Investigating the anticancer properties of six benzothiazolopyrimidine derivatives on colon carcinoma cells, in vitro and in vivo.

Colorectal cancer (CRC) is the third most common cancer in the world. Despite considerable improvements in the treatment of this cancer, further research to discover novel and more effective agents is ongoing. In this study, possible cytotoxic and apoptotic properties of six benzothiazolopyrimidine derivatives were studied. To assess the IC50 values of these agents, MTT assay was performed on HCT 116, CT26, and NIH/3T3 cells. Moreover, cell death mechanism induced by studied compounds was evaluated by PI/annexin V staining. Then, based on molecular docking results and in vitro experiments, the compounds with the highest anticancer properties were further analyzed in vivo in a mouse model of CRC. MTT results indicated that BTP(1) and BTP(4) had the highest selective cytotoxicity on colorectal cancer cells. Furthermore, flow cytometry results demonstrated a considerable increase in the percentage of the early apoptotic cells in BTP(1)- and BTP(4)-treated groups. In vivo studies confirmed the antitumor properties of the two compounds by a significant regression in tumor size of BTP(1)- and BTP(4)-treated mice compared to control groups. Histopathological examination of tumor tissues showed an increased number of apoptotic cells in these two groups compared to the control animals. Additionally, hematoxylin and eosin staining of the spleen and liver of treated mice did not exhibit considerable tissue damage. Thus, BTP(1) and BTP(4) can be considered promising agents in the treatment of colorectal cancer, although further experiments are required to assess their mechanism of action before their application in clinical studies.

Development of a bacterial cellulose-gelatin composite as a suitable scaffold for cardiac tissue engineering.

PURPOSE: Cardiac tissue engineering is suggested as a promising approach to overcome problems associated with impaired myocardium. This is the first study to investigate the use of BC and gelatin for cardiomyocyte adhesion and growth. METHODS: Bacterial cellulose (BC) membranes were produced by Komagataeibacter xylinus and coated or mixed with gelatin to make gelatin-coated BC (BCG) or gelatin-mixed BC (mBCG) scaffolds, respectively. BC based-scaffolds were characterized via SEM, FTIR, XRD, and AFM. Neonatal rat-ventricular cardiomyocytes (nr-vCMCs) were cultured on the scaffolds to check the capability of the composites for cardiomyocyte attachment, growth and expansion. RESULTS: The average nanofibrils diameter in all scaffolds was suitable (~ 30-65 nm) for nr-vCMCs culture. Pore diameter (≥ 10 µm), surface roughness (~ 182 nm), elastic modulus (0.075 ± 0.015 MPa) in mBCG were in accordance with cardiomyocyte requirements, so that mBCG could better support attachment of nr-vCMCs with high concentration of gelatin, and appropriate surface roughness. Also, it could better support growth and expansion of nr-vCMCs due to submicron scale of nanofibrils and proper elasticity (~ 0.075 MPa). The viability of nr-vCMCs on BC and BCG scaffolds was very low even at day 2 of culture (~ ≤ 40%), but, mBCG could promote a metabolic active state of nr-vCMCs until day 7 (~ ≥ 50%). CONCLUSION: According to our results, mBCG scaffold was the most suitable composite for cardiomyocyte culture, regarding its physicochemical and cell characteristics. It is suggested that improvement in mBCG stability and cell attachment features may provide a convenient scaffold for cardiac tissue engineering.

Elesclomol, a copper-transporting therapeutic agent targeting mitochondria: from discovery to its novel applications.

Copper (Cu) is an essential element that is involved in a variety of biochemical processes. Both deficiency and accumulation of Cu are associated with various diseases; and a high amount of accumulated Cu in cells can be fatal. The production of reactive oxygen species (ROS), oxidative stress, and cuproptosis are among the proposed mechanisms of copper toxicity at high concentrations. Elesclomol (ELC) is a mitochondrion-targeting agent discovered for the treatment of solid tumors. In this review, we summarize the synthesis of this drug, its mechanisms of action, and the current status of its applications in the treatment of various diseases such as cancer, tuberculosis, SARS-CoV-2 infection, and other copper-associated disorders. We also provide some detailed information about future directions to improve its clinical performance.

Assessing the biocompatibility of bovine tendon scaffold, a step forward in tendon tissue engineering.

Tendon is a collagen-enriched, tough, and intricately arranged connective tissue that connects muscle to the bone and transmits forces, resulting in joint movement. High mechanical demands can affect normal tissues and may lead to severe disorders, which usually require replacement of the damaged tendon. In recent decades, various decellularization methods have been studied for tissue engineering applications. One of the major challenges in tendon decellularization is preservation of the tendon extracellular matrix (ECM) architecture to maintain natural tissue characteristics. The aim of the present study was to create a decellularized bovine Achilles tendon scaffold to investigate its cytocompatibility with seeded hAd-MSCs (human adipose derived-mesenchymal stem cells) and blastema tissue in vitro. Here, we describe a reliable procedure to decellularize bovine Achilles tendon using a combination of physical and chemical treatments including repetitive freeze-thaw cycles and the ionic detergent SDS, respectively. The decellularization effectiveness and cytocompatibility of the tendon scaffolds were verified by histological studies and scanning electron microscopy for up to 30 days after culture. Histological studies revealed hAd-MSC attachment and penetration into the scaffolds at 5, 10, 15 and 20 days of culture. However, a decrease in cell number was observed on days 25 and 30 after culture in vitro. Moreover, migration of the blastema tissue cells into the scaffold were shown at 10 to 25 days post culture, however, destruction of the scaffolds and reduction in cell number were observed on 30th day after culture. Our results suggest that this decellularization protocol is an effective and biocompatible procedure which supports the maintenance and growth of both hAd-MSCs and blastema cells, and thus might be promising for tendon tissue engineering.

Tracing foreign sequences in plant transcriptomes and genomes using OCT4, a POU domain protein.

Contaminations in sequencing data, especially in reference genomes, lead to inevitable errors in downstream analyses. Similarly, presence of contaminants in transcriptomes, misrepresents the molecular basis of various interactions. In this study, we report the presence of a large number of plant transcriptomes contaminated with RNAs encoding POU domain proteins; a family of proteins that has not been reported in plants and fungi. Besides, our findings illustrated that there are four POU domain protein-coding sequences in the reference genome of Rhodamnia argentea. It turned out that the existing foreign fragments are related to arthropods that are considered as plant pests. We also identified two contaminated draft genomes, Humulus lupulus and Cannabis sativa that contained complete rDNA sequences originating from Tetranychus species. As a result, careful screening of sequencing data before releasing them in public databases or checking existing genomes for possible contaminations is recommended.

Design, synthesis and evaluation of PD-L1 peptide antagonists as new anticancer agents for immunotherapy.

Blocking the interaction of programmed cell death protein 1 (PD-1) and its ligand PD-L1 is known as a promising immunotherapy for treatment of a variety of tumors expressing PD-L1 on their cell surface. In the last decade, several antibodies against the PD-1/PD-L1 interaction have been approved, while there are few reports of small-molecule inhibitors against PD-1/PD-L1 axis. Due to many advantages of cancer treatment with small molecules over antibodies, we developed several peptidic PD-L1 antagonists using computational peptide design methods, and evaluated them both in vitro and in vivo. Importantly, among six peptides with best affinity to PD-L1, four peptides exhibited significant potency to block PD-1/PD-L1 axis at molecular level. Moreover, the PD-L1 expression in nine human colorectal cancer cell lines stimulated with interferon-γ was compared and LoVo cells with the highest expression were selected for further experiments. The peptides could also restore the function of activated Jurkat T cells, which had been suppressed by stimulated LoVo cells. A blockade assay in tumor-bearing mice experiments indicated that peptides HS5 and HS6 consisting of a d-amino acid in their structures, could also effectively reduce tumor growth in vivo, without induction of any observable liver or renal toxicity, tissue damages and loss of body weight. As new designed peptides showed no toxicity against murine colon cancer cells in vitro, the observed anti-tumor results in mice are most probably due to disrupting the PD-1/PD-L1 interaction. Thus, peptides described in this study can be considered as proper low molecular weight candidates for immunotherapy of cancer.

Investigating the association between rs6983267 polymorphism and susceptibility to gastrointestinal cancers in Iranian population.

Genome-wide association studies have revealed that some single nucleotide polymorphisms at 8q24, such as rs6983267, might be effective in susceptibility to various cancers in different populations. Therefore, rs6983267 might be useful as a marker for multiple cancers. In this study, we considered a population, including 478 gastrointestinal cancer cases from the Iranian population, to investigate the association between rs6983267 and susceptibility to gastrointestinal cancers. The samples were genotyped using the TaqMan real-time PCR method while 10% of them were also confirmed by sequencing. Higher frequency of G allele was associated with higher grades of tumors in esophageal cancer and the tumors located in the lower portion of the esophagus (OR 3.56; 95% CI 1.13-11.24; P = 0.03) and cardia (OR 5.24; 95% CI 1.26-21.83; P = 0.02), which both locations are involved in esophageal adenocarcinomas with poor prognosis. The results indicated that in the male subgroup, the rs6983267 GG genotype significantly enhanced the gastric cancer susceptibility (OR 4.76; 95% CI 1.57-14.45; P = 0.01). GG genotype also increased the risk of intestinal-type gastric cancer, located in non-cardia (OR 4.62; 95% CI 1.25-17.04; P = 0.02). Moreover, gastric cancer cases and controls with a family history of gastrointestinal tumors were mostly genotyped with the G allele (OR 3.61; 95% CI = 1.09-12.01; P = 0.04). There were no remarkable associations between rs6983267 and susceptibility to esophageal and colon cancers in the Iranian population. However, different genotypes of rs6983267 had significant correlations with tumor grade, cancer type, and family history of gastrointestinal cancers. Further investigations in a larger population and other ethnicities are required to confirm these results.

Ellagic acid and human cancers: a systems pharmacology and docking study to identify principal hub genes and main mechanisms of action.

Research on anticancer properties of natural compounds, as effective materials that are available while causing minimal side effects, is growing. Ellagic acid (EA) is a well-known polyphenolic compound, which has been found in both free and complex modes in several medicinal plants such as pomegranate, walnut, and berries. Although many articles have reported anticancer properties for this compound, its mechanism of action has not been fully elucidated. In this study, we used several online and offline bioinformatics tools and databases to identify the mechanism of action of EA on various types of human malignancies including bladder, blood, breast, cervical, colorectal, liver, pancreas, and prostate cancers. In this context, after identifying and extracting EA-affected human genes/proteins that have been reported in various references, we built the related gene networks and determined functional hub genes. In addition, docking was performed to recognize target proteins that react directly with EA and are in fact most affected by this compound. Our findings revealed that EA exerts its anticancer effects by influencing specific hub genes in various types of cancers. Moreover, different cellular signaling pathways are affected by this natural compound. Generally, it turned out that EA probably exerts most of its anticancer activities, through induction of apoptosis, as well as P53 and WNT signaling pathways, and also by affecting the expression of several hub genes such as CDKN1A, CDK4, CDK2, CDK6, TP53, JUN, CCNA2, MAPK14, CDK1, and CCNB1 and especially interactions with some related proteins including P53, CDK6, and MAPK14.

Improving anti-cancer drug delivery performance of magnetic mesoporous silica nanocarriers for more efficient colorectal cancer therapy.

BACKGROUND: Improving anti-cancer drug delivery performance can be achieved through designing smart and targeted drug delivery systems (DDSs). For this aim, it is important to evaluate overexpressed biomarkers in the tumor microenvironment (TME) for optimizing DDSs. MATERIALS AND METHODS: Herein, we designed a novel DDS based on magnetic mesoporous silica core-shell nanoparticles (SPION@MSNs) in which release of doxorubicin (DOX) at the physiologic pH was blocked with gold gatekeepers. In this platform, we conjugated heterofunctional polyethylene glycol (PEG) onto the outer surface of nanocarriers to increase their biocompatibility. At the final stage, an epithelial cell adhesion molecule (EpCAM) aptamer as an active targeting moiety was covalently attached (Apt-PEG-Au@NPs-DOX) for selective drug delivery to colorectal cancer (CRC) cells. The physicochemical properties of non-targeted and targeted nanocarriers were fully characterized. The anti-cancer activity, cellular internalization, and then the cell death mechanism of prepared nanocarriers were determined and compared in vitro. Finally, tumor inhibitory effects, biodistribution and possible side effects of the nanocarriers were evaluated in immunocompromised C57BL/6 mice bearing human HT-29 tumors. RESULTS: Nanocarriers were successfully synthesized with a mean final size diameter of 58.22 ± 8.54 nm. Higher cytotoxicity and cellular uptake of targeted nanocarriers were shown in the EpCAM-positive HT-29 cells as compared to the EpCAM-negative CHO cells, indicating the efficacy of aptamer as a targeting agent. In vivo results in a humanized mouse model showed that targeted nanocarriers could effectively increase DOX accumulation in the tumor site, inhibit tumor growth, and reduce the adverse side effects. CONCLUSION: These results suggest that corporation of a magnetic core, gold gatekeeper, PEG and aptamer can strongly improve drug delivery performance and provide a theranostic DDS for efficient CRC therapy.

In vivo bone regeneration using a bioactive nanocomposite scaffold and human mesenchymal stem cells.

Due to the osteoconductive role of bioceramics, use of these bioactive nanocomposite scaffolds that can maintain their structural integrity during bone tissue repair is one of the major goals of tissue engineering. Herein, a nanofibrous poly-L-lactic acid (PLLA) scaffold was fabricated by electrospinning and then gelatin and hydroxyapatite nanoparticles (nHA) were coated over the surface of the scaffold. Osteoconductivity of the fabricated nano-composite scaffolds was then studied while grafted on the rat calvarial defects. Our results indicated that the coating of PLLA scaffold with nHA and gelatin increased the adhesion and growth of the human bone marrow derived mesenchymal stem cells (BM-MSCs) and also significantly increased the level of mineralization over a week culture period. The results of radiographic and histological studies showed that the newly created bone tissue at the defect site was significantly higher in animals treated with nanocomposite scaffolds than the empty scaffolds and control groups. This increase in the defect reconstruction was significantly increased after culturing BM-MSCs on the scaffolds, especially nanocomposite scaffolds. It can be concluded that the combination of nanocomposite scaffolds and BM-MSCs could be a very good candidate for treatment of bone lesions and could be considered as a bony bioimplant.

Expression and Function of C1orf132 Long-Noncoding RNA in Breast Cancer Cell Lines and Tissues.

miR-29b2 and miR-29c play a suppressive role in breast cancer progression. C1orf132 (also named MIR29B2CHG) is the host gene for generating both microRNAs. However, the region also expresses longer transcripts with unknown functions. We employed bioinformatics and experimental approaches to decipher C1orf132 expression and function in breast cancer tissues. We also used the CRISPR/Cas9 technique to excise a predicted C1orf132 distal promoter and followed the behavior of the edited cells by real-time PCR, flow cytometry, migration assay, and RNA-seq techniques. We observed that C1orf132 long transcript is significantly downregulated in triple-negative breast cancer. We also identified a promoter for the longer transcripts of C1orf132 whose functionality was demonstrated by transfecting MCF7 cells with a C1orf132 promoter-GFP construct. Knocking-out the promoter by means of CRISPR/Cas9 revealed no alterations in the expression of the neighboring genes CD46 and CD34, while the expression of miR-29c was reduced by half. Furthermore, the promoter knockout elevated the migration ability of the edited cells. RNA sequencing revealed many up- and downregulated genes involved in various cellular pathways, including epithelial to mesenchymal transition and mammary gland development pathways. Altogether, we are reporting here the existence of an additional/distal promoter with an enhancer effect on miR-29 generation and an inhibitory effect on cell migration.

Potential of mesenchymal stem cells for bioengineered blood vessels in comparison with other eligible cell sources.

Application of stem cells in tissue engineering has proved to be effective in many cases due to great proliferation and differentiation potentials as well as possible paracrine effects of these cells. Human mesenchymal stem cells (MSCs) are recognized as a valuable source for vascular tissue engineering, which requires endothelial and perivascular cells. The goal of this review is to survey the potential of MSCs for engineering functional blood vessels in comparison with other cell types including bone marrow mononuclear cells, endothelial precursor cells, differentiated adult autologous smooth muscle cells, autologous endothelial cells, embryonic stem cells, and induced pluripotent stem cells. In conclusion, MSCs represent a preference in making autologous tissue-engineered vascular grafts (TEVGs) as well as off-the-shelf TEVGs for emergency vascular surgery cases.

Comparison the effects of hypoxia-mimicking agents on migration-related signaling pathways in mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (Ad-MSCs) have been designated as the promising agents for clinical applications for easy accessibility, multi-linage differentiation and immunomodulation capacity. Despite this, optimal cell delivery conditions have remained as a clinical challenge and improvement of stem cell homing to the target organs is being considered as a major strategy in cell therapy systemic injection. It has been shown that homing of mesenchymal stem cells are increased when treated with physical or chemical hypoxia-mimicking factors, however, efficiency of different agents remained to be determined. In this study, hypoxia-mimicking agents, including valproic acid (VPA), cobalt chloride (CoCl2) and deferoxamine (DFX) were examined to determine whether they are able to activate signaling molecules involved in migration of Ad-MSCs in vitro. We report that Ad-MSCs treated by DFX resulted in a significantly enhanced mRNA expression of MAPK4 (associated with MAPK signaling pathway), INPP4B (associated with Inositol polyphosphate pathway), VEGF-A and VEGF-C (associated with cytokine-cytokine receptor pathways), IL-8 and its receptor, CXCR2 (associated with IL-8 signaling pathway). While the cells treated with VPA did not show such effects and CoCl2 only upregulated VEGF-A and VEGF-C gene expression. Furthermore, results of wound-healing assays showed migration capacity of Ad-MSCs treated with DFX significantly increased 8 and 24 h of the treatment. This study provides credible evidence around DFX, which might be an effective drug for pharmacological preconditioning of Ad-MSCs to boost their homing capacity and regeneration of damaged tissues though, activation of the migration-related signaling pathways.

Exosomes and their importance in metastasis, diagnosis, and therapy of colorectal cancer.

Extracellular vesicles are known as actual intermediaries of intercellular communications, such as biological signals and cargo transfer between different cells. A variety of cells release the exosomes as nanovesicular bodies. Exosomes contain different compounds such as several types of nucleic acids and proteins. In this study, we focused on exosomes in colorectal cancer as good tools that can be involved in various cancer-related processes. Furthermore, we summarize the advantages and disadvantages of exosome extraction methods and review related studies on the role of exosomes in colorectal cancer. Finally, we focus on reports available on relations between mesenchymal stem cell-derived exosomes and colorectal cancer. Several cancer-related processes such as cancer progression, metastasis, and drug resistance of colorectal cancer are related to the cargoes of exosomes. A variety of molecules, especially proteins, microRNAs, and long noncoding RNAs, play important roles in these processes. The microenvironment features, such as hypoxia, also have very important effects on the properties of the origin cell-derived exosomes. On the other hand, exosomes derived from colorectal cancer cells also interfere with cancer chemoresistance. Furthermore, today it is known that exosomes and their contents can likely be very effective in noninvasive colorectal cancer diagnosis and therapy. Thus, exosomes, and especially their cargoes, play different key roles in various aspects of basic and clinical research related to both progression and therapy of colorectal cancer.

The importance of 15-lipoxygenase inhibitors in cancer treatment.

Cancer-targeted therapy is an expanding and successful approach in treatment of many types of cancers. One of the main categories of targeted therapy is use of small molecule inhibitors. 15-Lipoxygenase (15-LOX) is an enzyme which reacts with polyunsaturated fatty acids and produces metabolites that are implicated in many important human diseases, such as cancer. Considering the role of 15-LOX (mainly 15-LOX-1) in the progression of some cancers, the discovery of 15-LOX inhibitors could potentially lead to development of novel cancer therapeutics and it can be claimed that 15-LOX inhibitors might be suitable as chemotherapy agents in the near future. This article reviews relevant publications on 15-LOX inhibitors with focus on their anticancer activities in vitro and in vivo. Many 15-LOX inhibitors have been reported for which separate studies have shown their anticancer activities. This review paves the way to further explore the mechanism of their antiproliferative effects via 15-LOX inhibition.

Selenium nanoparticle as a bright promising anti-nanobacterial agent.

The use of nanotechnology for nanobacteria (or calcifying nanoparticles) treatment is a new creative approach. Use of selenium nanoparticles (SeNPs) as anti-nanobacterial agents might be considered as a bright promising approach due to their critical role in the inhibition of crystal growth and aggregation of calcium oxalate. Hence, in this study, we investigated the probable outcome of SeNPs inhibitory effects on growth of nanobacteria. Fragments of thirty urinary tract stones were chemically analyzed by X-ray diffraction (XRD) and urinary stones Kits for calcifying nanoparticles presence. Then powder of stone fragments were resuspended in Dulbecco's modified Eagle's medium (DMEM), sterilized by filtration and cultured in presence of 1, 5, 30, 60, and 90 µmol/L SeNPs concentrations. Besides, calcifying nanoparticles growth in the culture without SeNPs was measured spectrophotometrically. Also, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses were used, where calcifying nanoparticles formation occurred. Results showed that in the culture without SeNPs, the positive calcifying nanoparticles detection was 60% while after adding SeNPs at 90 µmol/L, not any calcifying nanoparticles were observed. Further confirmation came out when Energy-dispersive X-ray (EDX) analysis showed calcium and phosphate peaks in the culture medium without any SeNPs while in the culture containing 90 µm/L SeNPs a decrease in calcium and other minerals was obvious. Therefore, SeNPs clearly restricted the growth of nanobacteria due to their inhibitory effects on calcium oxalate deposition.

Efficacy of hyperthermia in human colon adenocarcinoma cells is improved by auraptene.

Colon adenocarcinoma is one of the most common cancers worldwide, and resistance to current therapeutic modalities is a serious drawback in its treatment. Auraptene is a natural coumarin with considerable anticancer effects. The goal of this study was to introduce a novel combinatorial approach for treatment against colon adenocarcinoma cells. To do so, HT29 cells were pretreated with nontoxic auraptene and then hyperthermia was induced. Afterwards, the viability of the cells was assessed, changes induced in the cell cycle were analyzed, and the expression patterns of candidate genes were studied. Results from the MTT assay demonstrated significant (p < 0.01) decreases in cell viability when 20 µg/mL auraptene was used for 72 h, heat shock was induced, and cells were allowed to recover for 24 h. Flow cytometry analysis also indicated considerable changes in the distribution of cells between the sub-G1/G1 and G2/M phases of cell cycle after the combinatorial treatment. Real-time RT-PCR studies revealed significant (p < 0.01) up-regulation of P21 in the cells pretreated with auraptene after heat shock, whereas no significant change was observed in HSP27 expression. Our findings not only indicate, for the first time, that the efficacy of hyperthermia was improved by auraptene pretreatment, but also suggest that this coumarin could be used in the future to achieve more effective therapeutic outcomes.

Suppression of dsRNA response genes and innate immunity following Oct4, Stella, and Nanos2 overexpression in mouse embryonic fibroblasts.

The self-renewal capacity of germline derived stem cells (GSCs) makes them an ideal source for research and use in clinics. Despite the presence of active gene network similarities between embryonic stem cells (ESCs) and GSCs, there are unanswered questions regarding the roles of evolutionary conserved genes in GSCs. To determine the reprogramming potential of germ cell- specific genes, we designed a polycistronic gene cassette expressing Stella, Oct4 and Nanos2 in a lentiviral-based vector. Deep transcriptome analysis showed the activation of a set of pluripotency and germ-cell-specific markers and the downregulation of innate immune system. The global shut down of antiviral genes included MHC class I, interferon response genes and dsRNA 2'-5'-oligoadenylate synthetase are critical pathways that has been affected . Individual expression of each factor highlighted suppressive effect of Nanos2 on genes such as Isg15 and Oasl2. Collectively, to our knowledge this is the first report showing that Nanos2 could be considered as an immunosuppressive factor. Furthermore, our results demonstrate suppression of endogenous retrotransposons that harbor immune response but further analysis require to uncover the correlation between transposon suppression and immune response in germ cell development.

Supportive properties of basement membrane layer of human amniotic membrane enable development of tissue engineering applications.

Human amniotic membrane (HAM) has been widely used as a natural scaffold in tissue engineering due to many of its unique biological properties such as providing growth factors, cytokines and tissue inhibitors of metalloproteinases. This study aimed at finding the most suitable and supportive layer of HAM as a delivery system for autologous or allogeneic cell transplantation. Three different layers of HAM were examined including basement membrane, epithelial and stromal layers. In order to prepare the basement membrane, de-epithelialization was performed using 0.5 M NaOH and its efficiency was investigated by histological stainings, DNA quantification, biomechanical testing and electron microscopy. Adipose-derived stromal cells (ASCs) and a human immortalized keratinocyte cell line (HaCaT) were seeded on the three different layers of HAM and cultured for 3 weeks. The potential of the three different layers of HAM to support the attachment and viability of cells were then monitored by histology, electron microscopy and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, mechanical strengths of the basement membrane were assessed before and after cell culture. The results indicated that the integrity of extra cellular matrix (ECM) components was preserved after de-epithelialization and resulted in producing an intact basement amniotic membrane (BAM). Moreover, all three layers of HAM could support the attachment and proliferation of cells with no visible cytotoxic effects. However, the growth and viability of both cell types on the BAM were significantly higher than the other two layers. We conclude that growth stimulating effectors of BAM and its increased mechanical strength after culturing of ASCs, besides lack of immunogenicity make it an ideal model for delivering allogeneic cells and tissue engineering applications.

Using paracrine effects of Ad-MSCs on keratinocyte cultivation and fabrication of epidermal sheets for improving clinical applications.

Recent advances in wound healing have made cell therapy a potential approach for the treatment of various types of skin defects such as trauma, burns, scars and diabetic leg ulcers. Cultured keratinocytes have been applied to burn patients since 1981. Patients with acute and chronic wounds can be treated with autologous/allograft cultured keratinocytes. There are various methods for cultivation of epidermal keratinocytes used in cell therapy. One of the important properties of an efficient cell therapy is the preservation of epidermal stem cells. Mesenchymal Stem Cells (MSCs) are major regulatory cells involved in the acceleration of wound healing via induction of cell proliferation, angiogenesis and stimulating the release of paracrine signaling molecules. Considering the beneficial effects of MSCs on wound healing, the main aim of the present study is investigating paracrine effects of Adipose-derived Mesenchymal Stem Cell (Ad-MSCs) on cultivation of keratinocytes with focusing on preservation of stem cells and their differentiation process. We further introduced a new approach for culturing isolated keratinocytes in vitro in order to generate epidermal keratinocyte sheets without using a feeder layer. To do so, Ad-MSC conditioned medium was applied as an alternative to commercial media for keratinocyte cultivation. In this study, the expression of several stem/progenitor cell (P63, K19 and K14) and differentition (K10, IVL and FLG) markers was examined using real time PCR on days 7, 14 and 21 of culture in keratinocytes in Ad-MSC conditioned medium. P63 and α6 integrin expression was also evaluated via flow cytometry. The results were compared with control group including keratinocytes cultured in EpiLife medium and our data indicated that this Ad-MSC conditioned medium is a good alternative for keratinocyte cultivation and producing epidermal sheets for therapeutic and clinical purposes. The reasons are the expression of stem cell and differentiation markers and overcoming the requirement for feeder layer which leads to a xenograft-free transplantation. Besides, this approach has low cost and is easier to perform. However, more in vitro and in vivo experiments as well as safety evaluation required before clinical applications.