Human-derived Tumor-On-Chip model to study the heterogeneity of breast cancer tissue.

One of the leading causes that complicate the treatment of some malignancies, including breast cancer, is tumor heterogeneity. In addition to inter-heterogeneity and intra-heterogeneity of tumors that reflect the differences between cancer cell characteristics, heterogeneity in the tumor microenvironment plays a critical role in tumor progression and could be considered an overlooked and a proper target for the effective selection of therapeutic approaches. Due to the difficulty of completely capturing tumor heterogeneity in conventional detection methods, Tumor-on-Chip (TOC) devices with culturing patient-derived spheroids could be an appropriate alternative. In this research, human-derived spheroids from breast cancer individuals were cultured for 6 days in microfluidic devices. To compare TOC data with conventional detection methods, immunohistochemistry (IHC) and ITRAQ data were employed, and various protein expressions were validated using the transcriptomic databases. The behavior of the spheroids in the collagen matrix and the cell viability were monitored over 6 days of culture. IHC and immunocytochemistry (ICC) results revealed that inter and intra-heterogeneity of tumor spheroids are associated with HER2/ER expression. HER2 expression levels revealed a more important biomarker associated with invasion in the 3D culturing of spheroids. The expression levels of CD163 (as a marker for Ma2 macrophages) and CD44 (a marker for cancer stem cells (CSCs)) were also evaluated. Interestingly, the levels of M2a macrophages and CSCs were higher in triple-negative specimens and samples that showed higher migration and invasion. Cell density and extracellular matrix (ECM) stiffness were also important factors affecting the migration and invasion of the spheroids through the matrix. Among these, rigid ECM revealed a more crucial role than cell density. To sum up, these research findings demonstrated that human-derived spheroids from breast cancer specimens in microfluidic devices provide a dynamic condition for predicting tumor heterogeneity in patients, which can help move the field forward for better and more accurate therapeutic strategies.

Potential therapeutic options for celiac Disease: An update on Current evidence from Gluten-Free diet to cell therapy.

Celiac disease (CD) is a chronic autoimmune enteropathy and multifactorial disease caused by inappropriate immune responses to gluten in the small intestine. Weight loss, anemia, osteoporosis, arthritis, and hepatitis are among the extraintestinal manifestations of active CD. Currently, a strict lifelong gluten-free diet (GFD) is the only safe, effective, and available treatment. Despite the social burden, high expenses, and challenges of following a GFD, 2 to 5 percent of patients do not demonstrate clinical or pathophysiological improvement. Therefore, we need novel and alternative therapeutic approaches for patients. Innovative approaches encompass a broad spectrum of strategies, including enzymatic degradation of gluten, inhibition of intestinal permeability, modulation of the immune response, inhibition of the transglutaminase 2 (TG2) enzyme, blocking antigen presentation by HLA-DQ2/8, and induction of tolerance. Hence, this review is focused on comprehensive therapeutic strategies ranging from dietary approaches to novel methods such as antigen-based immunotherapy, cell and gene therapy, and the usage of nanoparticles for CD treatment.

Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus.

INTRODUCTION: The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus. METHODS: T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5'- and 3'-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays. RESULTS: Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells. CONCLUSION: Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.

Single-cell Technology in Stem Cell Research.

Single-cell technology (SCT), which enables the examination of the fundamental units comprising biological organs, tissues, and cells, has emerged as a powerful tool, particularly in the field of biology, with a profound impact on stem cell research. This innovative technology opens new pathways for acquiring cell-specific data and gaining insights into the molecular pathways governing organ function and biology. SCT is not only frequently used to explore rare and diverse cell types, including stem cells, but it also unveils the intricacies of cellular diversity and dynamics. This perspective, crucial for advancing stem cell research, facilitates non-invasive analyses of molecular dynamics and cellular functions over time. Despite numerous investigations into potential stem cell therapies for genetic disorders, degenerative conditions, and severe injuries, the number of approved stem cell-based treatments remains limited. This limitation is attributed to the various heterogeneities present among stem cell sources, hindering their widespread clinical utilization. Furthermore, stem cell research is intimately connected with cutting-edge technologies, such as microfluidic organoids, CRISPR technology, and cell/tissue engineering. Each strategy developed to overcome the constraints of stem cell research has the potential to significantly impact advanced stem cell therapies. Drawing from the advantages and progress achieved through SCT-based approaches, this study aims to provide an overview of the advancements and concepts associated with the utilization of SCT in stem cell research and its related fields.

Current progress in engineered and nano-engineered mesenchymal stem cells for cancer: From mechanisms to therapy.

Mesenchymal stem cells (MSCs), as self-renewing multipotent stromal cells, have been considered promising agents for cancer treatment. A large number of studies have demonstrated the valuable properties of MSC-based treatment, such as low immunogenicity and intrinsic tumor-trophic migratory properties. To enhance the potency of MSCs for therapeutic purposes, equipping MSCs with targeted delivery functions using genetic engineering is highly beneficial. Genetically engineered MSCs can express tumor suppressor agents such as pro-apoptotic, anti-proliferative, anti-angiogenic factors and act as ideal delivery vehicles. MSCs can also be loaded with nanoparticle drugs for increased efficacy and externally moderated targeting. Moreover, exosomes secreted by MSCs have important physiological properties, so they can contribute to intercellular communication and transfer cargo into targeted tumor cells. The precise role of genetically modified MSCs in tumor environments is still up for debate, but the beginning of clinical trials has been confirmed by promising results from preclinical investigations of MSC-based gene therapy for a wide range of malignancies. This review highlights the advanced techniques of engineering/nano-engineering and MSC-derived exosomes in tumor-targeted therapy.

Bromelain- and Silver Nanoparticle-Loaded Polycaprolactone/Chitosan Nanofibrous Dressings for Skin Wound Healing.

A cutaneous wound is caused by various injuries in the skin, which can be wrapped with an efficient dressing. Electrospinning is a straightforward adjustable technique that quickly and continuously generates nanofibrous wound dressings containing antibacterial and anti-inflammatory agents to promote wound healing. The present study investigated the physicochemical and biological properties of bromelain (BRO)- and silver nanoparticle (Ag NPs)-loaded gel-based electrospun polycaprolactone/chitosan (PCL/CS) nanofibrous dressings for wound-healing applications. Electron microscopy results showed that the obtained nanofibers (NFs) had a uniform and homogeneous morphology without beads with an average diameter of 176 ± 63 nm. The FTIR (Fourier transform infrared) analysis exhibited the loading of the components. Moreover, adding BRO and Ag NPs increased the tensile strength of the NFs up to 4.59 MPa. BRO and Ag NPs did not significantly affect the hydrophilicity and toxicity of the obtained wound dressing; however, the antibacterial activity against E. coli and S. aureus bacteria was significantly improved. The in vivo study showed that the wound dressing containing BRO and Ag NPs improved the wound-healing process within one week compared to other groups. Therefore, gel-based PCL/CS nanofibrous dressings containing BRO and Ag NPs could be a promising solution for healing skin wounds.

Overexpression of SIRT6 alleviates apoptosis and enhances cell viability and monoclonal antibody expression in CHO-K1 cells.

BACKGROUND: Chinese hamster ovary (CHO) cells are the most predominantly utilized host for the production of monoclonal antibodies (mAbs) and other complex glycoproteins. A major challenge in the process of CHO cell culture is the occurrence of cell death following different stressful conditions, which hinders the production yield. Engineering genes involved in pathways related to cell death is a remarkable strategy to delay apoptosis, improve cell viability and enhance productivity. SIRT6 is a stress-responsive protein that regulates DNA repair, maintains genome integrity, and is critical for longevity and cell survival in organisms. METHODS AND RESULTS: In this study, SIRT6 was stably overexpressed in CHO-K1 cells and the impact of its expression on apoptosis related gene expression profile, viability, apoptosis, and mAb productivity was investigated. While a significant increase was observed in Bcl-2 mRNA level, caspase-3 and Bax mRNA levels were decreased in the SIRT6 engineered cells compared to the parental CHO-K1 cells. Moreover, improved cell viability and decreased rate of apoptotic progression was observed in a SIRT6-derived clone in comparision to the CHO-K1 cells during 5 days of batch culture. anti-CD52 IgG1 mAb titers were improved up to 1.7- and 2.8-fold in SIRT6-derived clone during transient and stable expression, respectively. CONCLUSIONS: This study indicates the positive effects of SIRT6 overexpression on cell viability and anti-CD52 IgG1 mAb expression in CHO-K1 cells. Further studies are needed to examine the potential of SIRT6-engineered host cells for the production of recombinant biotherapeutics in industrial settings.

Surface Coating of Polyurethane Films with Gelatin, Aspirin and Heparin to Increase the Hemocompatibility of Artificial Vascular Grafts.

Purpose: A hemocompatible substrate can offer a wonderful facility for nitric oxide (NO) production by vascular endothelial cells in reaction to the inflammation following injuries. NO inhibits platelet aggregation this is especially critical in small-diameter vessels. Methods: The substrate films were made of polyurethane (PU) in a casting process and after plasma treatments, their surface was chemically decorated with polyethylene glycol (PEG) 2000, gelatin, gelatin-aspirin, gelatin-heparin and gelatin-aspirin-heparin. The concentrations of these ingredients were optimized in order to achieve the biocompatible values and the resulting modifications were characterized by water contact angle and Fourier transform infra-red (FTIR) assays. The values of NO production and platelet adhesion were then examined. Results: The water contact angle of the modified surface was reduced to 26±4∘ and the newly developed hydrophilic chemical groups were confirmed by FTIR. The respective concentrations of 0.05 mg/ml and 100 mg/mL were found to be the IC50 values for aspirin and heparin. However, after the surface modification with aspirin, the bioactivity of the substrate increased in compared to the other experimental groups. In addition, there was a synergistic effect between these reagents for NO synthesis. While, heparin inhibited platelet adhesion more than aspirin. Conclusion: Because of the highly hydrophilic nature of heparin, this reagent was hydrolyzed faster than aspirin and therefore its influence on platelet aggregation and cell growth was greater. Taken together, the results give the biocompatible concentrations of both biomolecules that are required for endothelial cell proliferation, NO synthesis and platelet adhesion.

Recent Emerging Trend in Stem Cell Therapy Risk Factors.

Different types of stem cells have remarkable characteristics such as high proliferation rate, multi/pluripotency, self-renewal, and broad differentiation that can effectively treat diseases, cancers, and damage. Despite abundant therapeutic applications of stem cells in medical science, numerous risks threaten stem cell transplantation. Tumor development, immune response, cellular senescence, dosage effects, and administration timing are critical risks that should be considered in stem cell therapy. Hence, an investigation of possible risks is required before utilizing stem cell-based medicinal products in the clinical phase and human trials. This review aims to survey the literature and perspectives on the advantages and risks associated with pluripotent and multipotent stem cells.

Biological and clinical review of IORT-induced wound fluid in breast cancer patients.

Intraoperative radiotherapy (IORT) has become a growing therapy for early-stage breast cancer (BC). Some studies claim that wound fluid (seroma), a common consequence of surgical excision in the tumor cavity, can reflect the effects of IORT on cancer inhibition. However, further research by our team and other researchers, such as analysis of seroma composition, affected cell lines, and primary tissues in two-dimensional (2D) and three-dimensional (3D) culture systems, clarified that seroma could not address the questions about IORT effectiveness in the surgical site. In this review, we mention the factors involved in tumor recurrence, direct or indirect effects of IORT on BC, and all the studies associated with BC seroma to attain more information about the impact of IORT-induced seroma to make a better decision to remove or remain after surgery and IORT. Finally, we suggest that seroma studies cannot decipher the mechanisms underlying the effectiveness of IORT in BC patients. The question of whether IORT-seroma has a beneficial effect can only be answered in a trial with a clinical endpoint, which is not even ongoing.

Overexpression of VEGF in dermal fibroblast cells accelerates the angiogenesis and wound healing function: in vitro and in vivo studies.

Fibroblasts are the main cells of connective tissue and have pivotal roles in the proliferative and maturation phases of wound healing. These cells can secrete various cytokines, growth factors, and collagen. Vascular endothelial growth factor (VEGF) is a unique factor in the migration process of fibroblast cells through induces wound healing cascade components such as angiogenesis, collagen deposition, and epithelialization. This study aimed to create VEGF165 overexpressing fibroblast cells to evaluate angiogenesis function in wound healing. In vitro, a novel recombinant expression vector, pcDNA3.1(-)-VEGF, was produced and transfected into the fibroblast cells. Following selecting fibroblast cells with hygromycin, recombinant cells were investigated in terms of VEGF expression by quantifying and qualifying methods. Mechanical, physical, and survival properties of polyurethane-cellulose acetate (PU-CA) scaffold were investigated. Finally, in vivo, the angiogenic potential was evaluated in four groups containing control, PU-CA, PU-CA with fibroblast cells, and VEGF-expressing cells on days 0, 2, 5, 12 and 15. Wound biopsies were harvested and the healing process was histopathologically evaluated on different days. qRT-PCR showed VEGF overexpression (sevenfold) in genetically-manipulated cells compared to fibroblast cells. Recombinant VEGF expression was also confirmed by western blotting. Manipulated fibroblast cells represented more angiogenesis than other groups on the second day after surgery, which was also confirmed by the antiCD31 antibody. The percentage of wound closure area on day 5 in genetically-manipulated Hu02 and Hu02 groups showed a significant reduction of wound area compared to other groups. These findings indicate that overexpression of VEGF165 in fibroblast cells results in enhanced angiogenesis and formation of granulated tissue in the early stage of the healing process, which can show its therapeutic potential in patients with impaired wound healing and also provide functional support for gene therapy.

Advance trends in targeting homology-directed repair for accurate gene editing: An inclusive review of small molecules and modified CRISPR-Cas9 systems.

Introduction: Clustered regularly interspaced short palindromic repeat and its associated protein (CRISPR-Cas)-based technologies generate targeted modifications in host genome by inducing site-specific double-strand breaks (DSBs) that can serve as a substrate for homology-directed repair (HDR) in both in vitro and in vivo models. HDR pathway could enhance incorporation of exogenous DNA templates into the CRISPR-Cas9-mediated DSB site. Owing to low rate of HDR pathway, the efficiency of accurate genome editing is diminished. Enhancing the efficiency of HDR can provide fast, easy, and accurate technologies based on CRISPR-Cas9 technologies. Methods: The current study presents an overview of attempts conducted on the precise genome editing strategies based on small molecules and modified CRISPR-Cas9 systems. Results: In order to increase HDR rate in targeted cells, several logical strategies have been introduced such as generating CRISPR effector chimeric proteins, anti-CRISPR proteins, modified Cas9 with donor template, and using validated synthetic or natural small molecules for either inhibiting non-homologous end joining (NHEJ), stimulating HDR, or synchronizing cell cycle. Recently, high-throughput screening methods have been applied for identification of small molecules which along with the CRISPR system can regulate precise genome editing through HDR. Conclusion: The stimulation of HDR components or inhibiting NHEJ can increase the accuracy of CRISPR-Cas-mediated engineering systems. Generating chimeric programmable endonucleases provide this opportunity to direct DNA template close proximity of CRISPR-Cas-mediated DSB. Small molecules and their derivatives can also proficiently block or activate certain DNA repair pathways and bring up novel perspectives for increasing HDR efficiency, especially in human cells. Further, high throughput screening of small molecule libraries could result in more discoveries of promising chemicals that improve HDR efficiency and CRISPR-Cas9 systems.

Recent advances and challenges in graphene-based nanocomposite scaffolds for tissue engineering application.

Graphene-based nanocomposites have recently attracted increasing attention in tissue engineering because of their extraordinary features. These biocompatible substances, in the presence of an apt microenvironment, can stimulate and sustain the growth and differentiation of stem cells into different lineages. This review discusses the characteristics of graphene and its derivatives, such as their excellent electrical signal transduction, carrier mobility, outstanding mechanical strength with improving surface characteristics, self-lubrication, antiwear properties, enormous specific surface area, and ease of functional group modification. Moreover, safety issues in the application of graphene and its derivatives in terms of biocompatibility, toxicity, and interaction with immune cells are discussed. We also describe the applicability of graphene-based nanocomposites in tissue healing and organ regeneration, particularly in the bone, cartilage, teeth, neurons, heart, skeletal muscle, and skin. The impacts of special textural and structural characteristics of graphene-based nanomaterials on the regeneration of various tissues are highlighted. Finally, the present review gives some hints on future research for the transformation of these exciting materials in clinical studies.

Evaluation of in vitro fibroblast migration by electrospun triple-layered PU-CA/gelatin.PRGF/PU-CA scaffold using an AAVS1 targeted EGFP reporter cell line.

Introduction: Migration of fibroblast cells in wound areas is a critical aspect of the wound healing process. Employment of enhanced green fluorescent protein (EGFP) labeled fibroblast cells facilitates real-time monitoring and functional evaluation of these cells in both in vitro and in vivo settings. Plasma rich in growth factor (PRGF) is a potent accelerator of wound healing; therefore, in this study, a novel method to fabricate an electrospun bioactive scaffold containing PRGF was employed to induce in vitro cell proliferation and migration. Methods: First, the EGFP reporter gene was integrated into the AAVS1 locus of fibroblast cells using CRISPR/Cas9 system. Then, PRGF was obtained from platelet-rich plasma, and a multi-layered scaffold was fabricated using polyurethane-cellulose acetate (PU-CA) fibers as the outer layers and PRGF-containing gelatin fibers were located in the internal layer like a central strip. Scanning electron microscopy (SEM), tensile, water contact angle, and FTIR tests were performed to assess the characteristics of the scaffolds. The EGFP targeted cells were cultured on scaffolds with or without PRGF to investigate their viability, toxicity, and migration pattern in response to the release profile. Results: Fluorescence images showed that the number of migrating cells on scaffold containing PRGF was more significant than PU-CA scaffold up to day 6. Increased expression of SGPL1, DDR2, and VEGF genes was also observed on the scaffold containing PRGF compared to PU-CA using real-time polymerase chain reaction (PCR) analysis with around 3-, 2-, and 2-fold enhancement, respectively. Conclusion: The current scaffold provides the appropriate template for cell attachment and migration. In addition, the present results highlight the potential of reporter gene targeting for the in vitro analysis of biological processes such as migration.

Radiobiological effects of wound fluid on breast cancer cell lines and human-derived tumor spheroids in 2D and microfluidic culture.

Intraoperative radiotherapy (IORT) could abrogate cancer recurrences, but the underlying mechanisms are unclear. To clarify the effects of IORT-induced wound fluid on tumor progression, we treated breast cancer cell lines and human-derived tumor spheroids in 2D and microfluidic cell culture systems, respectively. The viability, migration, and invasion of the cells under treatment of IORT-induced wound fluid (WF-RT) and the cells under surgery-induced wound fluid (WF) were compared. Our findings showed that cell viability was increased in spheroids under both WF treatments, whereas viability of the cell lines depended on the type of cells and incubation times. Both WFs significantly increased sub-G1 and arrested the cells in G0/G1 phases associated with increased P16 and P21 expression levels. The expression level of Caspase 3 in both cell culture systems and for both WF-treated groups was significantly increased. Furthermore, our results revealed that although the migration was increased in both systems of WF-treated cells compared to cell culture media-treated cells, E-cadherin expression was significantly increased only in the WF-RT group. In conclusion, WF-RT could not effectively inhibit tumor progression in an ex vivo tumor-on-chip model. Moreover, our data suggest that a microfluidic system could be a suitable 3D system to mimic in vivo tumor conditions than 2D cell culture.

Combination Therapy of Stem Cell-derived Exosomes and Biomaterials in the Wound Healing.

Wound healing is a serious obstacle due to the complexity of evaluation and management. While novel approaches to promoting chronic wound healing are of critical interest at the moment, several studies have demonstrated that combination therapy is critical for the treatment of a variety of diseases, particularly chronic wounds. Among the various approaches that have been proposed for wound care, regenerative medicine-based methods have garnered the most attention. As is well known, regenerative medicine's three primary tools are gene/cell therapy, biomaterials, and tissue engineering. Multifunctional biomaterials composed of synthetic and natural components are highly advantageous for exosome carriers, which utilizing them is an exciting wound healing method. Recently, stem cell-secreted exosomes and certain biomaterials have been identified as critical components of the wound healing process, and their combination therapy appears to produce significant results. This paper presents a review of literature and perspectives on the use of stem cell-derived exosomes and biomaterials in wound healing, particularly chronic wounds, and discusses the possibility of future clinical applications.

The effect of Ccnb1ip1 insulator on monoclonal antibody expression in Chinese hamster ovary cells.

BACKGROUND: The increasing need for therapeutic monoclonal antibodies (mAbs) entails the development of innovative and improved expression strategies. Chromatin insulators have been utilized for the enhancement of the heterologous proteins in mammalian cells. METHODS AND RESULTS: In the current study the Ccnb1ip1 gene insulator element was utilized to construct a novel vector system for the expression of an anti-CD52 mAb in Chinese hamster ovary (CHO) cells. The insulator containing (pIns-mAb) and control (pmAb) vectors were generated and stable cell pools were established using these constructs. The expression level in the cells created with pIns-mAb vector was calculated to be 233 ng/mL, and the expression rate in the control vector was 210 ng/mL, which indicated a 10.9% increase in mAb expression in pIns-mAb pool. In addition, analysis of mAb expression in clonal cells established from each pool showed a 10% increase in antibody productivity in the highest mAb producing clone derived from the pIns-mAb pool compared to the clone isolated from pmAb pool. CONCLUSIONS: More studies are needed to fully elucidate the effects of Ccnb1ip1 gene insulator on recombinant therapeutic protein expression in mammalian cells. The combination of this element with other chromatin-modifying elements might improve its augmentation effect which could pave the way for efficient and cost-effective production of therapeutic drugs.

Nanotechnology-based products for cancer immunotherapy.

Currently, nanoscale materials and scaffolds carrying antitumor agents to the tumor target site are practical approaches for cancer treatment. Immunotherapy is a modern approach to cancer treatment in which the body's immune system adjusts to deal with cancer cells. Immuno-engineering is a new branch of regenerative medicine-based therapies that uses engineering principles by using biological tools to stimulate the immune system. Therefore, this branch's final aim is to regulate distribution, release, and simultaneous placement of several immune factors at the tumor site, so then upgrade the current treatment methods and subsequently improve the immune system's handling. In this paper, recent research and prospects of nanotechnology-based cancer immunotherapy have been presented and discussed. Furthermore, different encouraging nanotechnology-based plans for targeting various innate and adaptive immune systems will also be discussed. Due to novel views in nanotechnology strategies, this field can address some biological obstacles, although studies are ongoing.

The utility of dermal fibroblasts in treatment of skin disorders: A paradigm of recessive dystrophic epidermolysis bullosa.

Dermal fibroblasts are the most accessible cells in the skin that have gained significant attention in cell therapy. Applying dermal fibroblasts' regenerative capacity can introduce new patterns to develop cell-based therapies to treat skin disorders. Dermal fibroblasts originate from mesenchymal cells and are located within the dermis. These cells are mainly responsible for synthesizing glycosaminoglycans, collagens, and components of extracellular matrix supporting skin's structural integrity. Preclinical studies suggested that allogeneic and autologous dermal fibroblasts provide widespread and beneficial applications for wound healing, burn ulcers, and inherited skin disorders. In this regard, generating induced pluripotent stem cells (iPSCs) from fibroblasts and gene-edited fibroblasts are promising approaches for treating skin disorders. Here, we aimed to review literature about ongoing and completed clinical trials that applied fibroblasts and bioengineered fibroblasts as therapeutic agents for various skin disorders. This review explores cell therapy protocols from the earliest phase of allogeneic and autologous fibroblasts development in different benches to translating them into bedside-level treatment for skin disorders, particularly recessive dystrophic epidermolysis bullosa.

Utilization of the human gamma-satellite insulator for the enhancement of anti-PCSK9 monoclonal antibody expression in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are widely employed as invaluable therapeutics for a vast number of human disorders. Several approaches have been introduced for the improvement of mAb production in Chinese hamster ovary (CHO) cells due to the increasing demand for these products. In this regard, various chromatin-modifying elements such as insulators have been incorporated in the expression vectors to augment mAb expression. In this study, human gamma-satellite insulator containing vectors were utilized for the expression of an anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) mAb in CHO-K1 cells. To this aim, dual expression vectors encoding the antibody light chain (LC) and heavy chain (HC) with or without the insulator element were constructed, and mAb expression was evaluated in transient and stable expression. Based on the results, mAb expression significantly increased in the stable cell pool, and clonal cells developed using the human gamma-satellite insulator. In contrast, transient antibody expression was not affected by the insulator element. Finally, the enhancement of LC and HC mRNA levels was found in the insulator containing stable cell pools using the quantitative real-time-polymerase chain reaction (qRT-PCR). Our findings showed the positive effect of the human gamma-satellite insulator on the stable expression of an anti-PCSK9 immunoglobulin G1 (IgG1) mAb in CHO-K1 cells using dual expression vectors.