Search

Abstract Exosomes are extracellular vesicles well known for facilitating cell-to-cell communication by distributing essential macromolecules like proteins, DNA, mRNA, lipids, and miRNA. These vesicles are abundant in fluids distributed throughout the body, including urine, blood, saliva, and even bile. They are important diagnostic tools for breast, lung, gastrointestinal cancers, etc. However, their application as cancer biomarkers has not yet been implemented in most parts of the world. In this review, we discuss how OMICs profiling of exosomes can be practiced by substituting traditional imaging or biopsy methods for cancer detection. Previous methods like extensive imaging and biopsy used for screening were expensive, mostly invasive, and could not easily provide early detection for various types of cancer. Exosomal biomarkers can be utilized for routine screening by simply collecting body fluids from the individual. We anticipate that the use of exosomes will be brought to light by the success of clinical trials investigating their potential to enhance cancer detection and treatment in the upcoming years. Graphical Abstract

Because hair loss is a common concern for many individuals, potential regenerative therapies of hair follicles have been extensively researched. Induced pluripotent stem cells (iPSCs) are a promising avenue for hair follicle regeneration. This review explores current iPSC-based approaches and highlights their potential applications and challenges in hair restoration. The principles of iPSC technology, iPSC differentiation into hair follicle precursor cells, and potential clinical implications for hair follicle regeneration are also discussed. This overview of iPSCs and their applications aims to contribute to our understanding of their role in hair restoration and potential future therapeutic applications.

Alzheimer’s disease (AD) still prevails and continues to increase indiscriminately throughout the 21st century, and is thus responsible for the depreciating quality of health and associated sectors. AD is a progressive neurodegenerative disorder marked by a significant amassment of beta-amyloid plaques and neurofibrillary tangles near the hippocampus, leading to the consequent loss of cognitive abilities. Conventionally, amyloid and tau hypotheses have been established as the most prominent in providing detailed insight into the disease pathogenesis and revealing the associative biomarkers intricately involved in AD progression. Nanotheranostic deliberates rational thought toward designing efficacious nanosystems and strategic endeavors for AD diagnosis and therapeutic implications. The exceeding advancements in this field enable the scientific community to envisage and conceptualize pharmacokinetic monitoring of the drug, sustained and targeted drug delivery responses, fabrication of anti-amyloid therapeutics, and enhanced accumulation of the targeted drug across the blood–brain barrier (BBB), thus giving an optimistic approach towards personalized and precision medicine. Current methods idealized on the design and bioengineering of an array of nanoparticulate systems offer higher affinity towards neurocapillary endothelial cells and the BBB. They have recently attracted intriguing attention to the early diagnostic and therapeutic measures taken to manage the progression of the disease. In this article, we tend to furnish a comprehensive outlook, the detailed mechanism of conventional AD pathogenesis, and new findings. We also summarize the shortcomings in diagnostic, prognostic, and therapeutic approaches undertaken to alleviate AD, thus providing a unique window towards nanotheranostic advancements without disregarding potential drawbacks, side effects, and safety concerns.

Abstract Tumor development and metastasis are facilitated by the complex interactions between cancer cells and their microenvironment, which comprises stromal cells and extracellular matrix (ECM) components, among other factors. Stromal cells can adopt new phenotypes to promote tumor cell invasion. A deep understanding of the signaling pathways involved in cell‐to‐cell and cell‐to‐ECM interactions is needed to design effective intervention strategies that might interrupt these interactions. In this review, we describe the tumor microenvironment (TME) components and associated therapeutics. We discuss the clinical advances in the prevalent and newly discovered signaling pathways in the TME, the immune checkpoints and immunosuppressive chemokines, and currently used inhibitors targeting these pathways. These include both intrinsic and non‐autonomous tumor cell signaling pathways in the TME: protein kinase C (PKC) signaling, Notch, and transforming growth factor (TGF‐β) signaling, Endoplasmic Reticulum (ER) stress response, lactate signaling, Metabolic reprogramming, cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) and Siglec signaling pathways. We also discuss the recent advances in Programmed Cell Death Protein 1 (PD‐1), Cytotoxic T‐Lymphocyte Associated Protein 4 (CTLA4), T‐cell immunoglobulin mucin‐3 (TIM‐3) and Lymphocyte Activating Gene 3 (LAG3) immune checkpoint inhibitors along with the C‐C chemokine receptor 4 (CCR4)‐ C‐C class chemokines 22 (CCL22)/ and 17 (CCL17), C‐C chemokine receptor type 2 (CCR2)‐ chemokine (C‐C motif) ligand 2 (CCL2), C‐C chemokine receptor type 5 (CCR5)‐ chemokine (C‐C motif) ligand 3 (CCL3) chemokine signaling axis in the TME. In addition, this review provides a holistic understanding of the TME as we discuss the three‐dimensional and microfluidic models of the TME, which are believed to recapitulate the original characteristics of the patient tumor and hence may be used as a platform to study new mechanisms and screen for various anti‐cancer therapies. We further discuss the systemic influences of gut microbiota in TME reprogramming and treatment response. Overall, this review provides a comprehensive analysis of the diverse and most critical signaling pathways in the TME, highlighting the associated newest and critical preclinical and clinical studies along with their underlying biology. We highlight the importance of the most recent technologies of microfluidics and lab‐on‐chip models for TME research and also present an overview of extrinsic factors, such as the inhabitant human microbiome, which have the potential to modulate TME biology and drug responses.

Globally, neurological diseases pose a major burden to healthcare professionals in terms of the management and prevention of the disorder. Among neurological diseases, Alzheimer’s disease (AD) accounts for 50%–70% of dementia and is the fifth leading cause of mortality worldwide. AD is a progressive, degenerative neurological disease, with the loss of neurons and synapses in the cerebral cortex and subcortical regions. The management of AD remains a debate among physicians as no standard and specific “disease-modifying” modality is available. The concept of ‘Regenerative Medicine’ is aimed at regenerating the degenerated neural tissues to reverse the pathology in AD. Genetically modified engineered stem cells modify the course of AD after transplantation into the brain. Extracellular vesicles (EVs) are an emerging new approach in cell communication that involves the transfer of cellular materials from parental cells to recipient cells, resulting in changes at the molecular and signaling levels in the recipient cells. EVs are a type of vesicle that can be transported between cells. Many have proposed that EVs produced from mesenchymal stem cells (MSCs) may have therapeutic promise in the treatment of AD. The biology of AD, as well as the potential applications of stem cells and their derived EVs-based therapy, were explored in this paper.

Exosomes, which are nano-sized transport bio-vehicles, play a pivotal role in maintaining homeostasis by exchanging genetic or metabolic information between different cells. Exosomes can also play a vital role in transferring virulent factors between the host and parasite, thereby regulating host gene expression and the immune interphase. The association of inflammation with disease development and the potential of exosomes to enhance or mitigate inflammatory pathways support the notion that exosomes have the potential to alter the course of a disease. Clinical trials exploring the role of exosomes in cancer, osteoporosis, and renal, neurological, and pulmonary disorders are currently underway. Notably, the information available on the signatory efficacy of exosomes in immune-related disorders remains elusive and sporadic. In this review, we discuss immune cell-derived exosomes and their application in immunotherapy, including those against autoimmune connective tissue diseases. Further, we have elucidated our views on the major issues in immune-related pathophysiological processes. Therefore, the information presented in this review highlights the role of exosomes as promising strategies and clinical tools for immune regulation.

Chimeric antigen receptor (CAR)-T cell therapy is a progressive new pillar in immune cell therapy for cancer. It has yielded remarkable clinical responses in patients with B-cell leukemia or lymphoma. Unfortunately, many challenges remain to be addressed to overcome its ineffectiveness in the treatment of other hematological and solidtumor malignancies. The major hurdles of CAR T-cell therapy are the associated severe life-threatening toxicities such as cytokine release syndrome and limited anti-tumor efficacy. In this review, we briefly discuss cancer immunotherapy and the genetic engineering of T cells and, In detail, the current innovations in CAR T-cell strategies to improve efficacy in treating solid tumors and hematologic malignancies. Furthermore, we also discuss the current challenges in CAR T-cell therapy and new CAR T-cell-derived nanovesicle therapy. Finally, strategies to overcome the current clinical challenges associated with CAR T-cell therapy are included as well.

Hair loss is one of the most common disorders that affect both male and female patients. Cell-derived nanovesicles (CDVs) are natural extracellular vesicles and engineered nanovesicles that can carry various biologicals materials such as proteins, lipids, mRNA, miRNA, and DNA. These vesicles can communicate with local or distant cells and are capable of delivering endogenous materials and exogenous drugs for regenerative therapies. Recent studies revealed that CDVs can serve as new treatment strategies for hair growth. Herein, we review current knowledge on the role of CDVs in applications to hair growth. The in-depth understanding of the mechanisms by which CDVs enable therapeutic effects for hair growth may accelerate successful clinical translation of these vesicles for treating hair loss.

Abstract Biodistribution and role of extracellular vesicles (EVs) are still largely unknown. Reliable tracking methods for EVs are needed. In this study, nuclear imaging using radioiodine were developed and applied for tracking EVs derived from cell lines. EVs were obtained from supernatant of thyroid cancer cell (Cal62) and natural killer cells (NK92-MI) using sequential ultracentrifuges. Sulfosuccinimidyl-3-(4-hydroxypheynyl) propionate were labeled to membrane of Cal62 and NK92-MI cell derived EVs, then the EVs were labeled with radioiodine (I-131 and I-125) using pre-coated iodination tubes (RI-EVs). In vivo gamma camera images were obtained after intravenous injection of the RI-EVs, and ex vivo biodistribution study was also performed. EVs were labeled with radioiodine and radiochemical purity of the RI-EV was more than 98%. Results of nanoparticle tracking analysis and electron microscopy showed that there was no significant difference in EVs before and after the radioiodine labeling. After intravenous injection of RI-EVs to mice, gamma camera imaging well visualized the real-time biodistribution of the RI-EVs. RI-EVs were mainly visualized at liver, spleen, and lung. Nuclear imaging system of EVs derived from thyroid cancer and NK cells using radioiodine labeling of the EVs was established. Thus, this system might be helpful for in vivo tracking of EVs.

Over the past few decades, various forms of platelet concentrates have evolved with significant clinical utility. The newer generation products, including leukocyte-platelet-rich fibrin (L-PRF) and advanced platelet-rich fibrin (A-PRF), have shown superior biological properties in musculoskeletal regeneration than the first-generation concentrates, such as platelet-rich plasma (PRP) and plasma rich in growth factors. These newer platelet concentrates have a complete matrix of physiological fibrin that acts as a scaffold with a three-dimensional (3D) architecture. Further, it facilitates intercellular signaling and migration, thereby promoting angiogenic, chondrogenic, and osteogenic activities. A-PRF with higher leukocyte inclusion possesses antimicrobial activity than the first generations. Due to the presence of enormous amounts of growth factors and anti-inflammatory cytokines that are released, A-PRF has the potential to replicate the various physiological and immunological factors of wound healing. In addition, there are more neutrophils, monocytes, and macrophages, all of which secrete essential chemotactic molecules. As a result, both L-PRF and A-PRF are used in the management of musculoskeletal conditions, such as chondral injuries, tendinopathies, tissue regeneration, and other sports-related injuries. In addition to this, its applications have been expanded to include the fields of reconstructive cosmetic surgery, wound healing in diabetic patients, and maxillofacial surgeries.

Alopecia is a common medical condition affecting both sexes. Dermal papilla (DP) cells are the primary source of hair regeneration in alopecia patients. Therapeutic applications of extracellular vesicles (EVs) are restricted by low yields, high costs, and their time-consuming collection process. Thus, engineered nanovesicles (eNVs) have emerged as suitable therapeutic biomaterials in translational medicine. We isolated eNVs by the serial extrusion of fibroblasts (FBs) using polycarbonate membrane filters and serial and ultracentrifugation. We studied the internalization, proliferation, and migration of human DP cells in the presence and absence of FB-eNVs. The therapeutic potential of FB-eNVs was studied on ex vivo organ cultures of human hair follicles (HFs) from three human participants. FB-eNVs (2.5, 5, 7.5, and 10 µg/mL) significantly enhanced DP cell proliferation, with the maximum effect observed at 7.5 µg/mL. FB-eNVs (5 and 10 µg/mL) significantly enhanced the migration of DP cells at 36 h. Western blotting results suggested that FB-eNVs contain vascular endothelial growth factor (VEGF)-a. FB-eNV treatment increased the levels of PCNA, pAKT, pERK, and VEGF-receptor-2 (VEGFR2) in DP cells. Moreover, FB-eNVs increased the human HF shaft size in a short duration ex vivo. Altogether, FB-eNVs are promising therapeutic candidates for alopecia.

Adhesive capsulitis shoulder is a common problem of patients presenting with shoulder pain and disability. The approach to such patients includes a variety of modalities. This systematic review evaluates the efficacy of intra-articular injections of platelet-rich plasma (PRP) in the treatment. A literature search was performed between January 2010 and 30 May 2022. MeSH terms used were ‘Platelet-rich plasma’ OR ‘PRP’ AND ‘Frozen shoulder’ OR ‘Adhesive capsulitis shoulder’ OR ‘Periarthritis shoulder’. The search included published articles in the English language involving human subjects. Studies evaluating other types of shoulder disorders, in vitro studies, review articles, animal-model studies, and pre-clinical trials were excluded. The data regarding study characteristics, efficacy, and safety outcomes were analyzed. A total of 11 studies with 347 patients over 10 years were finally included in this review. Most publications were in 2019 and 2020, mostly from India. This review included seven comparative studies, three case series, and one case report. In seven studies, a single intra-articular PRP injection was administered, whereas in the rest of the studies two or multiple injections were given. Only one study demonstrated an equivocal efficacy of PRP and steroid intra-articular injection. The rest all depicted better clinical and functional outcomes with the PRP injection. Only one study compared the outcomes of hydro-dissection treatment in adhesive capsulitis with the intra-articular PRP injection. The rest all either examined PRP alone or compared it with the steroid intra-articular injection. None of the studies showed any major side effects. The intra-articular injections of PRP in the management of adhesive capsulitis of the shoulder provide a new treatment approach. Further studies are required to ascertain the efficacy and safety of the PRP intraarticular injection as a management alternative in adhesive capsulitis.

The two-dimensional (2D) monolayer culture as a conventional method has been widely applied in molecular biology fields, but it has limited capability to recapitulate real cell environments, being prone to misinterpretation with poor prediction of in vivo behavior. Recently, the three-dimensional (3D) spheroid culture has been studied extensively. Spheroids are self-assembled cell aggregates that have biomimicry capabilities. The behavior of thyroid cancer under the 3D spheroid culture environment has been studied; however, there are no reports regarding differences in the degree of thyroid cancer cell differentiation under 2D and 3D culture conditions. This study investigated the expression of thyroid differentiation proteins related to iodide-metabolizing mechanisms in thyroid cancer cells under different culture conditions. Four thyroid cancer cell lines and one thyroid follicular epithelial cell line were grown in adherent 2D cell culture and 3D spheroid culture with agarose-coated plates. We observed changes in proliferation, hypoxia, extracellular matrix (ECM), cytoskeleton, thyroid-specific proteins, and thyroid transcription factors. All cell lines were successfully established in the spheroid following cell aggregation. Proliferation considerably decreased, while hypoxia-inducible factor 1-α(HIF1-α) was promoted in 3D spheroids; moreover, 3D spheroids with thyroid cancers showed diminished thyroid differentiation markers, but thyroid follicular epithelial cells revealed either a maintenance or weak decline of protein expression. We verified that the 3D spheroid culture environment can be similar to in vivo conditions because of its alterations in numerous cellular and functional activities, including morphology, cellular proliferation, viability, hypoxia, ECM, cytoskeleton, and thyroid differentiation, compared to the conventional 2D monolayer culture environment. An in vitro experimental study using 3D spheroid culture is ideal for the faster discovery of new drugs.

Chalcones are a class of privileged scaffolds with high medicinal significance due to the presence of an α,β-unsaturated ketone functionality. Numerous functional modifications of chalcones have been reported, along with their pharmacological behavior. The present review aims to summarize the structures from natural sources, synthesis methods, biological characteristics against infectious and non-infectious diseases, and uses of chalcones over the past decade, and their structure–activity relationship studies are detailed in depth. This critical review provides guidelines for the future design and synthesis of various chalcones. In addition, this could be highly supportive for medicinal chemists to develop more promising candidates for various infectious and non-infectious diseases.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been demonstrated to deliver therapeutic drugs in preclinical studies. However, their use is limited, as they lack the ability to specifically deliver drugs to tumor tissues in vivo. In the present study, we propose the use of a targeting peptide, IL-4R-binding peptide (IL4RPep-1), to specifically deliver intravenously (i.v.) infused EVs to thyroid tumors. In vivo, a xenograft tumor model was treated with either the control peptide (NSSSVDK) or IL4RPep-1-Flamma; mice were fluorescently imaged (FLI) using an in vivo imaging system at 0–3 h post-treatment. EVs (labeled with DiD dye) were conjugated with IL4RPep-1 through a DOPE-NHS linker and administered to mice intravenously. FLI was performed 0–24 h post-injection, and the animals were sacrificed for further experiments. The morphology and size of EVs, the presence of EV markers such as CD63 and ALIX, and the absence of the markers GM130 and Cyto-C were confirmed. In vivo, FLI indicated an accumulation of i.v. injected IL4RPep-1-Flamma at the tumor site 90 min post-injection. No accumulation of NSSSVDK-Flamma was detected. In vivo, IL4RPep-1-EVs targeted the Cal-62 tumor 2 h post-injection. NSSSVDK-EVs were not even detected in the tumor 24 h post-injection. The quantification of FLI showed a significant accumulation of MSC-EVs in the tumor 2 h, 3 h, and 24 h post-injection. Furthermore, ex vivo imaging and an IF analysis confirmed the in vivo findings. Our results demonstrate the use of the IL4RPep-1 peptide as a targeting moiety of EVs for IL-4R-expressing anaplastic thyroid tumors.

A complete redevelopment of the skin remains a challenge in the management of acute and chronic wounds. Recently, the application of extracellular vesicles (EVs) for soft tissue wound healing has received much attention. As fibroblasts are fundamental cells for soft tissues and skin, we investigate the proangiogenic factors in human normal fibroblast-derived EVs (hNF-EVs) and their effects on wound healing. Normal fibroblasts were isolated from human skin tissues and characterized by immunofluorescence (IF) and Western blotting (WB). hNF-EVs were isolated by ultracentrifugation and characterized using transmission electron microscopy and WB. The proangiogenic cargos in hNF-EVs were identified by a TaqMan assay and a protein array. Other in vitro assays, including internalization assays, cell counting kit-8 analysis, scratch wound assays, WBs, and tube formation assays were conducted to assess the effects of hNF-EVs on fibroblasts and endothelial cells. A novel scaffold-free noninvasive delivery of hNF-EVs with or without fibrin glue was applied onto full-thickness skin wounds in mice. The wound healing therapeutical effect of hNF-EVs was assessed by calculating the rate of wound closure and through histological analysis. Isolated hNF was confirmed by verifying the expression of the fibroblast markers vimentin, αSMA, Hsp70, and S100A4. Isolated hNF-EVs showed intact EVs with round morphology, enriched in CD81 and CD63, and devoid of the cell markers GM130, Calnexin, and Cytochrome C. Our TaqMan assay showed that hNF-EVs were enriched in miR130a and miR210, and protein arrays showed enriched levels of the proangiogenic proteins’ vascular endothelial growth factor (VEGF)-D and CXCL8. Next, we found that the internalization of hNF-EVs into hNF increased the proliferation and migration of hNF, in addition to increasing the expression of bFGF, MMP2, and αSMA. The internalization of hNF-EVs into the endothelial cells increased their proliferation and tube formation. A scaffold-free noninvasive delivery of hNF-EVs with or without fibrin glue accelerated the wound healing rate in full-thickness skin wounds in mice, and the treatments increased the cellular density, deposition, and maturation of collagens in the wounds. Moreover, the scaffold-free noninvasive delivery of hNF-EVs with or without fibrin glue increased the VEGF and CD31 expression in the wounds, indicating that hNF-EVs have an angiogenic ability to achieve complete skin regeneration. These findings open up for new treatment strategies to be developed for wound healing. Further, we offer a new approach to the efficient, scaffold-free noninvasive delivery of hNF-EVs to wounds.

This study investigates the role of Sygen® in diabetic peripheral neuropathy, a severe disease that affects the peripheral nervous system in diabetic individuals. This disorder often impacts the lower limbs, causing significant discomfort and, if left untreated, progresses into more serious conditions involving chronic ulcers and even amputation in many cases. Although there are management strategies available, peripheral neuropathies are difficult to treat as they often present multiple causes, especially due to metabolic dysfunction in diabetic individuals. Gangliosides, however, have long been studied and appreciated for their role in neurological diseases. The monosialotetrahexosylganglioside (GM1) ganglioside, popularly known as Sygen, provides beneficial effects such as enhanced neuritic sprouting, neurotrophism, neuroprotection, anti-apoptosis, and anti-excitotoxic activity, being particularly useful in the treatment of neurological complications that arise from diabetes. This product mimics the roles displayed by neurotrophins, improving neuronal function and immunomodulation by attenuating exacerbated inflammation in neurons. Furthermore, Sygen assists in axonal stabilization and keeps nodal and paranodal regions of myelin fibers organized. This maintains an adequate propagation of action potentials and restores standard peripheral nerve function. Given the multifactorial nature of this complicated disorder, medical practitioners must carefully screen the patient to avoid confusion and misdiagnosis. There are several studies analyzing the role of Sygen in neurological disorders. However, the medical literature still needs more robust investigations such as randomized clinical trials regarding the administration of this compound for diabetic peripheral neuropathies, specifically.

Lately, microalgae-based value-added products have been gaining market value because they moderate the dependency on fossil fuel and high-value chemical products. To this end, the purpose of this study was to develop prebiotic products from the microalgae Spirulina sp. The microalgae were isolated from the fresh water and characterized at the molecular level. The dry biomass, chlorophyll content, phycocyanin, cytotoxicity and antimicrobial and antioxidant properties of the isolated strains were analyzed. Moreover, value-added products like Spirulina cake, chocolate, tea, vermicelli and Spirulina juice were made for a vulnerable population due to high nutritive value.

Tissue engineering and regenerative medicine (TERM) have paved a way for treating musculoskeletal diseases in a minimally invasive manner. The regenerative medicine cocktail involves the usage of mesenchymal stem/stromal cells (MSCs), either uncultured or culture-expanded cells along with growth factors, cytokines, exosomes, and secretomes to provide a better regenerative milieu in degenerative diseases. The successful regeneration of cartilage depends on the selection of the appropriate source of MSCs, the quality, quantity, and frequency of MSCs to be injected, and the selection of the patient at an appropriate stage of the disease. However, confirmation on the most favorable source of MSCs remains uncertain to clinicians. The lack of knowledge in the current cellular treatment is uncertain in terms of how beneficial MSCs are in the long-term or short-term (resolution of pain) and improved quality of life. Whether MSCs treatments have any superiority, exists due to sources of MSCs utilized in their potential to objectively regenerate the cartilage at the target area. Many questions on source and condition remain unanswered. Hence, in this review, we discuss the lineage differentiation potentials of various sources of MSCs used in the management of knee osteoarthritis and emphasize the role of tissue engineering in cartilage regeneration.

Stem cells can be defined as the cells that have the capacity to both self-renew and give rise to differentiated cells. Under the right conditions and signals, depending on their origin and bio-plasticity, stem cells can differentiate into multiple cell lineages and develop into various mature cells. Stem cell therapy is a fast-developing branch of medicine that includes the most innovative regenerative therapies for the restoration of cell and tissue function in individuals with severe diseases. Stem cell research has resulted in the emergence of cell-based therapies for disorders that are resistant to conventional drugs and therapies, and they are considered under the category of an Advanced Therapeutic Medicinal Product (ATMP). The FDA and the European Medicines Agency (EMA) devised a new strategy in 2017 with the aim of unifying the standards for development of ATMPs such that it is easy to exchange information at the international level. In this review, we discuss the evolution of mesenchymal stem cell-based therapy as an ATMP in the global and Indian scenarios, along with the guidelines governing their usage and clinical application of these therapeutics.

Orthobiologics are biological materials that are intended for the regeneration of bone, cartilage, and soft tissues. In this review, we discuss the application of orthobiologics in Achilles tendinopathy, more specifically. We explain the concepts and definitions of each orthobiologic and the literature regarding its use in tendon disorders. The biological potential of these materials can be harnessed and administered into injured tissues, particularly in areas where standard healing is disrupted, a typical feature of Achilles tendinopathy. These products contain a wide variety of cell populations, cytokines, and growth factors, which have been shown to modulate many other cells at local and distal sites in the body. Collectively, they can shift the state of escalated inflammation and degeneration to reestablish tissue homeostasis. The typical features of Achilles tendinopathy are failed healing responses, persistent inflammation, and predominant catabolic reactions. Therefore, the application of orthobiologic tools represents a viable solution, considering their demonstrated efficacy, safety, and relatively easy manipulation. Perhaps a synergistic approach regarding the combination of these orthobiologics may promote more significant clinical outcomes rather than individual application. Although numerous optimistic results have been registered in the literature, additional studies and clinical trials are still highly desired to further illuminate the clinical utility and efficacy of these therapeutic strategies in the management of tendinopathies.

Alopecia or baldness is a common diagnosis in clinical practice. Alopecia can be scarring or non-scarring, diffuse or patchy. The most prevalent type of alopecia is non-scarring alopecia, with the majority of cases being androgenetic alopecia (AGA) or alopecia areata (AA). AGA is traditionally treated with minoxidil and finasteride, while AA is treated with immune modulators; however, both treatments have significant downsides. These drawbacks compel us to explore regenerative therapies that are relatively devoid of adverse effects. A thorough literature review was conducted to explore the existing proven and experimental regenerative treatment modalities in non-scarring alopecia. Multiple treatment options compelled us to classify them into growth factor-rich and stem cell-rich. The growth factor-rich group included platelet-rich plasma, stem cell-conditioned medium, exosomes and placental extract whereas adult stem cells (adipose-derived stem cell-nano fat and stromal vascular fraction; bone marrow stem cell and hair follicle stem cells) and perinatal stem cells (umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), Wharton jelly-derived MSCs (WJ-MSCs), amniotic fluid-derived MSCs (AF-MSCs), and placental MSCs) were grouped into the stem cell-rich group. Because of its regenerative and proliferative capabilities, MSC lies at the heart of regenerative cellular treatment for hair restoration. A literature review revealed that both adult and perinatal MSCs are successful as a mesotherapy for hair regrowth. However, there is a lack of standardization in terms of preparation, dose, and route of administration. To better understand the source and mode of action of regenerative cellular therapies in hair restoration, we have proposed the “À La Mode Classification”. In addition, available evidence-based cellular treatments for hair regrowth have been thoroughly described.

Hepatotoxicity is a major global public health concern. Despite advances in modern medicine, the demerits of chemically prepared drugs outweigh their merits. In addition, the treatment of liver diseases based on modern medical principles has been found to produce several undesired side effects. Therefore, the exploration of medicinal plants has gained worldwide attention for treating various diseases, including liver diseases, owing to their potential efficacy and cost effectiveness. Several plants, including Andrographis paniculata, Bauhinia purpurea, Commelina nudiflora, Dillenia suffruticosa, Elaeis guineensis, Lygodium microphyllum, and Nephrolepis biserrata, have been reported with hepatoprotection. Moreover, these plants have been shown to play a vital role in ameliorating cellular damage because they contain several phytochemicals, including alkaloids, saponins, flavonoids, tannins, terpenoids, steroids, polyphenols, and diterpenoid lactones. The following antioxidant, anti-inflammatory, immunomodulatory, and hepatoprotective compounds have been found in these plants: andrographolide, rosmarinic acid, phenol, eugenol, 9,12-octadecadienoic, n-hexadecanoic acid, dihydroxy dimethoxy flavone, sitosterol, demethoxycurcumin, quercetin, linoleic acid, stigmasterol, kojic acid, indole-2-one, α-terpinol, linalool, kaempferol, catechin, ellagic acid, and oleanolic acid. This paper aimed to provide an in-depth review of in vivo studies on Malaysian medicinal plants possessing hepatoprotective properties, phytochemical ingredients, and antioxidant mechanisms, with an emphasis on the species proven particularly useful for treating hepatic disorders.

Human bone marrow (BM) has been highlighted as a promising source of mesenchymal stromal cells (MSCs) containing various growth factors and cytokines that can be potentially utilized in regenerative procedures involving cartilage and bone. However, the proportion of MSCs in the nucleated cell population of BM is only around 0.001% to 0.01% thereby making the harvesting and processing technique crucial for obtaining optimal results upon its use in various regenerative processes. Although several studies in the literature have given encouraging results on the utility of BM aspiration concentrate (BMAC) in various regenerative procedures, there is a lack of consensus concerning the harvesting variables such as choice of anesthetic agent to be used, site of harvest, size of the syringe to be used, anticoagulant of choice, and processing variables such as centrifugation time, and speed. In this review article, we aim to discuss the variables in the harvesting and processing technique of BMAC and their impact on the yield of MSCs in the final concentrate obtained from them.

Probiotics are living microbes that play a significant role in protecting the host in various ways. Gut microbiota is one of the key players in maintaining homeostasis. Cancer is considered one of the most significant causes of death worldwide. Although cancer treatment has received much attention in recent years, the number of people suffering from neoplastic syndrome continues to increase. Despite notable improvements in the field of cancer therapy, tackling cancer has been challenging due to the multiple properties of cancer cells and their ability to evade the immune system. Probiotics alter the immunological and cellular responses by enhancing the epithelial barrier and stimulating the production of anti-inflammatory, antioxidant, and anticarcinogenic compounds, thereby reducing cancer burden and growth. The present review focuses on the various mechanisms underlying the role of probiotics in the prevention and treatment of cancer.

Study Design: Meta-analysis. Objectives: We aimed to analyze the impact of cultured expansion of autologous mesenchymal stromal cells (MSCs) in the management of osteoarthritis of the knee from randomized controlled trials (RCTs) available in the literature. Materials and Methods: We conducted independent and duplicate electronic database searches including PubMed, Embase, Web of Science, and Cochrane Library until August 2021 for RCTs analyzing the efficacy and safety of culture-expanded compared to non-cultured autologous MSCs in the management of knee osteoarthritis. The Visual Analog Score (VAS) for pain, Western Ontario McMaster University’s Osteoarthritis Index (WOMAC), Lysholm score, Knee Osteoarthritis Outcome Score (KOOS), and adverse events were the analyzed outcomes. Analysis was performed in R-platform using OpenMeta [Analyst] software. Results: Overall, 17 studies involving 767 patients were included for analysis. None of the studies made a direct comparison of the culture expanded and non-cultured MSCs, hence we pooled the results of all the included studies of non-cultured and cultured types of MSC sources and made a comparative analysis of the outcomes. At six months, culture expanded MSCs showed significantly better improvement (p < 0.001) in VAS outcome. Uncultured MSCs, on the other hand, demonstrated significant VAS improvement in the long term (12 months) in VAS (p < 0.001), WOMAC (p = 0.025), KOOS score (p = 0.016) where cultured-expanded MSCs failed to demonstrate a significant change. Culturing of MSCs did not significantly increase the complications noted (p = 0.485). On sub-group analysis, adipose-derived uncultured MSCs outperformed culture-expanded MSCs at both short term (six months) and long term (12 months) in functional outcome parameters such as WOMAC (p < 0.001, p = 0.025), Lysholm (p < 0.006), and KOOS (p < 0.003) scores, respectively, compared to their controls. Conclusions: We identified a void in literature evaluating the impact of culture expansion of MSCs for use in knee osteoarthritis. Our indirect analysis of literature showed that culture expansion of autologous MSCs is not a necessary factor to obtain superior results in the management of knee osteoarthritis. Moreover, while using uncultured autologous MSCs, we recommend MSCs of adipose origin to obtain superior functional outcomes. However, we urge future trials of sufficient quality to validate our findings to arrive at a consensus on the need for culture expansion of MSCs for use in cellular therapy of knee osteoarthritis.

The periosteum, with its outer fibrous and inner cambium layer, lies in a dynamic environment with a niche of pluripotent stem cells for their reparative needs. The inner cambium layer is rich in mesenchymal progenitors, osteogenic progenitors, osteoblasts, and fibroblasts in a scant collagen matrix environment. Their role in union and remodeling of fracture is well known. However, the periosteum as a source of mesenchymal stem cells has not been explored in detail. Moreover, with the continuous expansion of techniques, newer insights have been acquired into the roles and regulation of these periosteal cells. From a therapeutic standpoint, the periosteum as a source of tissue engineering has gained much attraction. Apart from its role in bone repair, analysis of the bone-forming potential of periosteum-derived stem cells is lacking. Hence, this article elucidates the role of the periosteum as a potential source of mesenchymal stem cells along with their capacity for osteogenic and chondrogenic differentiation for therapeutic application in the future.

Stem cell therapy has been considered a promising strategy in the management of both type I and type II diabetes mellitus (DM) because of its immunomodulatory and regenerative capability to restore the beta cell number and function. Various modalities of cellular therapy like transplantation of pancreatic islet cells, transplantation of pancreatic ductal stem cells, and mesenchymal stromal cell transplantation have been tried, and the modality is undergoing rapid advancements that may become the reality in the near future. In the course of its evolution, it is essential to have a comprehensive summary of the progress for a greater capacity to refine our future directives. With technological developments like data mining, graphic drawing, and information analytics combined with computational statistics, visualization of scientific metrology has become a reality. With a newer perspective, we intend to use scientometric tools including text mining, co-word analysis, word frequency analysis, co-citation analysis, cluster network analysis, to perform a systematic and comprehensive analysis of the research trend in stem cell therapy in the management of DM over the past three decades (1990–2020) and to identify the future research hotspots.

Angiogenesis is defined as the generation of new blood vessels or the sprouting of endothelial cells from a pre-existing vascular network. Angiogenesis occurs during the growth and development of an organism, the response of organs or tissues to injury, and during cancer development and progression. The majority of studies on stem-cell-derived extracellular vesicles (EVs) have used cell lines, and have primarily focused on well-known solitary proteins. Here, we isolated stem cells from human adipose tissue (ADSCs), and we isolated EVs from them (ADSC-EVs). The ADSC-EVs were characterised and 20 angiogenic proteins were analysed using an angiogenic antibody array. Furthermore, we analysed the ability of ADSC-EVs to induce angiogenesis in vitro and in vivo. ADSC-EVs were positive for CD81 and negative for GM130, calnexin, and cytochrome-C. ADSC-EVs showed typical EV spherical morphology and were ~200 nm in size. ADSC-EVs were found to contain angiogenic proteins as cargo, among which interleukin 8 (IL-8) was the most abundant, followed by chemokine (C-C motif) ligand 2 (CCL2), a tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, and vascular endothelial growth factor-D (VEGF-D). ADSC-EVs treatment increased the proliferation, migration, total vessel length, total number of junctions, and junction density of endothelial cells in vitro. The results of an in vivo Matrigel plug assay revealed that ADSC-EVs induced more blood vessels in the Matrigel compared with the control. These results demonstrate that ADSC-EVs contain angiogenic proteins as cargo and promote angiogenesis in vitro and in vivo. Therefore, ADSC-EVs have potential for therapeutic use in ischaemia.

A new approach for using extracellular vesicles (EVs) to deliver tyrosine kinase inhibitors (TKIs) to enhance iodine avidity in radioactive iodine-refractory thyroid cancer is needed. We isolated and characterized primary human adipose-derived stem cells (ADSCs) and isolated their EVs. The EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. A new TKI was loaded into the EVs by incubation (37 °C; 10 min) or sonication (18 cycles; 4 s per cycle) with 2 s intervals and a 2 min ice bath every six cycles. TKI loading was confirmed and measured by mass spectrometry. EV uptake into radioactive iodine-refractory thyroid cancer cells (SW1736 cells) was confirmed by microscopy. We treated the SW1736 cells with vehicle, TKI, or TKI-loaded EVs (sonication TKI-loaded EVs [EVsTKI(S)]) and examined the expression of iodide-metabolizing proteins and radioiodine uptake in the SW1736 cells. ADSCs cells showed >99% of typical stem cell markers, such as CD90 and CD105. The EVs displayed a round morphology, had an average size of 211.4 ± 3.83 nm, and were positive for CD81 and Alix and negative for cytochrome c. The mass spectrometry results indicate that the sonication method loaded ~4 times more of the TKI than did the incubation method. The EVsTKI(S) were used for further experiments. Higher expression levels of iodide-metabolizing mRNA and proteins in the EVsTKI(S)-treated SW1736 cells than in TKI-treated SW1736 cells were confirmed. EVsTKI(S) treatment enhanced 125I uptake in the recipient SW1736 cells compared with free-TKI treatment. This is the first study that demonstrated successful delivery of a TKI to radioactive iodine-refractory thyroid cancer cells using EVs as the delivery vehicle. This approach can revert radioiodine-resistant thyroid cancer cells back to radioiodine-sensitive thyroid cancer cells.

Extracellular vesicles (EVs) are small membrane-based nanovesicles naturally released from cells. Extracellular vesicles mimetics (EVMs) are artificial vesicles engineered from cells or in combination with lipid materials, and they mimic certain characteristics of EVs. As such, EVs facilitate intracellular communication by carrying and delivering biological materials, such as proteins, lipids, and nucleic acids, and they have been found to find organ tropism in preclinical studies. Because of their native structure and characteristics, they are considered promising drug carriers for future clinical use. This review outlines the origin and composition of natural EVs and EVM engineering and internalization. It then details different loading approaches, with examples of the drug delivery of therapeutic molecules. In addition, the advantages and disadvantages of loading drugs into EVs or EVMs as a drug delivery system are discussed. Finally, the advantages of EVMs over EVs and the future clinical translation of EVM-based drug delivery platforms are outlined.

Hair loss is a common medical problem affecting both males and females. Dermal papilla (DP) cells are the ultimate reservoir of cells with the potential of hair regeneration in hair loss patients. Here, we analyzed the role of macrophage-derived Wnts (3a and 7b) and macrophage extracellular vesicles (MAC-EVs) in promoting hair growth. We studied the proliferation, migration, and expression of growth factors of human-DP cells in the presence or absence of MAC-EVs. Additionally, we tested the effect of MAC-EV treatment on hair growth in a mouse model and human hair follicles. Data from western blot and flow cytometry showed that MAC-EVs were enriched with Wnt3a and Wnt7b, and more than 95% were associated with their membrane. The results suggest that Wnt proteins in MAC-EVs activate the Wnt/β-catenin signaling pathways, which leads to activation of transcription factors (Axin2 and Lef1). The MAC-EVs significantly enhanced the proliferation, migration, and levels of hair-inductive markers of DP cells. Additionally, MAC-EVs phosphorylated AKT and increased the levels of the survival protein Bcl-2. The DP cells treated with MAC-EVs showed increased expression of vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF). Treatment of Balb/c mice with MAC-EVs promoted hair follicle (HF) growth in vivo and also increased hair shaft size in a short period in human HFs. Our findings suggest that MAC-EV treatment could be clinically used as a promising novel anagen inducer in the treatment of hair loss.

Exosomes derived from mesenchymal stem cells (MSCs) have been evaluated for their potential to be used as drug delivery vehicles. Synthetically personalized exosome mimetics (EMs) could be the alternative vesicles for drug delivery. In this study, we aimed to isolate EMs from human MSCs. Cells were mixed with paclitaxel (PTX) and PTX-loaded EMs (PTX-MSC-EMs) were isolated and evaluated for their anticancer effects against breast cancer. EMs were isolated from human bone marrow-derived MSCs. MSCs (4 × 106 cells/mL) were mixed with or without PTX at different concentrations in phosphate-buffered saline (PBS) and serially extruded through 10-, 5-, and 1-μm polycarbonate membrane filters using a mini-extruder. MSCs were centrifuged to remove debris and the supernatant was filtered through a 0.22-μm filter, followed by ultracentrifugation to isolate EMs and drug-loaded EMs. EMs without encapsulated drug (MSC-EMs) and those with encapsulated PTX (PTX-MSC-EMs) were characterized by western blotting, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). The anticancer effects of MSC-EMs and PTX-MSC-EMs were assessed with breast cancer (MDA-MB-231) cells both in vitro and in vivo using optical imaging. EMs were isolated by the extrusion method and ultracentrifugation. The isolated vesicles were positive for membrane markers (ALIX and CD63) and negative for golgi (GM130) and endoplasmic (calnexin) marker proteins. NTA revealed the size of MSC-EM to be around 149 nm, while TEM confirmed its morphology. PTX-MSC-EMs significantly (p < 0.05) decreased the viability of MDA-MB-231 cells in vitro at increasing concentrations of EM. The in vivo tumor growth was significantly inhibited by PTX-MSC-EMs as compared to control and/or MSC-EMs. Thus, MSC-EMs were successfully isolated using simple procedures and drug-loaded MSC-EMs were shown to be therapeutically efficient for the treatment of breast cancer both in vitro and in vivo. MSC-EMs may be used as drug delivery vehicles for breast cancers.

Exosomes are natural nano-sized membrane vesicles that have garnered recent interest owing to their potential as drug delivery vehicles. Though exosomes are effective drug carriers, their production and in vivo biodistribution are still not completely elucidated. We analyzed the production of exosome mimetics (EMs) from red blood cells (RBCs) and the radio-labeling of the RBC-EMs for in vivo imaging. Engineered EMs from RBCs were produced in large-scale by a one-step extrusion method, and further purified by density-gradient centrifugation. RBC-EMs were labeled with technetium-99m (99mTc). For non-invasive imaging, 99mTc (free) or 99mTc-RBC-EMs were injected in mice, and their biodistribution was analyzed by gamma camera imaging. Animals were sacrificed, and organs were collected for further biodistribution analysis. RBC-EMs have similar characteristics as the RBC exosomes but have a 130-fold higher production yield in terms of particle numbers. Radiochemical purity of 99mTc-RBC-EMs was almost 100% till 2 h reduced to 97% at 3 h. Radio-labeling did not affect the size and morphology of RBC-EMs. In contrast to free 99mTc, in vivo imaging of 99mTc-RBC-EMs in mice showed higher uptake in the liver and spleen, and no uptake in the thyroid. Ex vivo imaging confirmed the in vivo findings. Furthermore, fluorescent imaging confirmed the nuclear imaging findings. Immunofluorescent imaging revealed that the hepatic uptake of RBC-EMs was significantly mediated by kupffer cells (resident hepatic macrophages). Our results demonstrate a simple yet large-scale production method for a novel type of RBC-EMs, which can be effectively labeled with 99mTc, and feasibly monitored in vivo by nuclear imaging. The RBC-EMs may be used as in vivo drug delivery vehicles.

ObjectiveGlioblastoma is a highly aggressive primary brain tumor that is resistant to radiotherapy and chemotherapy. Natural killer (NK) cells have been used to treat incurable cancers. Recent studies have investigated the effectiveness of NK-cell-derived exosomes (NK-Exo) for treating incurable cancers such as melanoma, leukemia, and neuroblastoma; however, NK-Exo have not been used to treat glioblastoma. In the present study, we investigated the antitumor effects of NK-Exo against aggressive glioblastoma both in vitro and in vivo and determined the tumor-targeting ability of NK-Exo by performing fluorescence imaging.MethodsU87/MG cells were transfected with the enhanced firefly luciferase (effluc) and thy1.1 genes; thy1.1-positive cells were selected using microbeads. U87/MG/F cells were assessed by reverse transcription polymerase chain reaction (RT-PCR), western blotting, and luciferase-activity assays. NK-Exo were isolated by ultracentrifugation, purified by density gradient centrifugation, and characterized by transmission electron microscopy, dynamic light scattering (DLS), nanoparticle-tracking analysis (NTA), and western blotting. Cytokine levels in NK-Exo were compared to those in NK cells and NK-cell medium by performing an enzyme-linked immunosorbent assay (ELISA). NK-Exo-induced apoptosis of cancer cells was confirmed by flow cytometry and western blotting. In vivo therapeutic effects and specificity of NK-Exo against glioblastoma were assessed in a xenograft mouse model by fluorescence imaging. Xenograft mice were treated with NK-Exo, which was administered seven times through the tail vein. Tumor growth was monitored by bioluminescence imaging (BLI), and tumor volume was measured by ultrasound imaging. The mice were intraperitoneally injected with dextran sulfate 2 h before NK-Exo injection to decrease the liver uptake and increase the tumor specificity of NK-Exo.ResultsRT-PCR and western blotting confirmed the gene and protein expression of effluc in U87/MG/F cells, with the bioluminescence activity of U87/MG/F cells increasing with an increase in cell number. NTA and DLS results indicated that the size of NK-Exo was ~100 nm, and the western blot results confirmed that NK-Exo expressed exosome markers CD63 and Alix. We confirmed the in vitro cytotoxic effects of NK-Exo on U87/MG/F cells by performing BLI, and the killing effect on U87/MG and U87MG/F cells was measured by CCK-8 and MTT assays (p

Extracellular vesicles (EVs) are currently being considered as promising drug delivery vehicles. EVs are naturally occurring vesicles that exhibit many characteristics favorable to serve as drug delivery vehicles. In addition, EVs have inherent properties for treatment of cancers and other diseases. For research and clinical translation of use of EVs as drug delivery vehicles, in vivo tracking of EVs is essential. The latest molecular imaging techniques enable the tracking of EVs in living animals. However, each molecular imaging technique has its certain advantages and limitations for the in vivo imaging of EVs; therefore, understanding the molecular imaging techniques is essential to select the most appropriate imaging technology to achieve the desired imaging goal. In this review, we summarize the characteristics of EVs as drug delivery vehicles and the molecular imaging techniques used in visualizing and monitoring EVs in in vivo environments. Furthermore, we provide a perceptual vision of EVs as drug delivery vehicles and in vivo monitoring of EVs using molecular imaging technologies.

Molecular imaging is a relatively new discipline that allows visualization, characterization, and measurement of the biological processes in living subjects, including humans, at a cellular and molecular level. The interaction between cancer cells and natural killer (NK) cells is complex and incompletely understood. Despite our limited knowledge, progress in the search for immune cell therapies against cancer could be significantly improved by dynamic and non-invasive visualization and tracking of immune cells and by visualization of the response of cancer cells to therapies in preclinical and clinical studies. Molecular imaging is an essential tool for these studies, and a multimodal molecular imaging approach can be applied to monitor immune cells in vivo, for instance, to visualize therapeutic effects. In this review, we discuss the usefulness of NK cells in cancer therapies and the preclinical and clinical usefulness of molecular imaging in NK cell-based therapies. Furthermore, we discuss different molecular imaging modalities for use with NK cell-based therapies, and their preclinical and clinical applications in animal and human subjects. Molecular imaging has contributed to the development of NK cell-based therapies against cancers in animal models and to the refinement of current cell-based cancer immunotherapies. Developing sensitive and reproducible non-invasive molecular imaging technologies for in vivo NK cell monitoring and for real-time assessment of therapeutic effects will accelerate the development of NK cell therapies.

ObjectiveNatural killer (NK) cells represent the third largest population of lymphocytes, and they play an important role in immune surveillance against tumors. The lungs are a common metastatic site for anaplastic thyroid cancer (ATC), and metastasis is one of the most frequent causes of mortality in this type of cancer. In the current study, we evaluated the effects of NK cell-based immunotherapy for pulmonary metastasis of ATC and determined how it affects the effector molecules of NK cells.MethodsHuman NK cells (NK-92MI) were retrovirally transduced to express the effluc gene. Human ATC cells (CAL-62) were transduced with the effluc and Rluc genes. The cytotoxicity of NK cells against CAL-62 cells was assessed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay system. Pulmonary metastases of ATC were developed by i.v. injection of CAL-62, and metastasis growth was monitored using bioluminescence imaging (BLI). To treat the metastases, five million NK-92MI cells were injected twice into the caudal vein of nude mice. To assess the targetability of NK cells to ATC tumors, NK-92MI cells expressing the effluc gene (NK/F) were administered through the tail vein of nude mice with a pulmonary metastasis or tumor xenograft. BLI was subsequently performed at 1, 3, 24, and 48 h.ResultsNK/F and CAL-62 cells expressing the effluc or Rluc gene (CAL-62/F, CAL-62/R) were successfully established. Expression of the effluc and Rluc genes in NK/F, CAL-62/F, and CAL-62/R cells was verified by RT-polymerase chain reaction, western blotting, and luciferase assay. After coculture of NK-92MI and CAL-62/F cells for 24 h, the BLI signal intensity of CAL-62/F cells proportionally decreased with the number of cocultured NK cells. An ATC pulmonary metastasis mouse model was successfully generated, and NK cells significantly inhibited the growth of the metastasis (p

CXCR4, the stromal cell-derived factor-1 receptor, plays an important role in the migration of hematopoietic progenitor/stem cells to injured and inflamed areas. Noninvasive cell tracking methods could be useful for monitoring cell fate. Therefore, in this study, we evaluated the efficacy of an intravenous infusion of genetically engineered mesenchymal stem cells (MSCs) overexpressing CXC chemokine receptor 4 (CXCR4) to home to the tumor, by optical imaging. We constructed a retroviral vector containing CXCR with dual reporter genes, eGFP and Fluc2, under the control of an EF1α promoter (pBABE-EF1α-CXCR4-eGFP-IRES-Fluc2). We also developed an eGFP-Fluc2 construct in the Retro-X retroviral vector (Retro-X-eGFP-Fluc2). MSCs were transduced with retroviruses to generate CXCR4-overexpressing MSCs (MSC-CXCR4/Fluc2) and MSCs (MSC/Fluc2). CXCR4 mRNA and protein expression was confirmed by RT-PCR and Western blotting, respectively, and it was higher in MSC-CXCR4/Fluc2 than in naive MSCs. eGFP expression was confirmed by confocal microscopy. The transfected MSC-CXCR4/Fluc2 cells showed higher migratory capacity than naive MSCs observed in Transwell migration assay. The in vivo migration of CXCR4-overexpressing MSCs to MDAMB231/Rluc tumor model by BLI imaging was also confirmed. Intravenous delivery of genetically modified MSCs overexpressing CXCR4 with a Fluc2 reporter gene may be a useful, noninvasive BLI imaging tool for tracking cell fate.

Anaplastic thyroid cancer (ATC) is the most aggressive malignancy of the thyroid, during which undifferentiated tumors arise from the thyroid follicular epithelium. ATC has a very poor prognosis due to its aggressive behavior and poor response to conventional therapies. Gene-directed enzyme/prodrug therapy using genetically engineered mesenchymal stromal cells (MSC) is a promising therapeutic strategy. The doxycycline (DOX)-controlled Tet inducible system is the most widely utilized regulatory system and could be a useful tool for therapeutic gene-based therapies. For example, use a synthetic "tetracycline-on" switch system to control the expression of the therapeutic gene thymidine kinase, which converts prodrugs to active drugs. The aim of this study was to develop therapeutic MSCs, harboring an inducible suicide gene, and to validate therapeutic gene expression using optical molecular imaging of ATC. We designed the Tet-On system using a retroviral vector expressing herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Mouse bone marrow-derived mesenchymal stromal cells (BM-MSC) were transduced using this system with (MSC-Tet-TK/Fluc2) or without (MSC-TK/Fluc) the Tet-On system. Transduced cells were screened and characterized. Engineered MSCs were co-cultured with ATC (CAL62/Rluc) cells in the presence of the prodrug ganciclovir (GCV) and stimulated with DOX. The efficiency of cell killing monitored by assessing Rluc (CAL62/Rluc) and Fluc (MSC-Tet-TK/Fluc and MSC-TK/Fluc) activities using IVIS imaging. Fluc activity increased in MSC-Tet-TK/Fluc cells in a dose dependent manner following DOX treatment (R2 = 0.95), whereas no signal was observed in untreated cells. eGFP could also be visualized after induction with DOX, and the HSV1-TK protein could be detected by western blotting. In MSC-TK/Fluc cells, the Fluc activity increased with increasing cell number (R2 = 0.98), and eGFP could be visualized by fluorescence microscopy. The Fluc activity and cell viability of MSC-Tet-TK/Fluc and MSC-TK/Fluc cells decreased significantly following GCV treatment. A bystander effect of the therapeutic cells confirmed in co-cultures of CAL62 cells, an anaplastic thyroid cancer cell line, with either MSC-Tet-TK/Fluc cells or MSC-TK/Fluc cells. The Rluc activity in MSC-Tet-TK/Fluc co-cultures, derived from the CAL62/Rluc cells, decreased significantly with GCV treatment of DOX treated cultures, whereas no significant changes were observed in untreated cultures. In addition, the Fluc activity of MSC-Tet-TK/Fluc cells also decreased significantly with DOX treatment whereas no signal was present in untreated cultures. A bystander effect also be demonstrated in co-cultures with MSC-TK/Fluc cells and CAL62/Rluc; both the Rluc activity and the Fluc activity were significantly decreased following GCV treatment. We have successfully developed a Tet-On system of gene-directed enzyme/prodrug delivery using MSCs. We confirmed the therapeutic bystander effect in CAL62/Rluc cells with respect to MSC-Tet-TK/Fluc and MSC-TK/Fluc cells after GCV treatment with and without DOX. Our results confirm the therapeutic efficiency of a suicide gene, with or without the Tet-On system, for ATC therapy. In addition, our findings provide an innovative therapeutic approach for using the Tet-On system to eradicate tumors by simple, repeated administration of MSC-Tet-TK/Fluc cells with DOX and GCV.

Vimentin is an intermediate filament protein, predominantly expressed in cells of mesenchymal origin, although its aberrant expression is seen in many carcinomas during epithelial mesenchymal transition. In cancer, vimentin expression is associated with the transition from a more differentiated epithelial phenotype to a dedifferentiated state. In view of the perceived role of keratins (Ks) as regulators of differentiation in epithelia, it was important to understand whether vimentin modulates differentiation through the reprogramming of keratins, in transformed cells. To address this, vimentin was stably downregulated in oral cancer derived cells. Further, global keratin profiling was performed after high salt keratin extraction. K5/K14 pair was found to be significantly downregulated, both at protein and mRNA levels upon vimentin downregulation. The previous study from our laboratory has shown a role of the K5/K14 pair in proliferation and differentiation of squamous epithelial cells. Vimentin depleted cells showed an increase in the differentiation state, marked by an increase in the levels of differentiation specific markers K1, involucrin, filaggrin and loricrin while its proliferation status remained unchanged. Rescue experiments with the K5/K14 pair overexpressed in vimentin knockdown background resulted in decreased differentiation state. ΔNp63 emerged as one of the indirect targets of vimentin, through which it modulates the expression levels of K5/K14. Further, immunohistochemistry showed a significant correlation between high vimentin-K14 expression and recurrence/poor survival in oral cancer patients. Thus, in conclusion, vimentin regulates the differentiation switch via modulation of K5/K14 expression. Moreover, vimentin-K14 together may prove to be the novel markers for the prognostication of human oral cancer.

Colorectal cancer is the most common cancer in both men and women and the second most common cause of cancer-related deaths. Suicide gene-based therapy with suicide gene-transduced mesenchymal stem cells (MSCs) is a promising therapeutic strategy. A tetracycline-controlled Tet-On inducible system used to regulate gene expression may be a useful tool for gene-based therapies. The aim of this study was to develop therapeutic MSCs with a suicide gene that is induced by an artificial stimulus, to validate therapeutic gene expression, and to monitor the MSC therapy for colon cancer using optical molecular imaging. For our study, we designed the Tet-On system using a retroviral vector and developed a response plasmid RetroX-TRE (tetracycline response element) expressing a mutant form of herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Bone marrow-derived MSCs were transduced using a RetroX-Tet3G (Clontech, CA, USA) regulatory plasmid and RetroX-TRE-HSV1-sr39TK-eGFP-IRES-Fluc2, for a system with a Tet-On (MSC-Tet-TK/Fluc2 or MSC-Tet-TK) or without a Tet-On (MSC-TK/Fluc2 or MSC-TK) function. Suicide gene engineered MSCs were co-cultured with colon cancer cells (CT26/Rluc) in the presence of the prodrug ganciclovir (GCV) after stimulation with or without doxycycline (DOX). Treatment efficiency was monitored by assessing Rluc (CT26/Rluc) and Fluc (MSC-Tet-TK and MSC-TK) activity using optical imaging. The bystander effect of therapeutic MSCs was confirmed in CT26/Rluc cells after GCV treatment. Rluc activity in CT26/Rluc cells decreased significantly with GCV treatment of DOX(+) cells (p < 0.05 and 0.01) whereas no significant changes were observed in DOX(−) cells. In addition, Fluc activity in also decreased significantly with DOX(+) MSC-Tet-TK cells, but no signal was observed in DOX(−) cells. In addition, an MSC-TK bystander effect was also confirmed. We assessed therapy with this system in a colon cancer xenograft model (CT26/Rluc). We successfully transduced cells and developed a Tet-On system with the suicide gene HSV1-sr39TK. Our results confirmed the therapeutic efficiency of a suicide gene with the Tet-On system for colon cancer. In addition, our results provide an innovative therapeutic approach using the Tet-On system to eradicate tumors by administration of MSC-Tet-TK cells with DOX and GCV.

BACKGROUND: Keratins are cytoplasmic intermediate filament proteins expressed in tissue specific and differentiation dependent manner. Keratins 8 and 18 (K8 and K18) are predominantly expressed in simple epithelial tissues and perform both mechanical and regulatory functions. Aberrant expression of K8 and K18 is associated with neoplastic progression, invasion and poor prognosis in human oral squamous cell carcinomas (OSCCs). K8 and K18 undergo several post-translational modifications including phosphorylation, which are known to regulate their functions in various cellular processes. Although, K8 and K18 phosphorylation is known to regulate cell cycle, cell growth and apoptosis, its significance in cell migration and/or neoplastic progression is largely unknown. In the present study we have investigated the role of K8 phosphorylation in cell migration and/or neoplastic progression in OSCC. METHODOLOGY AND PRINCIPAL FINDINGS: To understand the role of K8 phosphorylation in neoplastic progression of OSCC, shRNA-resistant K8 phospho-mutants of Ser73 and Ser431 were overexpressed in K8-knockdown human AW13516 cells (derived from SCC of tongue; generated previously). Wound healing assays and tumor growth in NOD-SCID mice were performed to analyze the cell motility and tumorigenicity respectively in overexpressed clones. The overexpressed K8 phospho-mutants clones showed significant increase in cell migration and tumorigenicity as compared with K8 wild type clones. Furthermore, loss of K8 Ser73 and Ser431 phosphorylation was also observed in human OSCC tissues analyzed by immunohistochemistry, where their dephosphorylation significantly correlated with size, lymph node metastasis and stage of the tumor. CONCLUSION AND SIGNIFICANCE: Our results provide first evidence of a potential role of K8 phosphorylation in cell migration and/or tumorigenicity in OSCC. Moreover, correlation studies of K8 dephosphorylation with clinico-pathological parameters of OSCC patients also suggest its possible use in prognostication of human OSCC.