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ABSTRACT Natural Killer cells are immune leukocytes required for responses against tumor cells and virus-infected cells. In the last decade, natural killer cells have emerged as promising tools in cancer therapy, and clinical studies on patients treated with natural killer cells have revealed increased rates of disease-free survival. In this article, we review results from the major clinical trials that have used natural killer cells for cancer treatment, including their global distribution. We also discuss the major mechanisms of natural killer cell activation and expansion and focus on the advantages and disadvantages of each mechanism for clinical applications. Although natural killer cells can be isolated from several sources, primary natural killer cells are most commonly used in clinical trials. However, the frequency of natural killer cells available in peripheral and cord blood is low, necessitating development of methods for expansion of natural killer cells for clinical use. The development of a platform for the expansion of large-scale good manufacturing practice-compliant natural killer cells has limitations as several methods for natural killer cell activation and expansion yield conflicting results. Only techniques using feeder cells can produce large numbers of cells, allowing the “off-the-shelf” use of natural killer cells. However, advances in cell culture have supported the development of feeder-free platforms for natural killer cell expansion, which is fundamental for improving the safety of this type of cell therapy.

Mucopolysaccharidosis type I (MPS I) is a metabolic disorder characterized by a deficiency in α-l-iduronidase (IDUA), leading to impaired glycosaminoglycan degradation. Current approved treatments seek to restore IDUA levels via enzyme replacement therapy (ERT) and/or hematopoietic stem cell transplantation (HSCT). The effectiveness of these treatment strategies in preventing neurodegeneration is limited due to the inability of ERT to penetrate the blood-brain barrier (BBB) and HSCT’s limited CNS reconstitution of IDUA levels. We reprogrammed human cord blood cells into induced pluripotent stem cells (iPSCs), differentiated them into human induced neural stem cells (hiNSCs), and sorted them using fluorescence-activated cell sorting (FACS). Our in vitro studies showed that these hiNSCs can migrate and cross-correct IDUA deficiency. Purified hiNSCs were then transplanted into neonatal immunodeficient MPS I mice (Idua−/−). Analysis of brain tissue obtained 8 months after transplantation showed partially restored IDUA activity, with distribution and differentiation of engrafted hiNSCs throughout the brain into glial cell types. The presence of engrafted hiNSCs was associated with decreased levels of biomarkers commonly elevated in the Idua−/− mouse brain, such as β-hexosaminidase, CD68, and LAMP1, suggesting physiological efficacy. These results highlight the potential of hiNSCs for use as a patient-specific cellular therapy for MPS I.

Nanorobots, also known as nanobots, are promising medical structures with significant potential for revolutionizing medical treatments and preventing their onset. Nanorobotics shows promise for diagnosing, treating, and regenerating age-related diseases. These tiny machines can repair cellular damage, deliver drugs, stimulate tissue regeneration, and monitor treatments. However, their application in controlling cellular aging is in its early stages, requiring more research to fully understand their potential and address safety concerns. Despite this, the benefits of nanorobots in age-related disease management justify further investigation. This narrative review article provides an overview of recent developments in nanorobotics, specifically focusing on its medical applications. PubMed and Google Scholar were utilized to conduct a comprehensive search using relevant keywords including "nanorobots", "cellular aging", "drug delivery systems", "cellular repair", "therapeutics", and "diagnosis". Only articles published between 2005-2023 were selected to support the arguments with up-to-date evidence. The extracted information includes nanorobot design, fabrication methods, applications, and key findings. A quality assessment was performed, and the data were categorized thematically to identify patterns and trends. The article concludes with an examination of the current state and future prospects of nanorobotics in different fields. Our research showed that nanorobots have significant potential in the diagnosis, treatment, and regenerative medicine of age-related diseases. They can detect and repair cellular damage caused by aging, targeting DNA damage and cellular protein misfolding. Nanorobots can also deliver drugs directly to affected areas, reducing side effects. Furthermore, they stimulate tissue regeneration and the growth of new blood vessels by delivering growth factors. Nanorobots can also monitor treatment effectiveness by measuring drug concentration in tumor cells. However, there are concerns about their safety in long-term use that require further research. Overall, nanorobots present an innovative approach to addressing cellular changes in aging, but more research is needed to understand their full potential and address safety concerns. The use of nanorobots in treating cellular aging has opened new possibilities in diagnosing and treating age-related diseases and it is a rapidly evolving field with the potential to revolutionize age-related disease treatment and improve the quality of life globally.

Exosomes are nano-sized membrane-bound extracellular vesicles containing various biological molecules, such as nucleic acids, proteins, and lipids, which can be used to modulate physiological processes. The exosomal molecules secreted by cells can be extensively used as tools for diagnosis and therapy. Exosomes carry specific molecules released by the cells they originate from, which can be transferred to surrounding cells or tissues by the exosome. For these reasons, exosomes can be exploited as biomarkers for diagnosis, carriers for drug delivery, as well as therapeutics. In stem cell technology, exosomes have been an attractive option because they can be used as safer therapeutic agents for stem cell-based cell-free therapy. Recently, studies have demonstrated the safety and efficacy of mesenchymal stem cell-derived exosomes in alleviating symptoms associated with coronavirus disease 2019 as they have anti-inflammatory and immunomodulatory potential. Performing multiple studies on exosomes would provide innovative next-generation options for clinical diagnostics and therapy. This review summarizes the use of exosomes focusing on their diverse roles. In addition, the potential of exosomes is illustrated with a focus on how exosomes can be exploited as powerful tools in the days to come.

Abstract Background Chimeric antigen receptor CAR-T cell therapies have ushered in a new era of treatment for specific blood cancers, offering unparalleled efficacy in cases of treatment resistance or relapse. However, the emergence of cytokine release syndrome (CRS) as a side effect poses a challenge to the widespread application of CAR-T cell therapies. Melatonin, a natural hormone produced by the pineal gland known for its antioxidant and anti-inflammatory properties, has been explored for its potential immunomodulatory effects. Despite this, its specific role in mitigating CAR-T cell-induced CRS remains poorly understood. Methods In this study, our aim was to investigate the potential of melatonin as an immunomodulatory agent in the context of CD19-targeting CAR-T cell therapy and its impact on associated side effects. Using a mouse model, we evaluated the effects of melatonin on CAR-T cell-induced CRS and overall survival. Additionally, we assessed whether melatonin administration had any detrimental effects on the antitumor efficacy and persistence of CD19 CAR-T cells. Results Our findings demonstrate that melatonin effectively mitigated the severity of CAR-T cell-induced CRS in the mouse model, leading to improved overall survival outcomes. Remarkably, melatonin administration did not compromise the antitumor effectiveness or persistence of CD19 CAR-T cells, indicating its compatibility with therapeutic goals. These results suggest melatonin's potential as an immunomodulatory compound to alleviate CRS without compromising the therapeutic benefits of CAR-T cell therapy. Conclusion The study's outcomes shed light on melatonin's promise as a valuable addition to the existing treatment protocols for CAR-T cell therapies. By attenuating CAR-T cell-induced CRS while preserving the therapeutic impact of CAR-T cells, melatonin offers a potential strategy for optimizing and refining the safety and efficacy profile of CAR-T cell therapy. This research contributes to the evolving understanding of how to harness immunomodulatory agents to enhance the clinical application of innovative cancer treatments.

In December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China. This virus rapidly spread worldwide and was declared a global pandemic by the World Health Organization (WHO) in March 2020. High incidence, long incubation period, and diverse clinical signs of the disease posed a huge challenge globally. The efforts of health systems have been focused on repurposing existing drugs or developing innovative therapies to reduce the morbidity and mortality associated with SARS-CoV-2. In addition, most of the large pharmaceutical companies are intensely working on vaccine development to swiftly deliver safe and effective vaccines to prevent further spread of the virus. In this review, we will discuss the latest data on therapeutic strategies undergoing clinical trials. Additionally, we will provide a summary of vaccines currently under development.

The paradigm of pediatric drug development has been evolving in a “carrot-and-stick”-based tactic to address population-specific issues. However, the off-label prescription of adult medicines to pediatric patients remains a feature of clinical practice, which may compromise the age-appropriate evaluation of treatments. Therefore, the United States and the European Pediatric Formulation Initiative have recommended applying nanotechnology-based delivery systems to tackle some of these challenges, particularly applying inorganic, polymeric, and lipid-based nanoparticles. Connected with these, advanced therapy medicinal products (ATMPs) have also been highlighted, with optimistic perspectives for the pediatric population. Despite the results achieved using these innovative therapies, a workforce that congregates pediatric patients and/or caregivers, healthcare stakeholders, drug developers, and physicians continues to be of utmost relevance to promote standardized guidelines for pediatric drug development, enabling a fast lab-to-clinical translation. Therefore, taking into consideration the significance of this topic, this work aims to compile the current landscape of pediatric drug development by (1) outlining the historic regulatory panorama, (2) summarizing the challenges in the development of pediatric drug formulation, and (3) delineating the advantages/disadvantages of using innovative approaches, such as nanomedicines and ATMPs in pediatrics. Moreover, some attention will be given to the role of pharmaceutical technologists and developers in conceiving pediatric medicines.

Advanced therapy medicinal products (ATMPs) are innovative therapies that mainly target orphan diseases and high unmet medical needs. The uncertainty about the product's benefit-risk balance at the time of approval, the limitations of nonclinical development, and the complex quality aspects of those highly individualized advanced therapies are playing a key role in the clinical development, approval, and post-marketing setting for these therapies. This article reviews the current landscape of clinical development of advanced therapies, its challenges, and some of the efforts several stakeholders are conducting to move forward within this field. Progressive iteration of the science, methodologically sound clinical developments, establishing new standards for ATMPs development with the aim to ensure consistency in clinical development, and the reproducibility of knowledge is required, not only to increase the evidence generation for approval but to set principles to achieve translational success in this field.

Abstract Cell therapies for neonatal morbidities are progressing to early phase clinical trials. However, protocols for intravenous (IV) delivery of cell therapies to infants have not been evaluated. It has been assumed the cell dose prescribed is the dose delivered. Early in our clinical trial of human amnion epithelial cells (hAECs), we observed cells settling in the syringe and IV tubing used to deliver the suspension. The effect on dose delivery was unknown. We aimed to quantify this observation and determine an optimal protocol for IV delivery of hAECs to extremely preterm infants. A standard pediatric infusion protocol was modeled in the laboratory. A syringe pump delivered the hAEC suspension over 60 minutes via a pediatric blood transfusion set (200‐μm filter and 2.2 mL IV line). The infusion protocol was varied by agitation methods, IV‐line volumes (0.2‐2.2 mL), albumin concentrations (2% vs 4%), and syringe orientations (horizontal vs vertical) to assess whether these variables influenced the dose delivered. The influence of flow rate (3‐15 mL/h) was assessed after other variables were optimized. The standard infusion protocol delivered 17.6% ± 9% of the intended hAEC dose. Increasing albumin concentration to 4%, positioning the syringe and IV line vertically, and decreasing IV‐line volume to 0.6 mL delivered 99.7% ± 13% of the intended hAEC dose. Flow rate did not affect dose delivery. Cell therapy infusion protocols must be considered. We describe the refinement of a cell infusion protocol that delivers intended cell doses and could form the basis of future neonatal cell delivery protocols.

ABSTRACT Objective Phase 1 clinical trial to determine feasibility, safety, and efficacy of a new advanced cell therapy product for treatment of knee articular cartilage injuries. Methods Three participants with knee focal chondral lesions were included, with no signs of osteoarthritis. Chondrocytes were obtained through knee arthroscopy, cultured in collagen membrane for 3 weeks at the laboratory, subjected to tests to release the cell therapy product, and implanted. All patients underwent a specific 3-month rehabilitation protocol, followed by assessments using functional and imaging scales. The main outcome was the incidence of severe adverse events. Results Three participants were included and completed the 2-year follow-up. There was one severe adverse event, venous thrombosis of distal leg veins, which was no associated with therapy, was treated and left no sequelae. The clinical and radiological scales showed improvement in the three cases. Conclusion The preliminary results, obtained with the described methodology, allow concluding that this product of advanced cell therapy is safe and feasible. ReBEC platform registration number: RBR-6fgy76

Abstract Background Chronic kidney disease (CKD) is a global public health problem. Cell therapy using pluripotent stem cells represents an attractive therapeutic approach for the treatment of CKD. Methods We transplanted mitomycin C (MMC)-treated human induced pluripotent stem cells (hiPSCs) and renal progenitor cells (RPCs) into a CKD rat model system. The RPC and hiPSC cells were characterized by immunofluorescence and qRT-PCR. Untreated 5/6 nephrectomized rats were compared to CKD animals receiving the same amount of MMC-treated hiPSCs or RPCs. Renal function, histology, and immunohistochemistry were evaluated 45 days post-surgery. Results We successfully generated hiPSCs from peripheral blood and differentiated them into RPCs expressing renal progenitor genes (PAX2, WT1, SIX2, and SALL1) and podocyte-related genes (SYNPO, NPHS1). RPCs also exhibited reduced OCT4 expression, confirming the loss of pluripotency. After cell transplantation into CKD rats, the body weight change was significantly increased in both hiPSC and RPC groups, in comparison with the control group. Creatinine clearance (CCr) was preserved only in the hiPSC group. Similarly, the number of macrophages in the kidneys of the hiPSC group reached a statistically significant reduction, when compared to control rats. Both treatments reduced positive staining for the marker α-smooth muscle actin. Histological features showed decreased tubulointerstitial damage (interstitial fibrosis and tubular atrophy) as well as a reduction in glomerulosclerosis in both iPSC and RPC groups. Conclusions In conclusion, we describe that both MMC-treated hiPSCs and RPCs exert beneficial effects in attenuating CKD progression. Both cell types were equally efficient to reduce histological damage and weight loss caused by CKD. hiPSCs seem to be more efficient than RPCs, possibly due to a paracrine effect triggered by hiPSCs. These results demonstrate that the use of MMC-treated hiPSCs and RPCs improves clinical and histological CKD parameters, avoided tumor formation, and therefore may be a promising cell therapy strategy for CKD. Graphical abstract

Discogenic back pain is a common condition without approved intervertebral disc (IVD) repair therapies. Cell delivery using injectable biomaterial carriers offers promise to restore disc height and biomechanical function, while providing a functional niche for delivered cells to repair degenerated tissues. This systematic review advances the injectable IVD cell delivery biomaterials field by characterising its current state and identifying themes of promising strategies. Preferred Reporting Items for Systematic Reviews and Meta- Analyses (PRISMA) guidelines were used to screen the literature and 183 manuscripts met the inclusion criteria. Cellular and biomaterial inputs, and biological and biomechanical outcomes were extracted from each study. Most identified studies targeted nucleus pulposus (NP) repair. No consensus exists on cell type or biomaterial carrier, yet most common strategies used mesenchymal stem cell (MSC) delivery with interpenetrating network/co-polymeric (IPN/CoP) biomaterials composed of natural biomaterials. All studies reported biological outcomes with about half the studies reporting biomechanical outcomes. Since the IVD is a load-bearing tissue, studies reporting compressive and shear moduli were analysed and two major themes were found. First, a competitive balance, or ‘seesaw’ effect, between biomechanical and biological performance was observed. Formulations with higher moduli had inferior cellular performance, and vice versa. Second, several low-modulus biomaterials had favourable biological performance and matured throughout culture duration with enhanced extracellular matrix synthesis and biomechanical moduli. Findings identify an opportunity to develop next-generation biomaterials that provide high initial biomechanical competence to stabilise and repair damaged IVDs with a capacity to promote cell function for long-term healing.

Abstract Mesenchymal stem cells (MSCs), also referred to as multipotent stromal cells or mesenchymal stromal cells, are present in multiple tissues and capable of differentiating into diverse cell lineages, holding a great promise in developing cell-based therapy for a wide range of conditions. Pelvic floor disorders (PFDs) is a common degenerative disease in women and may diminish a woman’s quality of life at any age. Since the treatments for this disease are limited by the high rates of recurrence and surgical complications, seeking an ideal therapy in the restoration of pelvic floor function is an urgent issue at present. Herein, we summarize the cell sources of MSCs used for PFDs and discuss the potential mechanisms of MSCs in treating PFDs. Specifically, we also provide a comprehensive review of current preclinical and clinical trials dedicated to investigating MSC-based therapy for PFDs. The novel therapy has presented promising therapeutic effects which include relieving the symptoms of urinary or fecal incontinence, improving the biological properties of implanted meshes and promoting the injured tissue repair. Nevertheless, MSC-based therapies for PFDs are still experimental and the unstated issues on their safety and efficacy should be carefully addressed before their clinical applications.

Objective: to study the viability of a tissue-engineered graft (TEG) based on a devitalized tracheal scaffold (DTS) seeded with mesenchymal stromal and epithelial cells in an experiment on rabbits with assessment of cytocompatibility and biocompatibility in vivo. Materials and methods. Syngeneic mesenchymal stromal bone marrow cells (MSBMCs) and syngeneic lung epithelial cells of rabbit were obtained. The morphology and phenotype of the MSBMC culture were confirmed via immunofluorescence staining for CD90 and CD271 markers. Pulmonary epithelial cells obtained by enzymatic treatment of minced rabbit lung tissue were stained with CKPan, CK8/18 and CK14 markers characteristic of epithelial cells. The donor trachea was devitalized in three successive freezethawing cycles. Double-layer cell seeding of DTS was performed under static and dynamic culturing. Orthotopic implantation of TEGs was performed at the site of the anterolateral wall defect in the rabbit that was formed as a result of tracheal resection over four rings. Results were evaluated by computed tomography, histological and immunohistochemical analyzes. Results. A TEG implant, based on DTS, with bilayer colonization by cell cultures of rabbit MSBMC and epithelial cells was obtained. Three months after implantation, TEG engraftment was noted, no tracheal wall stenosis was observed. However, slight narrowing of the lumen in the implantation site was noted. Six months after implantation, viability of TEG was confirmed by histological method. Epithelialization and vascularization of the tracheal wall, absence of signs of purulent inflammation and aseptic necrosis were shown. The small narrowing of the lumen of trachea was found to have been caused by chronic inflammation due to irritation of the mucous membrane with suture material. Conclusion. A new model for assessing the viability of a tissue engineering implant when closing a critical airway defect was created. The developed TEG – based on DTS seeded (bilayer) by lung epithelial cells and BMSCs – was successfully used to replace non-extended tracheal defects in an in vivo experiment. The use of tracheal tissue-engineered graft for orthotopic implantation showed biocompatibility with minimal tissue response.

Abstract A wide range of cancer immunotherapy approaches has been developed including non-specific immune-stimulants such as cytokines, cancer vaccines, immune checkpoint inhibitors (ICIs), and adoptive T cell therapy. Among them, ICIs are the most commonly used and intensively studied. Since 2011, these drugs have received marketing authorisation for melanoma, lung, bladder, renal, and head and neck cancers, with remarkable and long-lasting treatment response in some patients. The novel mechanism of action of ICIs, with immune and T cell activation, leads to unusual patterns of response on imaging, with the advent of so-called pseudoprogression being more pronounced and frequently observed when compared to other anticancer therapies. Pseudoprogression, described in about 2–10% of patients treated with ICIs, corresponds to an increase of tumour burden and/or the appearance of new lesions due to infiltration by activated T cells before the disease responds to therapy. To overcome the limitation of response evaluation criteria in solid tumors (RECIST) to assess these specific changes, new imaging criteria—so-called immune-related response criteria and then immune-related RECIST (irRECIST)—were proposed. The major modification involved the inclusion of the measurements of new target lesions into disease assessments and the need for a 4-week re-assessment to confirm or not confirm progression. The RECIST working group introduced the new concept of “unconfirmed progression”, into the irRECIST. This paper reviews current immunotherapeutic approaches and summarises radiologic criteria to evaluate new patterns of response to immunotherapy. Furthermore, imaging features of immunotherapy-related adverse events and available predictive biomarkers of response are presented.

Objective(s): Tendon healing is substantially slow and often associated with suboptimal repair. Cell therapy is one of the promising methods to improve tendon repair. Blastema, a population of undifferentiated cells, represents characteristics of pluripotent mesenchymal stem cells and has the potentials to be used in regenerative medicine. The aim of this study was to investigate the use of blastema allotransplantation in rabbit tendon healing.Materials and Methods: In this study, one rabbit was used as a blastema donor, and twenty-four rabbits were divided into control and treatment groups. Blastema cells were obtained from ear pinna upon punch hole injury in the donor rabbit. Under general anesthesia, a complete transverse tenotomy was performed on the midsubstance of deep digital flexor tendon followed by suture-repair. In the treatment group, 1 × 106 blastema cells suspended in buffer saline were injected intratendinously at the repair site, while the control group received only the buffer saline. Cast coaptation was maintained for two weeks. Eight weeks after the operation, tendons were harvested, and histopathological, biomechanical, and biochemical assays were performed on samples. Results: Mechanical testing showed a significant increase in ultimate load, energy absorption, stiffness, yield load, stress, and strain in blastema-treated tendons compared to controls. Also, higher hydroxyproline content and improved collagen alignment along with lower inflammatory cell infiltration and decreased angiogenesis were observed in blastema-treated tendons. Conclusion: Increased levels of hydroxyproline and improved histopathological and biomechanical parameters in the treatment group suggest that blastema cells could be considered an adjunct to tendon repair in rabbits.

The choice of an appropriate autogenous source of stem cells has not been adequately addressed especially for intraoral bone regeneration. The current review aims to assess the clinical success of various human stem cells in oral bone regeneration. Articles studying the potential of various stem cells utilized for reconstruction of intraoral bone defects in humans were included in this review. Relevant articles were electronically searched in MEDLINE-PubMed database using keywords with different combinations. Only the articles published in English between 2006 and 2020 were included in this review. It was concluded that intra and extraoral stem cells can be successfully used for bone regeneration of various jaw defects. Depending on the origin, quantity, and quality, each cell type has its own advantages and disadvantages. Also, it brings to the fore the need for more clinical studies to validate and adopt the use of stem cells in regular clinical practice.

Abstract Current regenerative strategies for alveolar bone and periodontal tissues are effective and well adopted. These are mainly based on the use of a combination of synthetic/natural scaffolds and bioactive agents, obviating the incorporation of cells. However, there are some inherent limitations associated with traditional techniques, and we hypothesized that the use of cell‐based therapies as part of comprehensive regenerative protocols may help overcome these hurdles to enhance clinical outcomes. We conducted a systematic review of human controlled clinical trials investigating the clinical and/or histological effect of the use of cell‐based therapies for alveolar bone and periodontal regeneration and explored the translational potential of the different cell‐based strategies identified in the included trials. A total of 16 studies (11 randomized controlled trials, 5 controlled clinical trials) were included for data synthesis and qualitative analysis with meta‐analyses performed when appropriate. The results suggest a clinical benefit from the use of cell therapy. Improved outcomes were shown for alveolar ridge preservation, lateral ridge augmentation, and periodontal regeneration. However, there was insufficient evidence to identify best‐performing treatment modalities amongst the different cell‐based techniques. In light of the clinical and histological outcomes, we identify extraction socket and challenging lateral and vertical bone defects requiring bone block grafts as strong candidates for the adjuvant application of mesenchymal stem cells. Given the complexity, invasiveness, and costs associated with techniques that include “substantial manipulation” of tissues and cells, their additional clinical benefit when compared with “minimal manipulation” must be elucidated in future trials. Stem Cells Translational Medicine 2019;8:1286&1295

Abstract The stromal vascular fraction (SVF) is a heterogeneous population of stem/stromal cells isolated from perivascular and extracellular matrix (ECM) of adipose tissue complex (ATC). Administration of SVF holds a strong therapeutic potential for regenerative and wound healing medicine applications aimed at functional restoration of tissues damaged by injuries or chronic diseases. SVF is commonly divided into cellular stromal vascular fraction (cSVF) and tissue stromal vascular fraction (tSVF). Cellular SVF is obtained from ATC by collagenase digestion, incubation/isolation, and pelletized by centrifugation. Enzymatic disaggregation may alter the relevant biological characteristics of adipose tissue, while providing release of complex, multiattachment of cell‐to‐cell and cell‐to‐matrix, effectively eliminating the bioactive ECM and periadventitial attachments. In many countries, the isolation of cellular elements is considered as a “more than minimal” manipulation, and is most often limited to controlled clinical trials and subject to regulatory review. Several alternative, nonenzymatic methods of adipose tissue processing have been developed to obtain via minimal mechanical manipulation an autologous tSVF product intended for delivery, reducing the procedure duration, lowering production costs, decreasing regulatory burden, and shortening the translation into the clinical setting. Ideally, these procedures might allow for the integration of harvesting and processing of adipose tissue for ease of injection, in a single procedure utilizing a nonexpanded cellular product at the point of care, while permitting intraoperative autologous cellular and tissue‐based therapies. Here, we review and discuss the options, advantages, and limitations of the major strategies alternative to enzymatic processing currently developed for minimal manipulation of adipose tissue. Stem Cells Translational Medicine 2019;8:1265&1271

Objectives: Stem cell therapy is a promising approach in the treatment of acute myocardial infarction (AMI). Mesenchymal stem cells (MSC) from bone marrow (BM-MSC) and adipose tissue (AT-MSC) are attractive and feasible for preclinical and clinical trials. In this study, we compared the therapeutic potential of BM-MSC and AT-MSC in repairing the hearts of rats with isoproterenol (ISO)-induced AMI. Methods: Forty-two female rats were assigned into two groups; the optimization and the experimental group. The optimization groups were further subdivided into control group and the AMI induced group (using ISO). The experimental group was subdivided into AMI+cell-free media injected in the tail vein, AMI+BM-MSC, and AMI+AT-MSC groups treated with the intravenous injection of their respective cell types. Twenty-eight days after induction, electrocardiogram (ECG) was performed, and heart tissue samples were collected for histological assessment and cells tracing. Results: MSC therapy repaired cardiac functions shown by the restoration of ST segment, QT and QRS intervals in the ECG when compared to the AMI group. Infarct area was significantly decreased, and cardiac tissue regeneration signs were shown on histopathological examination. Conclusions: Both MSC sources proved to be equally efficient in the assessed parameters.

Objective (s): Several methods have been proposed for repairing defects and damages, one of which is cell therapy. Bone marrow stromal cells seem to be suitable for this purpose. On the other hand, many biometric materials are used to improve and correct the defects in the body. Nanofibers are widely used in the medical industry, especially in tissue engineering, as scaffolds in wound healing and wound dressing. Chitosan/polyethylene oxide nanofibers can be a suitable replacement for routine wound coverages. Hence, this study was conducted to present a combination of these methods.Materials and Methods: Chitosan/polyethylene oxide nanofibers and thin films of chitosan were produced and optimized by electron microscopy, on which the bone marrow stromal cells were then cultivated. Interactions between the cells and these biomaterials were investigated through viability, morphology, immunocytochemistry and electron microscopy of cells after 6 days. All data were analyzed using Student’s t-test and one-way ANOVA tests in SPSS version 16. PResults: It seems that the high viscosity of chitosan prevents the formation of nanofibers, while chitosan/polyethylene oxide solutions with 80/20 and 90/10 ratios produce perfect, regular, bead free and non-toxic nanofibers with average diameter of 240±10 and 220±10 nm, respectively. The results of immunocytochemistry and viability showed that the cells had relatively high proliferation on the thin chitosan membranes, while the results of the electron microscopy showed that the morphology of cells was better on the nanofibers than on the thin membrane of chitosan.Conclusion: Since bone marrow stromal cells were grown well on chitosan-nanofibers, each of them alone was used in the therapeutic methods. It is better to consider a combination of two methods as the treatment method, especially in tissue engineering and cell therapy.

An arteriovenous fistula (AVF) is the preferred vascular access for chronic hemodialysis, but high failure rates restrict its use. Optimizing patients’ perioperative status and the surgical technique, among other methods for preventing primary AVF failure, continue to fall short in lowering failure rates in clinical practice. One of the predominant causes of AVF failure is neointimal hyperplasia (NIH), a process that results from the synergistic effects of inflammation, hypoxia, and hemodynamic shear stress on vascular tissue. Although several systemic therapies have aimed at suppressing NIH, none has shown a clear benefit towards this goal. Localized therapeutic approaches may improve rates of AVF maturation by providing direct structural and functional support to the maturating fistula, as well as by delivering higher doses of pharmacologic agents while avoiding the adverse effects associated with systemic administration of therapeutic agents. Novel materials—such as polymeric scaffolds and nanoparticles—have enabled the development of different perivascular therapies, such as supportive mechanical devices, targeted drug delivery, and cell-based therapeutics. In this review, we summarize various perivascular therapeutic approaches, available data on their effectiveness, and the outlook for localized therapies targeting NIH in the setting of AVF for hemodialysis use. Highlights: Most systemic therapies do not improve AVF patency outcomes; therefore, localized therapeutic approaches may be beneficial. Locally delivered drugs and medical devices may improve AVF patency outcomes by providing biological and mechanical support. Cell-based therapies have shown promise in suppressing NIH by delivering a more extensive array of bioactive substances in response to the biochemical changes in the AVF microenvironment.

T-lymphocytes have critical functions in the immune responses against viral and intracellular bacterial infections as well as cancers. Antigen (Ag)-specific T-lymphocyte clones enriched and expanded in vitro are valuable tools in the study of immune responses in animal models and adoptive T-cell therapy of patients with cancer or infection. We described a method for inducing, enriching, and replicating Ag-specific poly-clonal T-cells from BALB/c mice infected with live Bacillus Calmette Guérin (BCG) bacterium. During a 7-8 days procedure, T-lymphocytes were purified from immune cells of lymph nodes stimulated with immunodominant Ag of BCG, TB10.4, and expanded by interleukin -2 cytokine. We evaluated the effect of Ag doses (1, 10, and 100 μg/mL) and exposure method of Ag presenting cells (APCs) to T-cells, on T-cells’ proliferation, viability, and Interferon-gamma (IFN-γ) secretion at 2, 5, and 7 days after Ag stimulation. Increasing Ag concentration increased the average cell division, but at the highest dose of Ag (100 μg/mL), T-cell viability is decreased. Only clones induced by 10 μg/mL Ag produced a desirable amount of IFN-γ. Incubation of Ag and APCs, 24 h before T-lymphocytes addition, increased the proliferation and viability of cells. T cells are in a more favorable condition around day 5 of Ag stimulation in terms of proliferation and survival, and it is the desired time for T cell restimulation. For optimal preparation of specific T-cells for adoptive cell transfer, optimization of Ag dose, the order of APCs and T-cells exposure with Ag, and the duration of initial Ag stimulation, as well as the time for restimulation, is essential.

Natural killer (NK) cell therapy has proven to be a promising approach for the treatment of malignancies. Osaki method for ex-vivo autologous NK cell expansion has been recently introduced in Japan. To start clinical trial phase I at Shiraz University of Medical Sciences in collaboration with the Japanese group, this preclinical setting study aimed to evaluate the proliferative efficacy of the method, the activation status of expanded autologous NK cells, and the likely unwanted contamination of the final cell product. Peripheral blood mononuclear cells (PBMCs) were isolated from 5 healthy individuals' peripheral blood and transferred directly to the specified initial culture bag containing anti-CD52 and anti-CD3 and Interleukin (IL)-2. The cells were cultured for 14-17 days in an incubator, during which the cells received condition media, and underwent several passages into bigger culture bags. All the procedures were carried out in a cleanroom and associated facilities. Before and after activation PBMCs were analyzed for their phenotype and cytotoxic activity; using flow cytometry and cytokine release assay. Our results indicated that NK (CD3-CD16+/-CD56+) cells were expanded 510-fold on average (range 200-1100 fold), and the purity of NK cells per whole lymphocytes exceeded 68%. The expanded cells were highly lytic as indicated by in-vitro cytotoxic assay, with a strong expression of Natural killer group 2 member D (NKG2D) and CD16. The prepared final cell products were negative for HCV, HBV, HIV, mycoplasma, and endotoxin. In the preclinical phase, large numbers of activated and un-contaminated NK cells from healthy individuals' peripheral blood were successfully generated. The method seems to provide ample clean cell product with no contamination and has the potential to be used for NK cell therapy in future clinical trials, suitable to be infused back to the donors in phase I clinical trial.

Tooth losses due to pathological processes continue to be a reality in daily clinical dentistry, inducing functional and psychological complications in patients. In view of this, a new option for the management of this problem - tissue engineering - has been studied in Dentistry. This field, considered multidisciplinary, uses three key elements for tissue regeneration: scaffolds (extracellular matrices) - natural or synthetic; cells, and growth factors. In this sense, combination of these three elements may induce regeneration of the dental pulp, bone and periodontal tissue, among others. Therefore, the aim of this study was to conduct a literature review, describing the main elements of tissue engineering and their applicability in Dentistry, as a means of updating dental surgeons about this subject.

The combination of gene therapy and tissue engineering is one of the most promising strategies for the treatment of recessive dystrophic epidermolysis bullosa (RDEB). RDEB is a rare genetic disease characterised by mutations in the COL7A1 gene, encoding type VII collagen (COLVII), which forms anchoring fibrils at the dermal-epidermal junction of the skin. This disease causes severe blistering and only palliative treatments are offered. In this study, the base of a strategy combining gene therapy and a tissue-engineered skin substitute (TES), which would be suitable for the permanent closure of skin wounds, was set-up. As a high transduction efficiency into fibroblasts and/or keratinocytes seems to be a prerequisite for a robust and sustained correction of RDEB, different envelope pseudotyped retroviral vectors and the transduction enhancer EF-C were tested. When green fluorescent protein (GFP) was used as a reporter gene to evaluate the retroviral-mediated gene transfer, the fibroblast infection efficiency was 30 % higher with the Ampho pseudotyped vector as compared with the other pseudotypes. At least a 3.1-fold and a 1.3-fold increased transduction were obtained in fibroblasts and keratinocytes, respectively, with EF-C as compared with polybrene. A continuous and intense deposit of haemagglutinin (HA)-COLVII was observed at the dermal-epidermal junction of self-assembled TESs made of cells transduced with a HA-tagged COL7A1 vector. Furthermore, HA-tagged basal epidermal cells expressing keratin 19 were observed in TESs, suggesting stem cell transduction. This approach could be a valuable therapeutic option to further develop, in order to improve the long-term life quality of RDEB patients.

Abstract Purpose: To investigate the therapeutic potential of human immature dental pulp stem cells in the treatment of chronic spinal cord injury in dogs. Methods: Three dogs of different breeds with chronic SCI were presented as animal clinical cases. Human immature dental pulp stem cells were injected at three points into the spinal cord, and the animals were evaluated by limb function and magnetic resonance imaging (MRI) pre and post-operative. Results: There was significant improvement from the limb function evaluated by Olby Scale, though it was not supported by the imaging data provided by MRI and clinical sign and evaluation. Conclusion: Human dental pulp stem cell therapy presents promising clinical results in dogs with chronic spinal cord injuries, if used in association with physical therapy.

Abstract Purpose: To evaluate the effects of mesenchymal stem cells on liver regeneration in rats following a 70% hepatectomy. Methods: Forty rats were subjected to 70% hepatectomy and then ~106 mesenchymal stem cells (test group), or saline solution (control group), were infused into their livers via the portal vein. Each treatment group was divided into early and late subgroups (euthanized 3 d and 5 d following the operation, respectively). Group comparisons of Albumin, aminotransaminases (AST, ALT), and Alcaline Phosphatase (AP) levels, proliferative index (ki-67+ straining), and mitotic cell counts were conducted. Results: No significant differences in liver regeneration rate, number of mitoses, proliferative index, or serum levels of albumin, AST, or AP were observed. ALT levels were higher in the test group than in the control group (p

The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor β2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1−/− mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.

Advanced therapy medicinal products (ATMPs) are a fast-growing field of innovative therapies. The European Union (EU) and the United States (US) are fostering their development. For both regions, ATMPs fall under the regulatory framework of biological products, which determines the legal basis for their development. Sub-classifications of advanced therapies are different between regions, while in EU, there are four major groups, i.e., gene therapy, somatic cell therapy, tissue-engineered therapies, and combined advanced therapies; in US, the sub-classification covers two major groups of products, i.e., gene therapy and cellular therapy. The inclusion criteria that define a gene therapy are equivalent in both regions, and the exclusion criteria are directly related to the indications of the product. In the EU, there is a clear differentiation between cell- and tissue-based products regarding their classification as advanced therapies or coverage by other legal frameworks, whereas in US, there is a broader classification about whether or not these products can be categorized as biologic products. Both in EU and in US, in order to classify a cell- or a tissue-based product as an advanced therapy, it must be ensured that the processing of the cells implies a manipulation that alters their biological characteristics, although the term of manipulation in US differentiates between structural and non-structural cells and tissues. The regulatory terminology used to define ATMPs and their sub-classification reveals some differences between EU and US.

The cell therapy industry has identified the inability to reliably characterize cells as possibly its greatest challenge and has called for standards and reference materials to provide assurance for measurements of cell properties. The challenges in characterization of cell therapy products can be largely addressed with systematic approaches for assessing sources of uncertainty and improving confidence in key measurements. This article presents the many strategies that can be used to ensure measurement confidence and discusses them in terms of how they can be applied to characterization of cell therapy products. Application of these strategies to cell measurements will help to establish qualified assays for cell characterization, which may help streamline regulatory approval and enable more efficient development of cell therapy products. Significance The regenerative medicine industry has identified the lack of reliable methods for measuring critical cell attributes as possibly the single greatest challenge facing the field. There are many strategies for achieving measurement assurance, or confidence in cell assays, which can streamline regulatory approval and enable more efficient development of cell‐based therapies.

Mesenchymal stem cells (MSCs) are currently studied and used in numerous clinical trials. Nevertheless, some concerns have been raised regarding the safety of these infusions and the thrombogenic risk they induce. MSCs express procoagulant activity (PCA) linked to the expression of tissue factor (TF) that, when in contact with blood, initiates coagulation. Some even describe a dual activation of both the coagulation and the complement pathway, called Instant Blood-Mediated Inflammatory Reaction (IBMIR), explaining the disappointing results and low engraftment rates in clinical trials. However, nowadays, different approaches to modulate the PCA of MSCs and thus control the thrombogenic risk after cell infusion are being studied. This review summarizes both in vitro and in vivo studies on the PCA of MSC of various origins. It further emphasizes the crucial role of TF linked to the PCA of MSCs. Furthermore, optimization of MSC therapy protocols using different methods to control the PCA of MSCs are described.

The efficacy of mesenchymal stem cell infusion is currently tested in numerous clinical trials. However, therapy-induced thrombotic consequences have been reported in several patients. The aim of this study was to optimize protocols for heterologous human adult liver-derived progenitor cell (HHALPC) infusion, in order to eliminate acute thrombogenesis in liver-based metabolic or acute decompensated cirrhotic (ADC) patients. In rats, thrombotic effects were absent when HHALPCs were infused at low cell dose (5 × 106 cells/kg), or at high cell dose (5 × 107 cells/kg) when combined with anticoagulants. When HHALPCs were exposed to human blood in a whole blood perfusion assay, blocking of the tissue factor (TF) coagulation pathway suppressed fibrin generation and platelet activation. In a Chandler tubing loop model, HHALPCs induced less explosive activation of coagulation with blood from ADC patients, when compared to blood from healthy controls, without alterations in coagulation factor levels other than fibrinogen. These studies confirm a link between TF and thrombogenesis, when TF-expressing cells are exposed to human blood. This phenomenon however, could be controlled using either a low, or a high cell dose combined with anticoagulants. In clinical practice, this points to the suitability of a low HHALPC dose infusion to cirrhotic patients, provided that platelet and fibrinogen levels are monitored.

Parkinson’s Disease (PD) is an intractable disease resulting in localized neurodegeneration of dopaminergic neurons of the substantia nigra pars compacta. Many current therapies of PD can only address the symptoms and not the underlying neurodegeneration of PD. To better understand the pathophysiological condition, researchers continue to seek models that mirror PD’s phenotypic manifestations as closely as possible. Recent advances in the field of cellular reprogramming and personalized medicine now allow for previously unattainable cell therapies and patient-specific modeling of PD using induced pluripotent stem cells (iPSCs). iPSCs can be selectively differentiated into a dopaminergic neuron fate naturally susceptible to neurodegeneration. In iPSC models, unlike other artificially-induced models, endogenous cellular machinery and transcriptional feedback are preserved, a fundamental step in accurately modeling this genetically complex disease. In addition to accurately modeling PD, iPSC lines can also be established with specific genetic risk factors to assess genetic sub-populations’ differing response to treatment. iPS cell lines can then be genetically corrected and subsequently transplanted back into the patient in hopes of re-establishing function. Current techniques focus on iPSCs because they are patient-specific, thereby reducing the risk of immune rejection. The year 2018 marked history as the year that the first human trial for PD iPSC transplantation began in Japan. This form of cell therapy has shown promising results in other model organisms and is currently one of our best options in slowing or even halting the progression of PD. Here, we examine the genetic contributions that have reshaped our understanding of PD, as well as the advantages and applications of iPSCs for modeling disease and personalized therapies.

Background: Periodontitis is an important oral disease. Stem cell therapy has found its way in treatment of many diseases. Objective: To evaluate the regenerative potential of periodontal ligament-derived stem cells (PDLSCs) and osteoblast differentiated from PDLSC in comparison with bone marrow-derived mesenchymal stem cells (BM-MSCs) and pre-osteoblasts in calvarial defects. Methods: After proving the existence of surface markers by flow cytometry, BM-MSCs were differentiated into osteoblasts. 5 defects were made on rabbit calvaria. 3 of them were first covered with collagen membrane and then with BM-MSCs, PDLSCs, and pre-osteoblasts. The 4th defect was filled with collagen membrane and the 5th one was served as control. After 4 weeks, histological (quantitative) and histomorphological (qualitative) surveys were performed. Results: Both cell lineages were positive for CD-90 cell marker, which was specifically related to stem cells. Alizarin red staining was done for showing mineral material. RT-PCR set up for the expression of Cbfa1 gene, BMP4 gene, and PGLAP gene, confirmed osteoblast differentiation. The findings indicated that although PDLSCs and pre-osteoblasts could be used for bone regeneration, the rate of regeneration in BM-MSCs-treated cavities was more significant (p

After more than a decade of preclinical and clinical development, therapeutic infusion of mesenchymal stromal cells is now a leading investigational strategy for the treatment of acute graft-versus-host disease (GVHD). While their clinical use continues to expand, it is still unknown which of their immunomodulatory properties contributes most to their therapeutic activity. Herein we describe the proposed mechanisms, focusing on the inhibitory activity of mesenchymal stromal cells (MSCs) at immunologic checkpoints. A deeper understanding of the mechanism of action will allow us to design more effective treatment strategies.

OBJECTIVE: To evaluate the importance of stem cells derived from adipose tissue in reducing graft inflammation in a murine model of allogeneic heterotopic tracheal transplant.METHODS: We performed a heterotopic tracheal allografting in dorsal subcutaneous pouch and systemically injected 5x105 mesenchymal stem cells derived from adipose tissue. The animals were divided into two groups according to the time of sacrifice: T7 and T21. We also carried out histological analysis and digital morphometry.RESULTS: The T7 animals treated with cell therapy had median obstructed graft area of 0 versus 0.54 of controls (p = 0.635). The treated T21 subjects had median obstructed graft area of 0.25 versus 0 in controls (p = 0.041).CONCLUSION: The systemically injected cell therapy in experimental murine model of bronchiolitis obliterans did not reduce the severity of the allograft inflammation in a statistically significant way in seven days; Conversely, in 21 days, it increased the allograft inflammatory process.

Abstract Cell therapy associated with guided bone regeneration (GBR) can be used to treat bone defects under challenging conditions such as osteoporosis. This study aimed to evaluate the effect of mesenchymal stem cells (MSCs) in combination with a poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT) membrane on bone repair in osteoporotic rats. Osteoporosis was induced in female rats by bilateral removal of the ovaries (OVX) or sham surgery (SHAM), and the osteoporotic condition was characterized after 5 months by microtomographic and morphometric analyses. Calvarial defects were created in osteoporotic rats that immediately received the PVDF-TrFE/BT membrane. After 2 weeks, bone marrow-derived MSCs from healthy rats, characterized by the expression of surface markers using flow cytometry, or phosphate-buffered saline (PBS) (Control) were injected into the defects and bone formation was evaluated 4 weeks post-injection by microtomographic, morphometric, and histological analyses. A reduction in the amount of bone tissue in the femurs of OVX compared with SHAM rats confirmed the osteoporotic condition of the experimental model. More bone formation was observed when the defects were injected with MSCs compared to that with PBS. The modification that we are proposing in this study for the classical GBR approach where cells are locally injected after a membrane implantation may be a promising therapeutic strategy to increase bone formation under osteoporotic condition.