAR T cell therapy in gastric adenocarcinoma. The proposed antigenic targets might respond to the need to simultaneously target multiple antigens in a tumor matrix to prevent resistance.

ANTXR1 (TEM8) overexpression in gastric adenocarcinoma makes the protein a potential target of immunotherapy.

BACKGROUND: Despite the promise of immunotherapy for gastric adenocarcinoma, choices for the selection of effective antigenic targets are very limited. Previously published data and our own in-house computational analysis have suggested that ANTXR1 is a potential target, simultaneously expressed in malignant tumor cells and the endothelial cells of the tumors. However, the expression pattern of ANTXR1 protein in clinical samples of gastric adenocarcinoma has not been fully evaluated. METHODS: Using immunohistochemistry (IHC), we recorded the percentage of ANTXR1 positive cells separately in tumor cells and endothelial cells in the primary tumor, non-tumor gastric tissue adjacent to the primary tumor, and tumor in metastatic sites of 140 gastric adenocarcinoma patients. We also evaluated the association of ANTXR1 expression with the Lauren histological classification of the primary tumors, the patient's history of neoadjuvant chemotherapy and/or radiotherapy, and the patient's overall survival. RESULTS: ANTXR1 was expressed in a mean of 73.89 ± 30.12% of tumor cells and 13.55 ± 20.53% of endothelial cells in the primary tumors. Intestinal adenocarcinomas had lower ANTXR1 expression in the tumor cells and higher ANTXR1 expression in the endothelial cells of the tumor regions, and a history of neoadjuvant therapy was associated with increased ANTXR1 expression in the endothelial cells of the tumor regions. Finally, above median expression of ANTXR1 in the tumor cells of the tumor regions was associated with significantly lower overall patient survival. CONCLUSIONS: Our findings suggest that ANTXR1 is a promising candidate for preclinical and clinical evaluation for gastric adenocarcinoma immunotherapy.

Safety and efficacy of allogenic placental mesenchymal stem cells for treating knee osteoarthritis: a pilot study.

OBJECTIVE: Knee osteoarthritis (OA) is a common skeletal impairment that can cause many limitations in normal life activities. Stem cell therapy has been studied for decades for its regenerative potency in various diseases. We investigated the safety and efficacy of intra-articular injection of placental mesenchymal stem cells (MSCs) in knee OA healing. METHODS: In this double-blind, placebo-controlled clinical trial, 20 patients with symptomatic knee OA were randomly divided into two groups to receive intra-articular injection of either 0.5-0.6 × 108 allogenic placenta-derived MSCs or normal saline. The visual analogue scale, Knee OA Outcome Score (KOOS) questionnaire, knee flexion range of motion (ROM) and magnetic resonance arthrography were evaluated for 24 weeks post-treatment. Blood laboratory tests were performed before and 2 weeks after treatment. RESULTS: Four patients in the MSC group showed mild effusion and increased local pain, which resolved safely within 48-72 h. In 2 weeks post-injection there was no serious adverse effect and all of the laboratory test results were unchanged. Early after treatment, there was a significant knee ROM improvement and pain reduction (effect size, 1.4). Significant improvements were seen in quality of life, activity of daily living, sport/recreational activity and decreased OA symptoms in the MSC-injected group until 8 weeks (P < 0.05). These clinical improvements were also noted in 24 weeks post-treatment but were not statistically significant. Chondral thickness was improved in about 10% of the total knee joint area in the intervention group in 24 weeks (effect size, 0.3). There was no significant healing in the medial/lateral meniscus or anterior cruciate ligament. There was no internal organ impairment at 24 weeks follow-up. CONCLUSION: Single intra-articular allogenic placental MSC injection in knee OA is safe and can result in clinical improvements in 24 weeks follow-up. TRIAL REGISTRATION NUMBER: IRCT2015101823298N.

Antigenic targets of CAR T Cell Therapy. A retrospective view on clinical trials.

Chimeric antigen receptor (CAR) T cell therapy is anticipated to be increasingly implemented in the context of cancer treatment after two current FDA approval of anti-CD19 CAR-T cells (Kymriah™ & Yescarta™). The success of CD19 is mainly attributable to the proper selection of the antigen, CD19, as the target of the disease, highlighting the importance of target selection for other CAR therapies. Therefore, here we performed a global analysis of targets that are the prime focus for various CAR T cell therapies in human clinical trials.

Addressing cancer immunotherapy research in Iran: adoptive cell therapy on the horizon.

Recent advances in immunotherapeutic modalities have profoundly changed the prospect of cancer treatment. These modalities mainly focus on modulating the immune response toward tumor cells by using monoclonal antibodies, cancer vaccines, adoptive cell transfer or combination of these methods. In the last few years, Iranian scientists have conducted several projects in these arenas. Here, we provide an overview of these studies and analyze the quality and trend of publications in each sub-specialty of the field. In addition, the contribution of different universities and scientific institutes is assessed. This study may benefit scientific community and policymakers to plan future cancer immunotherapies in Iran and other countries.

Osteoconduction of Unrestricted Somatic Stem Cells on an Electrospun Polylactic-Co-Glycolic Acid Scaffold Coated with Nanohydroxyapatite.

The limitation of traditional bone grafts could be overcome by applying engineered bone constructs, which are mainly produced by seeding suitable stem cells on appropriate scaffolds. So far, bone marrow-derived stromal cells have been the most applied cells in bone tissue engineering, but current data show that unrestricted somatic stem cells (USSCs) from human cord blood might actually be a better stem cell source due to the accessibility and noninvasive procedure of collection. In this study, we cultured USSCs on a plasma-treated electrospun polylactic-co-glycolic acid (PLGA) scaffold coated with nanohydroxyapatite (nHA). Adhesion and proliferation of USSCs on PLGA/nHA were assessed by scanning electron microscopy and MTT assay. Osteogenic differentiation of USSCs into osteoblast lineage cells was evaluated via alkaline phosphatase (ALP) activity and real-time polymerase chain reaction. Our observation showed that USSCs attached and proliferated on PLGA/nHA. Osteogenic differentiation was confirmed by increased ALP activity and OSTEONECTIN expression in USSCs on PLGA/nHA after the 1st week of the osteogenic period. Therefore, using USSCs on electrospun PLGA/nHA is a promising approach in bone tissue engineering.

Directly injected native bone-marrow stem cells cannot incorporate into acetaminophen-induced liver injury.

The paucity of liver donation highlights the use of cell-based strategies for end-stage liver failure. We recently showed that bone marrow-derived aggregates (BMDAs) can completely restore the hematopoietic system in gamma-irradiated mice. These aggregates are stem and progenitor cells in the bone marrow (BM), composed of both hematopoietic and non-hematopoietic lineages. Furthermore, reports showed that resident BM cells migrate to the liver and integrate themselves into the tissue in small numbers. Hence, we hypothesized that direct delivery of BMDAs to the damaged liver might enhance the integration of BM cells in the liver because of its stemness property, intact BM architecture, the physical proximity of these niche-like structures to the damaged sites and the existence of liver paracrine factors. To this aim, we made an acute liver model by intraperitoneal injection of acetaminophen. Then, GFP-expressing BMDAs were intrahepatically injected. Despite the detection of GFP-expressing cells five days after intrahepatic injection, these cells were not detectable at days 15 and 60, indicating that the puzzle of BM cell integration in the liver still has more missing pieces other than stemness, physical proximity, and paracrine factors. Actually, it seems that even intact BM structures need further signals to be qualified for integration.

A new design for electrospinner collecting device facilitates the removal of small diameter tubular scaffolds and paves the way for tissue engineering of capillaries.

Electrospinning is a technique widely used for tissue engineering. Despite hurdles, electrospun vascular tissue scaffolds has shown great promise in in vitro studies. One problem is the removal of tubular scaffolds from a electrospinning collection device with no unwanted crumpling or tearing, especially for small diameter scaffolds. To tackle this problem we designed a collection device for simple removal of the scaffold from the collector while no chemical pretreatment was required. The scaffolds fabricated on this collecting device maintained their tubular structure and showed favorable surface properties, mechanical strength and biocompatibility. The device offers a new opportunity for tissue engineering researchers to fabricate tubular scaffolds from materials which have not been possible to date and help them improve the quality of synthesized scaffolds.

A review of animal models utilized in preclinical studies of approved gene therapy products: trends and insights.

Scientific progress heavily relies on rigorous research, adherence to scientific standards, and transparent reporting. Animal models play a crucial role in advancing biomedical research, especially in the field of gene therapy. Animal models are vital tools in preclinical research, allowing scientists to predict outcomes and understand complex biological processes. The selection of appropriate animal models is critical, considering factors such as physiological and pathophysiological similarities, availability, and ethical considerations. Animal models continue to be indispensable tools in preclinical gene therapy research. Advancements in genetic engineering and model selection have improved the fidelity and relevance of these models. As gene therapy research progresses, careful consideration of animal models and transparent reporting will contribute to the development of effective therapies for various genetic disorders and diseases. This comprehensive review explores the use of animal models in preclinical gene therapy studies for approved products up to September 2023. The study encompasses 47 approved gene therapy products, with a focus on preclinical trials. This comprehensive analysis serves as a valuable reference for researchers in the gene therapy field, aiding in the selection of suitable animal models for their preclinical investigations.

Gene therapy in glioblastoma multiforme: Can it be a role changer?

Glioblastoma multiforme (GBM) is one of the most lethal cancers with a poor prognosis. Over the past century since its initial discovery and medical description, the development of effective treatments for this condition has seen limited progress. Despite numerous efforts, only a handful of drugs have gained approval for its treatment. However, these treatments have not yielded substantial improvements in both overall survival and progression-free survival rates. One reason for this is its unique features such as heterogeneity and difficulty of drug delivery because of two formidable barriers, namely the blood-brain barrier and the tumor-blood barrier. Over the past few years, significant developments in therapeutic approaches have given rise to promising novel and advanced therapies. Target-specific therapies, such as monoclonal antibodies (mAbs) and small molecules, stand as two important examples; however, they have not yielded a significant improvement in survival among GBM patients. Gene therapy, a relatively nascent advanced approach, holds promise as a potential treatment for cancer, particularly GBM. It possesses the potential to address the limitations of previous treatments and even newer advanced therapies like mAbs, owing to its distinct properties. This review aims to elucidate the current status and advancements in gene therapy for GBM treatment, while also presenting its future prospects.

Exploring non-viral methods for the delivery of CRISPR-Cas ribonucleoprotein to hematopoietic stem cells.

Gene manipulation of hematopoietic stem cells (HSCs) using the CRISPR/Cas system as a potent genome editing tool holds immense promise for addressing hematologic disorders. An essential hurdle in advancing this treatment lies in effectively delivering CRISPR/Cas to HSCs. While various delivery formats exist, Ribonucleoprotein complex (RNP) emerges as a particularly efficient option. RNP complexes offer enhanced gene editing capabilities, devoid of viral vectors, with rapid activity and minimized off-target effects. Nevertheless, novel delivery methods such as microfluidic-based techniques, filtroporation, nanoparticles, and cell-penetrating peptides are continually evolving. This study aims to provide a comprehensive review of these methods and the recent research on delivery approaches of RNP complexes to HSCs.

Progress and pitfalls of gene editing technology in CAR-T cell therapy: a state-of-the-art review.

CAR-T cell therapy has shown remarkable promise in treating B-cell malignancies, which has sparked optimism about its potential to treat other types of cancer as well. Nevertheless, the Expectations of CAR-T cell therapy in solid tumors and non-B cell hematologic malignancies have not been met. Furthermore, safety concerns regarding the use of viral vectors and the current personalized production process are other bottlenecks that limit its widespread use. In recent years the use of gene editing technology in CAR-T cell therapy has opened a new way to unleash the latent potentials of CAR-T cell therapy and lessen its associated challenges. Moreover, gene editing tools have paved the way to manufacturing CAR-T cells in a fully non-viral approach as well as providing a universal, off-the-shelf product. Despite all the advantages of gene editing strategies, the off-target activity of classical gene editing tools (ZFNs, TALENs, and CRISPR/Cas9) remains a major concern. Accordingly, several efforts have been made in recent years to reduce their off-target activity and genotoxicity, leading to the introduction of advanced gene editing tools with an improved safety profile. In this review, we begin by examining advanced gene editing tools, providing an overview of how these technologies are currently being applied in clinical trials of CAR-T cell therapies. Following this, we explore various gene editing strategies aimed at enhancing the safety and efficacy of CAR-T cell therapy.

Human unrestricted somatic stem cell administration fails to protect nude mice from cisplatin-induced acute kidney injury.

BACKGROUND: Kidney failure is a debilitating disorder with limited treatment options. The kidney-protective effects of stem cells have been vastly investigated and promising results have been achieved with various sources of stem cells. However, in spite of beneficial effects on other disease models, the renoprotective potential of human cord blood-derived unrestricted somatic stem cells (USSC) has not been examined so far. METHODS: In the present study, acute kidney failure was induced in female nude mice and the effect of USSC transplantation on kidney function and structure was assessed. Furthermore, the expression of some cytokine genes was examined by real-time PCR. Homing of the transplanted cells into kidneys was assessed by flow cytometry, immunohistochemistry, and real-time PCR. RESULTS: USSC-conditioned medium did not attenuate the in vitro nephrotoxic effects of cisplatin. Transplantation of USSC to nude mice did not protect kidney function and was associated with worsened kidney structural damage. USSC transplantation was also associated with a decline in the renal expression of VEGF-A gene. In spite of these effects, the transplanted cells could not be detected in the kidneys by any of the exploited methods and they were mainly entrapped in the lungs. CONCLUSION: These data indicate that USSC are not suitable for cell therapy in the setting of acute kidney injury. Also, this study shows that these stem cells are able to affect damaged kidneys even if they are not homed there.

Analysis of microRNA signatures using size-coded ligation-mediated PCR.

The expression pattern and regulatory functions of microRNAs (miRNAs) are intensively investigated in various tissues, cell types and disorders. Differential miRNA expression signatures have been revealed in healthy and unhealthy tissues using high-throughput profiling methods. For further analyses of miRNA signatures in biological samples, we describe here a simple and efficient method to detect multiple miRNAs simultaneously in total RNA. The size-coded ligation-mediated polymerase chain reaction (SL-PCR) method is based on size-coded DNA probe hybridization in solution, followed-by ligation, PCR amplification and gel fractionation. The new method shows quantitative and specific detection of miRNAs. We profiled miRNAs of the let-7 family in a number of organisms, tissues and cell types and the results correspond with their incidence in the genome and reported expression levels. Finally, SL-PCR detected let-7 expression changes in human embryonic stem cells as they differentiate to neuron and also in young and aged mice brain and bone marrow. We conclude that the method can efficiently reveal miRNA signatures in a range of biological samples.

The paths and challenges of "off-the-shelf" CAR-T cell therapy: An overview of clinical trials.

The advent of chimeric antigen receptor T cells (CAR-T cells) has made a tremendous revolution in the era of cancer immunotherapy, so that since 2017 eight CAR-T cell products have been granted marketing authorization. All of these approved products are generated from autologous sources, but this strategy faces several challenges such as time-consuming and expensive manufacturing process and reduced anti-tumor potency of patients' T cells due to the disease or previous therapies. The use of an allogeneic source can overcome these issues and provide an industrial, scalable, and standardized manufacturing process that reduces costs and provides faster treatment for patients. Nevertheless, for using allogeneic CAR-T cells, we are faced with the challenge of overcoming two formidable impediments: severe life-threatening graft-versus-host-disease (GvHD) caused by allogeneic CAR-T cells, and allorejection of allogeneic CAR-T cells by host immune cells which is called "host versus graft" (HvG). In this study, we reviewed recent registered clinical trials of allogeneic CAR-T cell therapy to analyze different approaches to achieve a safe and efficacious "off-the-shelf" source for chimeric antigen receptor (CAR) based immunotherapy.

Challenges in Mesenchymal Stromal Cell-based Therapies.

Over 50 years have passed since discovering mesenchymal stromal cells (MSCs). Initially, despite gaps in the knowledge of the identity of these cells, their therapeutic aspects were recognized. Consequently, MSCs became candidates for treating a wide range of diseases. However, the therapeutic effects of MSCs are not stable in the long term, and there are inconsistent data on their clinical efficacy. Even though more than 1000 MSC-based clinical trials have been registered, and the safety of MSCbased cell therapies has been proven, data on the clinical efficacy of MSCs have not been enough to warrant FDA approval for clinical treatment and marketing purposes. The available information on MSCs still contains some controversies, perhaps owing to little progress in understanding their in vivo identity. MSCs have been used for therapeutic purposes despite poor knowledge of their in vivo origin or functions. Hence, perhaps we need to go back to the basics of MSCs and spend more time understanding the biology of these cells. An improved understanding of MSCs' location and function within tissues may improve their therapeutic efficacy and, consequently, their establishment as a cell therapy product.