Antimicrobial peptide-loaded decellularized placental sponge as an excellent antibacterial skin substitute against XDR clinical isolates.

Post-wound infections have remained a serious threat to society and healthcare worldwide. Attempts are still being made to develop an ideal antibacterial wound dressing with high wound-healing potential and strong antibacterial activity against extensively drug-resistant bacteria (XDR). In this study, a biological-based sponge was made from decellularized human placenta (DPS) and then loaded with different concentrations (0, 16 µg/mL, 32 µg/mL, 64 µg/mL) of an antimicrobial peptide (AMP, CM11) to optimize an ideal antibacterial wound dressing. The decellularization of DPS was confirmed by histological evaluations and DNA content assay. The DPS loaded with different contents of antimicrobial peptides (AMPs) showed uniform morphology under a scanning electron microscope (SEM) and cytobiocompatibility for human adipose tissue-derived mesenchymal stem cells. Antibacterial assays indicated that the DPS/AMPs had antibacterial behavior against both standard strain and XDR Acinetobacter baumannii in a dose-dependent manner, as DPS loaded with 64 µg/mL showed the highest bacterial growth inhibition zone and elimination of bacteria under SEM than DPS alone and DPS loaded with 16 µg/mL and 32 µg/mL AMP concentrations. The subcutaneous implantation of all constructs in the animal model demonstrated no sign of acute immune system reaction and graft rejection, indicating in vivo biocompatibility of the scaffolds. Our findings suggest the DPS loaded with 64 µg/mL as an excellent antibacterial skin substitute, and now promises to proceed with pre-clinical and clinical investigations.

3D-printed placental-derived bioinks for skin tissue regeneration with improved angiogenesis and wound healing properties.

Extracellular matrix (ECM)-based bioinks has attracted much attention in recent years for 3D printing of native-like tissue constructs. Due to organ unavailability, human placental ECM can be an alternative source for the construction of 3D print composite scaffolds for the treatment of deep wounds. In this study, we use different concentrations (1.5%, 3% and 5%w/v) of ECM derived from the placenta, sodium-alginate and gelatin to prepare a printable bioink biomimicking natural skin. The printed hydrogels' morphology, physical structure, mechanical behavior, biocompatibility, and angiogenic property are investigated. The optimized ECM (5%w/v) 3D printed scaffold is applied on full-thickness wounds created in a mouse model. Due to their unique native-like structure, the ECM-based scaffolds provide a non-cytotoxic microenvironment for cell adhesion, infiltration, angiogenesis, and proliferation. In contrast, they do not show any sign of immune response to the host. Notably, the biodegradation, swelling rate, mechanical property, cell adhesion and angiogenesis properties increase with the increase of ECM concentrations in the construct. The ECM 3D printed scaffold implanted into deep wounds increases granulation tissue formation, angiogenesis, and re-epithelialization due to the presence of ECM components in the construct, when compared with printed scaffold with no ECM and no treatment wound. Overall, our findings demonstrate that the 5% ECM 3D scaffold supports the best deep wound regeneration in vivo, produces a skin replacement with a cellular structure comparable to native skin.

Melatonin ameliorates testes against forced treadmill exercise training on spermatogenesis in rats.

INTRODUCTION: It is well documented that some forced exercises can have bad effects on the genital system. Melatonin is a potent antioxidant that is effective in reducing the physical stress.

Transdifferentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Dopaminergic Neurons in a Three-Dimensional Culture.

Introduction: The induction of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) toward dopaminergic neurons is a major challenge in tissue engineering and experimental and clinical treatments of various neurodegenerative diseases, including Parkinson disease. This study aims to differentiate HUC-MSCs into dopaminergic neuron-like cells. Methods: Following the isolation and characterization of HUC-MSCs, they were transferred to Matrigel-coated plates and incubated with a cocktail of dopaminergic neuronal differentiation factors. The capacity of differentiation into dopaminergic neuron-like cells in 2-dimensional culture and on Matrigel was assessed by real-time polymerase chain reaction, immunocytochemistry, and high-performance liquid chromatography. Results: Our results showed that dopaminergic neuronal markers' transcript and protein levels were significantly increased on the Matrigel differentiated cells compared to 2D culture plates. Conclusion: Overall, the results of this study suggest that HUC-MSCs can successfully differentiate toward dopaminergic neuron-like cells on Matrigel, having great potential for the treatment of dopaminergic neuron-related diseases.

Evaluation of Differential Gene Expression during Transdifferentiation of Bone Marrow Stromal Cells to Glial Phenotype in the Presence of Cerebrospinal Fluid.

BACKGROUND: The present study assessed the alteration of gene expression during transdifferentiation of Bone Marrow Stromal Cells (BMSCs) into oligodendrocyte in the presence of Cerebrospinal Fluid (CSF). METHODS: BMSCs were collected from female Sprague-Dawley rats and were cultured in DMEM/F12 medium supplemented with Retinoic Acid (RA), basic Fibroblast Growth Factor (bFGF), and Epidermal Growth Factor (EGF). CSF was added daily to the culture media. The oligoprogenitor and oligodendrocyte generation was assessed by immunocytochemistry for Oligo 2, A2B5, CNP and MBP markers. RESULTS: The mean percentages of immunopositive cells for Olig2 and A2B5 were 52.1±1.74 and 56.34±2.55%, respectively. The number of immunopositive cells for glial markers CNP and MBP were 48.8±3.12 and 40.5±8.92%, respectively. Alteration of gene expression of Oct4, Olig 2, PDGFR-α and PLP were examined by real time PCR during transdifferentiation of BMSC to oligodendrocyte. Immunocytochemical results indicate that oligoprogenitor cells were immunopositive for Oligo2 and A2B5 markers. Also, oligodendrocytes expressed the mature glial markers of CNP and MBP indicating successful differentiation. CONCLUSION: In conclusion, CSF promotes the transdifferentiation of BMSC into mature oligodendrocyte via providing an appropriate niche for glial maturation.

Trans-Differentiation of Human Dental Pulp Stem Cells Into Cholinergic-Like Neurons Via Nerve Growth Factor.

INTRODUCTION: Cell therapy has been widely considered as a therapeutic approach for neurodegenerative diseases and nervous system damage. Cholinergic neurons as one of the most important neurons that play a significant role in controlling emotions, mobility, and autonomic systems. In this study, Human Dental Pulp Stem Cells (hDPSCs) were differentiated into the cholinergic neurons by ß-mercaptoethanol in the preinduction phase and also by the nerve growth factor (NGF) in the induction phase. METHODS: The hDPSCs were evaluated for CD73, CD31, CD34, and Oct-4. Concentration-time relationships for NGF were assessed by evaluating the viability rate of cells and the immune response to nestin, neurofilament 160, microtubule-associated protein-2, and choline acetyltransferase. RESULTS: The hDPSCs had a negative response to CD34 and CD31. The optimal dose for the NGF was 50 ng/mL seven days after the induction when the highest percentage of expressing markers for the Cholinergic neurons (ChAT) was detected. CONCLUSION: The results of this study provided a method for producing cholinergic neurons by hDPSCs, which can be used in cytotherapy for degenerative diseases of the nervous system and also spinal cord injury.

Transdifferentiation of Human Dental Pulp Stem Cells Into Oligoprogenitor Cells.

INTRODUCTION: The nerve fibers in central nervous system are surrounded by myelin sheet which is formed by oligodendrocytes. Cell therapy based on oligodendrocytes and their precursors transplantation can hold a promising alternative treatment for myelin sheet repair in demyelinating diseases. METHODS: Human Dental Pulp Stem Cells (hDPSCs) are noninvasive, autologous and easy available source with multipotency characteristics, so they are in focus of interest in regenerative medicine. In the present study, hDPSCs were differentiated into oligoprogenitor using glial induction media, containing Retinoic Acid (RA), basic Fibroblast Growth Factor (bFGF), Platelet-Derived Growth Factor (PDGF), N2 and B27. The differentiated Oligoprogenitor Cells (OPCs) were evaluated for nestin, Olig2, NG2 and O4 using immunocytochemistry. Also, the expression of nestin, Olig2 and PDGFR-α gens (neuroprogenitor and oligoprogenitor markers) were investigated via RT-PCR technique. RESULTS: The results indicate that glial differentiation medium induces the generation of oligoprogenitor cells as revealed via exhibition of specific glial markers, including Olig2, NG2 and O4. The expersion of nestin gene (neuroprogenitor marker) and Olig2 and PDGFR-α genes (oligoprogentor markers) were detected in treated hDPSCs at the end of the induction stage. CONCLUSION: hDPSCs can be induced to transdifferentiate into oligoprogenitor cells and respond to the routinely applied regents for glial differentiation of mesanchymal stem cells. These data suggest the hDPSCs as a valuable source for cell therapy in neurodegenerative diseases.

Condition medium of cerebrospinal fluid and retinoic acid induces the transdifferentiation of human dental pulp stem cells into neuroglia and neural like cells.

Cerebrospinal fluid (CSF) contains several molecules which are essential for neurogenesis. Human dental pulp stem cells (hDPSCs) are putatively neural crest cell-derived that can differentiate into neurons and glial cells under appropriate neurotrophic factors. The aim of this study was to induce differentiation of hDPSCs into neuroglial phenotypes using retinoic acid (RA) and CSF. The hDPSCs from an impacted third molar were isolated by mechanical and digestion and cultured. The cells have treated by 10-7 µM RA (RA group) for 8 days, 10% CSF (CSF group) for 8 days and RA with CSF for 8 days (RA/CSF group). Nestin, microtubule-associated protein 2 (MAP2), and glial fibrillary acidic protein immunostaining were used to examine the differentiated cells. Axonal outgrowth was detected using Bielschowsky's silver impregnation method and Nissl bodies were stained in differentiated cells by Cresyl violet. The morphology of differentiated cells in treated groups was significantly changed after 3-5 days. The results of immunocytochemistry showed the presence of neuroprogenitor marker nestin was seen in all groups. However, the high percentage of nestin positive cells and MAP2, as mature neural markers, were observed at the pre-induction and induction stage, respectively. Nissl bodies were detected as dark-blue particles in the cytoplasm of treated cells. Our findings showed the RA as pre-inducer and CSF as inducer for using in vitro differentiation of neuron-like cells and neuroglial cells from hDPSCs.

Chitosan-Intercalated Montmorillonite/Poly(vinyl alcohol) Nanofibers as a Platform to Guide Neuronlike Differentiation of Human Dental Pulp Stem Cells.

In this study, we present a novel chitosan-intercalated montmorillonite/poly(vinyl alcohol) (OMMT/PVA) nanofibrous mesh as a microenvironment for guiding differentiation of human dental pulp stem cells (hDPSCs) toward neuronlike cells. The OMMT was prepared through ion exchange reaction between the montmorillonite (MMT) and chitosan. The PVA solutions containing various concentrations of OMMT were electrospun to form 3D OMMT-PVA nanofibrous meshes. The biomechanical and biological characteristics of the nanofibrous meshes were evaluated by ATR-FTIR, XRD, SEM, MTT, and LDH specific activity, contact angle, and DAPI staining. They were carried out for mechanical properties, overall viability, and toxicity of the cells. The hDPSCs were seeded on the prepared scaffolds and induced with neuronal specific differentiation media at two differentiation stages (2 days at preinduction stage and 6 days at induction stage). The neural differentiation of the cells cultured on the meshes was evaluated by determining the expression of Oct-4, Nestin, NF-M, NF-H, MAP2, and ßIII-tubulin in the cells after preinduction, at induction stages by real-time PCR (RT-PCR) and immunostaining. All the synthesized nanofibers exhibited a homogeneous morphology with a favorable mechanical behavior. The population of the cells differentiated into neuronlike cells in all the experimental groups was significantly higher than that in control group. The expression level of the neuronal specific markers in the cells cultured on 5% OMMT/PVA meshes was significantly higher than the other groups. This study demonstrates the feasibility of the OMMT/PVA artificial nerve graft cultured with hDPSCs for regeneration of damaged neural tissues. These fabricated matrices may have a potential in neural tissue engineering applications.

Multipotent SSEA1 Positive Cells Population Differentiation into Primordial Germ Cells and Subsequently Progress into Oocyte-like Cells.

BACKGROUND: Stage-specific embryonic antigen-1 (SSEA1) is a cell surface carbohydrate that its pattern expression is changed during induction of mouse embryonic stem cell differentiation. In this study, the spatial distribution of SSEA1 on primordial germ cells differentiation and subsequent progression into oocyte-like cells from mouse embryonic stem cells in vitro was evaluated. METHODS: Embryoid bodies from mouse embryonic stem cells were cultured for two days with 5 ng/mL BMP4. SSEA1 positive and negative cells were separated using the MACS system and cultured separately in a conditioned medium consist of in vitro maturation medium diluted in DMEM [1:1] for 10 days. We assayed viability, colony formation and alkaline phosphatase activity (ALP) of sorted cells. Also, germ cell markers were analyzed by flow cytometry, immunocytochemistry and RT-PCR. RESULTS: Viability percent SSEA1 positive cells were more than SSEA1 negative cells. SSEA1 positive cells and SSEA1 negative cells formed compact and flat colonies respectively. Unlike the SSEA1 positive population, the SSEA1 negative colonies showed a weak ALP activity. SSEA1 positive cells expressed Oct4, Stella, Mvh, c-kit, Scp3, Desmin, GFAP and Albumin. Interestingly, SSEA1 negative cells expressed Desmin and GFAP. The population of Mvh-positive cells in SSEA1 positive was 17.74%. All specific oocyte mentioned genes were detected in the SSEA1 positive. Also, oocyte specific proteins GDF9 and ZP3 were detected using immunocytochemistry. CONCLUSION: Our results suggest that conditioned medium provides a suitable niche to differentiation and progression putative primordial germ cells derived from the SSEA1 positive toward oocyte-like cells.

In vitro and in vivo evaluations of three-dimensional hydroxyapatite/silk fibroin nanocomposite scaffolds.

In this study, three-dimensional hydroxyapatite/silk fibroin (HAp/SF) nanocomposite scaffolds were successfully prepared through layer solvent casting combined with the freeze-drying technique for tissue engineering applications. Various SF aqueous concentrations, ranging from 2.5% to 10%, were used to control the physicochemical properties of the prepared scaffolds. Biologic responses of the rat bone marrow stromal cells (rBMSCs) to the HAp/SF scaffolds were examined by culturing the cells within them. In addition, biodegradation and biocompatibility of the scaffolds were evaluated in vitro and in vivo, respectively. Among the prepared scaffolds, HAp/SF-2.5% was the most brittle sample and showed porous structure with lowest mechanical properties. The average pore diameters were 350 ± 67 and 112 ± 89 µm and decreased with the increase in the SF concentration from 5% to 10%, respectively. The pores formed in the scaffolds, made up of the 5% SF, were more uniform and regular than those of the scaffolds made up of 5% and 10% SF. The HAp/SF scaffolds did not change the rBMSCs viability and were not cytotoxic compared with the control sample. The scanning electron microscopy micrographs showed that the cells migrated into the pores and well attached to the scaffolds and their cytoplasm was extended in all directions, indicating a promising cell adhesion, high biocompatibility, and no cytotoxicity of the HAp/SF-5% nanocomposite scaffolds. Subcutaneous implantation of the HAp/SF-5% scaffolds in rat models suggested an excellent biocompatibility. All data obtained from this study suggest the potential use of the HAp/SF-5% for hard tissue engineering.

Alteration in genes expression patterns during in vitro differentiation of mouse spermatogonial cells into neuroepithelial-like cells.

Pluripotent stem cells derived from testis is a new, natural, and unlimited source for cell therapy in regenerative medicine and represent a possible alternative to replacing of all cells in the body. Here, we designed a simple co-culture system of spermatogonia cells with Sertoli cells for the generation of embryonic stem-like cells from mouse testis. The importance of our simple method will be clear when we compared it with other complex and time-consuming methods. Embryonic stem-like colonies with sharp border confirmed by real-time PCR, immunocytochemistry and flow cytometry assessments. Embryonic stem-like colonies were immunopositive for pluripotency markers. Transition of spermatogonia cells to embryonic stem-like cells was accompanied by extensive changes in gene expression. These changes included significant increase in pluripotency genes expression and significant decrease in germ cell-specific genes expression. Also, we proved the differentiation capacity of embryonic stem-like cells to neuroepithelial-like cells which were immunoreactive to Nestin and Neurofilament 68. Evaluation of genes expression during in vitro differentiation into neuroepithelial-like cells showed high-level expression of Nestin whether this gene approximately has no expression in undifferentiated embryonic stem-like cells. Also, expression of pluripotency genes has significantly decreased in neuroepithelial-like cells compared with embryonic stem-like cells. This study shows that embryonic stem-like cells derived from testis are capable to differentiate into neuroepithelial-like cells that may provide a cellular reservoir usable for neurodegenerative disorders.

A Simple Co-culture System for Generation of Embryonic Stem-Like Cells From Testis.

BACKGROUND: New research proposes the pluripotency of spermatogonial cells obtained from testis. These spermatogonia-derived stem cells are called embryonic stem-like cells that express embryonic stem cell markers and differentiate to the three germ layers. OBJECTIVES: The aim of the present study was to generate embryonic stem-like cells from neonatal mouse testis. MATERIALS AND METHODS: The Testis cells were collected from neonatal mouse. After decapsulation, testis was mechanically dissected and dissociated via a two-step mechanical and enzymatic digestion. The spermatogonia and sertoli cells were cultured together in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% FBS and LIF. Before one week, several small spermatogonia colonies were observed on top of the monolayer of sertoli cells. These colonies were passaged every four days. ES-Like cells colonies that resembled ES cell was appeared within 2-3 weeks (at passages 5). Real time PCR was performed to analyze the expression of a subset of pluripotency markers, as well as germ cell-specific genes. ES Like cells were confirmed with SSEA1, SOX2 and Oct4 immunofluorescence stainng as pluripotency markers. RESULTS: The Results showed that at fifth passages, the pluripotency genes; Nanog and c-myc have significant increase in ES-Like cells in compare with spermatogonia cells, whereas the spermatogonial markers; Stra8, mvh, and piwill2 became downregulated. In addition to these pluripotency genes, the ES cell marker SSEA-1, SOX2 and Oct4 were expressed in the ES-like cells, similar to ES cells. CONCLUSIONS: This researh indicates pluripotency evidence of ES-like cells derived from testis. ES-like cells shows some molecular characteristics with embryonic stem cells.