The Story of Nanoparticles in Differentiation of Stem Cells into Neural Cells.

Stem cells have been long looked at as possible therapeutic vehicles in regenerative medicine largely due to their multi-lineage differentiation potential and paracrine actions. Therefore, development of new procedures for the differentiation of stem cells into different cell types holds great potential for opening new opportunities in regenerative medicine. In addition to various methods for inducing stem cell differentiation, the utilization of nanomaterials for differentiation of stem cells has recently received considerable attention and has become a potential tool for such purpose. Multiple lines of evidence revealed that nanomaterial-based scaffolds, inorganic nanoparticles (NPs), and biodegradable polymers have led to significant progress in regulation of stem cell differentiation. Several studies indicated that different NPs including selenium, gold, graphene quantum dots (QDs) and silica could be employed for the regulation of differentiation of stem cells such as human mesenchymal stem cells (hMSCs). In addition, magnetic core-shell NPs could be applied for the regulation of neural stem cell (NSC) differentiation. Taken together, these findings suggested that NPs are potential candidates which could be utilized for the differentiation of stem cells into various cell types such as neural cells. Herein, we summarized the application of NPs for differentiation of stem cells into various cells in particular neural cells.

Time- and Concentration-Dependent Effects of the Stem Cells Derived from Human Exfoliated Deciduous Teeth on Osteosarcoma Cells.

Background: Stem cells have been proposed to be one of the potent sources for treatment applications. Among diverse types of stem cells, stem cells derived from human exfoliated deciduous teeth (SHEDs) are known as the immature stem cell population, which are easily isolated, fast, and without ethical implications. SHEDs could induce pluripotent stem cells and show differentiation in chondrocytes, adipocytes, osteoblasts, neural cells, hepatocytes, myocytes, odontoblasts, and skin cells. Materials and Methods: In the current study, we investigated the effects of SHED on osteosarcoma cells (Saos-II) following 3 and 5 days indirect coculture. Results: Our results showed that indirect coculture of SHED with Saos-II cells could promote or inhibit Saos-II cells' growth in a concentration (the number of SHED vs. Saos-II cells) and time (days of indirect co-culture) dependent manner. Conclusion: Our findings suggested that, indirectly, SHED co-culture with the Soas-II cells might functions as a tumor suppressor where a higher number of SHEDs are used in the culture in comparison with the one cultured in the absence of/or fewer SHED incubation.

An animal model study of osteochondral defect repair by human adipose stem cells and pomegranate fruit hydroalchoholic extract.

Objective: Articular cartilage damages do not repair spontaneously. Tissue engineering is a promising approach to repair cartilage damage. Transforming growth factor-beta (TGF-ß) members are the known induction factors in chondrogenic differentiation. However, hypertrophy of the chondrocytes resulting from mesenchymal stem cells (MSCs) induction by TGF-ß is inevitable. Pomegranate fruit contains many ingredients which are useful in ensuring the health of organs. This study was designed to investigate the Pomegranate Fruit hydroalchoholic Extract (PFE) capability in human adipose derived stem cells (hASCs) differentiation into the chondrocytes on fibrin scaffold. Materials and Methods: Pomegranate fruit hydroalchoholic extract (PFE) was prepared. hASCs were isolated, expanded, labeled, and seeded on the fibrin scaffold. The constructs were divided into three groups including TGF-ß3, PFE, and control. The constructs were induced for 14 days, then, the MTT assay, Real-Time Polymerase Chain Reaction (PCR), and histochemistry assessments were run, and finally, the constructs were transplanted into the knee defect of rats. The gross and histological assessments of the transplants were done after 8 weeks. Results: The viability rate, COL2A1, Aggrecan (ACAN) and COL10A1 genes expression levels, and histological criterion of the PFE samples were significantly higher than that of the control. The macroscopic grades and histological results of the PFE samples were close to that of the TGF-ß3. The number of positive cells for COLІI protein were higher significantly in the PFE group than the control. Conclusion: PFE was effective in the chondrogenic induction of hASCs. Further studies are needed to find out the events of the chondrogenic induction using PFE.

Investigation and Comparison of the Effect of TGF-ß3, kartogenin and Avocado/Soybean Unsaponifiables on the In-vitro and In-vivo Chondrogenesis of Human Adipose-Derived Stem Cells on Fibrin Scaffold.

Due to the lack of suitable therapeutic approaches to cartilage defect, the objective of this study was to determine the effect of Transforming growth factor-ß3 (TGF-ß3), avocado/soybean (ASU) and Kartogenin (KGN) on chondrogenic differentiation in human adipose-derived stem cells (hADSCs) on fibrin scaffold. hADSCs seeded in fibrin scaffold and cultured in chondrogenic media. These cells were divided into 4 groups (control, TGF-ß3, ASU and KGN). Cell viability was estimated by MTT assay. Differentiated cells were evaluated by histological and immunohistochemical (IHC) techniques. Expression genes [sex determining region Y-box 9 (SOX9), Aggrecan (AGG), type II collagen (Coll II) and type X collagen (Coll X)] were assessed by real-time PCR. For a study on an animal model, differentiated cells in fibrin scaffolds were subcutaneously transplanted in rats. Histological and immunohistochemistry were done in the animal model. The results of the real-time PCR indicated that SOX9, AGG and Col II genes expression in TGF-ß3, KGN and ASU groups were significantly higher (p < 0.01) compared to the control group, Col X gene expression only in the TGF-ß3 group was significantly higher (p < 0.01) compared to the control group. The glycosaminoglycan (GAG) deposition was higher in TGF-ß3, KGN and ASU groups compared to the control group. The immunohistological analysis showed the distribution of collagen type X in the extracellular matrix in the fibrin scaffold TGF-ß3 group was significantly higher in control, KGN and ASU groups, and (p < 0.001). ASU, particularly KGN, was suitable for successful chondrogenic differentiation of hADSCs and a suppressor of the consequent hypertrophy.

Evaluation of Fas positive sperm and complement mediated lysis in subfertile individuals.

PURPOSE: Evaluation of Fas receptor on surface of sperm, as an apoptotic marker, using flow cytometry and confirming the results using an antibody-antigen complex through the classic complement pathway. MATERIALS AND METHODS: Semen samples were obtained from 10 fertile and 73 infertile individuals with diagnoses of male factor infertility. Expressions of Fas receptor and phosphatidyl serine on sperm were assessed by flow cytometry. Fas expression was further assessed by antibody-antigen complex through the complement pathway. Lysis was detected via PI (propidium iodide) staining. RESULTS: The mean Fas expression was considerably lower than previously reported values. No significant differences in the percentage of PI were detected before and after activation of the classic complement pathway. Annexin V positive samples showed low Fas expression. CONCLUSION: Our results have confirmed the presence of selected apoptotic markers such as Fas or phosphatidyl serine on ejaculate sperm, but suggest that Fas expression is low. Further studies are required to investigate the "abortive apoptosis" mechanism through Fas/Fas L.

Chondrogenic activity of two herbal products; pomegranate fruit extract and avocado/soybean unsaponifiable.

BACKGROUND AND PURPOSE: Articular cartilage defects aren't repaired by itself. Numerous studies have been conducted in the area of cartilage tissue engineering and some of them considered herbal products. An attempt was made in this study to compare the effects of pomegranate fruit extract (PFE), avocado/soybean unsaponifiable (ASU), and their equal proportional mixture on the chondrogenesis of human adipose-derived stem cells (hADSCs). EXPERIMENTAL APPROACH: PFE was prepared through the percolation method. ASU powder was dissolved in ethanol at 10 µg/mL concentration and was sterilized. The hADSCs first were isolated, expanded in monolayer culture and identified, and next seeded on fibrin scaffolds. The hADSCs/fibrin scaffolds were divided into 4 groups of control, ASU, PFE, and PFE+ ASU and subjected to in vitro induction for 2 weeks. The control group received chondrogenic medium, other groups received chondrogenic medium plus ASU, PFE, or PFE + ASU, respectively. The MTT assay was performed for cell viability evaluation, real-time polymerase chain reaction for expression of cartilage genes, and the toluidine blue, safranin-O, and immunohistochemistry for staining of the constructs. FINDINGS / RESULTS: Cell viability, cartilage genes expression, matrix staining density, and collagen II protein levels in PFE samples were significantly higher than those of the other groups (P < 0.05). Histological assessments revealed more chondrogenic centers (P < 0.05) in the PFE group compared to the other groups. CONCLUSION AND IMPLICATIONS: In this study, it was revealed that PFE can be considered as an induction factor for future chondrogenic studies.

Direct Conjugation of Retinoic Acid with Gold Nanoparticles to Improve Neural Differentiation of Human Adipose Stem Cells.

Gold nanoparticles (AuNPs) have been proposed as useful medical carriers in the field of regenerative medicine. This study aimed to assess the direct conjugation ability of retinoic acid (RA) with AuNPs and to develop a strategy to differentiate the human adipose-derived stromal/stem cells (hADSCs) into neurons using AuNPs-RA. The physical properties of this nanocarrier were characterized using FT-IR, TEM, and FE-SEM. Moreover, the efficiency of RA conjugation on AuNPs was determined at 99% using UV-Vis spectroscopy. According to the MTT assay, an RA concentration of 66 µM caused a 50% inhibition of cell viability and AuNPs were not cytotoxic in concentrations below 5 µg/ml. Real-time PCR and immunocytochemistry proved that AuNPs-RA is able to increase the expression of neuronal marker genes and the number of neuronal protein (GFAP and MAP2)-positive cells, 14 days post-induction of hADSCs. Taken together, these results confirmed that the AuNPs-RA promote the neuronal differentiation of hADSCs.

Effect of Thymoquinone on P53 Gene Expression and Consequence Apoptosis in Breast Cancer Cell Line.

BACKGROUND: Nigella sativa has been a nutritional flavoring factor and natural treatment for many ailments for so many years in medical science. Earlier studies have been reported that thymoquinone (TQ), an active compound of its seed, contains anticancer properties. Previous studies have shown that TQ induces apoptosis in breast cancer cells but it is unclear the role of P53 in the apoptotic pathway. Hereby, this study reports the potency of TQ on expression of tumor suppressor gene P53 and apoptosis induction in breast cancer cell line Michigan Cancer Foundation-7 (MCF-7). METHODS: MCF-7 cell line was cultured and treated with TQ, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out for evaluating the half-maximal inhibitory concentration (IC50) values after 24 h of treatment. The percentage of apoptotic cells was measured by flow cytometry. Real-time polymerase chain reaction (PCR) was performed to estimate the messenger RNA expression of P53 in MCF-7 cell line at different times. RESULTS: The IC50 value for the TQ in MCF-7 cells was 25 µM that determined using MTT assay. The flow cytometry and real-time PCR results showed that TQ could induce apoptosis in MCF-7 cells, and the P53 gene expression was dramatically up-regulated by ascending time, respectively. Hence, there was significant difference in 48 and 72 h. CONCLUSIONS: Our results demonstrated that TQ could induce apoptosis in MCF-7 cells through up-regulation of P53 expression in breast cancer cell line (MCF-7) by time-dependent manner.

Bone Repair with Differentiated Osteoblasts from Adipose-derived Stem Cells in Hydroxyapatite/Tricalcium Phosphate In vivo.

BACKGROUND: Recently, tissue engineering has developed approaches for repair and restoration of damaged skeletal system based on different scaffolds and cells. This study evaluated the ability of differentiated osteoblasts from adipose-derived stem cells (ADSCs) seeded into hydroxyapatite/tricalcium phosphate (HA-TCP) to repair bone. METHODS: In this study, ADSCs of 6 canines were seeded in HA-TCP and differentiated into osteoblasts in osteogenic medium in vitro and bone markers evaluated by reverse transcription polymerase chain reaction (RT-PCR). Scanning electron microscopy (SEM) was applied for detection of cells in the pores of scaffold. HA-TCP with differentiated cells as the test group and without cells as the cell-free group were implanted in separate defected sites of canine's tibia. After 8 weeks, specimens were evaluated by histological, immunohistochemical methods, and densitometry test. The data were analyzed using the SPSS 18 version software. RESULTS: The expression of Type I collagen and osteocalcin genes in differentiated cells were indicated by RT-PCR. SEM results revealed the adhesion of cells in scaffold pores. Formation of trabecular bone confirmed by histological sections that revealed the thickness of bone trabecular was more in the test group. Production of osteopontin in extracellular matrix was indicated in both groups. Densitometry method indicated that strength in the test group was similar to cell-free group and natural bone (P > 0.05). CONCLUSIONS: This research suggests that ADSCs-derived osteoblasts in HA-TCP could be used for bone tissue engineering and repairing.

Evaluation of the proliferation and viability rates of nucleus pulposus cells of human intervertebral disk in fabricated chitosan-gelatin scaffolds by freeze drying and freeze gelation methods.

BACKGROUND: Low back pain is one of the most significant musculoskeletal diseases of our time. Intervertebral disk herniation and central degeneration of the disk are two major reasons for low back pain, which occur because of structural impairment of the disk. The reduction of cell count and extracellular matrix, especially in the nucleus pulposus, causes disk degeneration. Different scaffolds have been used for tissue repairing and regeneration of the intervertebral disk in tissue engineering. Various methods are used for fabrication of the porosity scaffolds in tissue engineering. The freeze drying method has disadvantages such as: It is time consuming, needs high energy, and so on. The freeze-gelation method can save a great deal of time and energy, and large-sized porous scaffolds can be fabricated by this method. In this study, proliferation of the nucleus pulposus (NP) cells of the human intervertebral disk are compromised in the fabricated Chitosan-gelatin scaffolds by freeze drying and freeze gelation methods. MATERIALS AND METHODS: The cells were obtained from the nucleus pulposus by collagenase enzymatic hydrolysis. They were obtained from patients who were undergoing open surgery for discectomy in the Isfahan Alzahra Hospital. Chitosan was blended with gelatin. Chitosan polymer, solution after freezing at -80°C, was immersed in sodium hydroxide (NaOH) solution. The cellular suspension was transferred to each scaffold and cultured in plate for 14 days. Cell viability and proliferation were investigated by Trypan blue and MTT assays. RESULTS: The MTT and Trypan blue assays demonstrated that cell viability and the mean of the cell number showed a significant difference between three and fourteen days, in both scaffolds. Accordingly, there was a significantly decrease in the fabricated chitosan-gelatin scaffold by the freeze-drying method. CONCLUSION: The fabricated chitosan-gelatin scaffold by the freeze-gelation method prepared a better condition for proliferation of NP cells when compared with the fabricated chitosan-gelatin scaffold by the freeze drying method.

Changes of neural markers expression during late neurogenic differentiation of human adipose-derived stem cells.

BACKGROUND: Different studies have been done to obtain sufficient number of neural cells for treatment of neurodegenerative diseases, spinal cord, and traumatic brain injury because neural stem cells are limited in central nerves system. Recently, several studies have shown that adipose-derived stem cells (ADSCs) are the appropriate source of multipotent stem cells. Furthermore, these cells are found in large quantities. The aim of this study was an assessment of proliferation and potential of neurogenic differentiation of ADSCs with passing time. MATERIALS AND METHODS: Neurosphere formation was used for neural induction in isolated human ADSCs (hADSCs). The rate of proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and potential of neural differentiation of induced hADSCs was evaluated by immunocytochemical and real-time reverse transcription polymerase chain reaction analysis after 10 and 14 days post-induction. RESULTS: The rate of proliferation of induced hADSCs increased after 14 days while the expression of nestin, glial fibrillary acidic protein, and microtubule-associated protein 2 was decreased with passing time during neurogenic differentiation. CONCLUSION: These findings showed that the proliferation of induced cells increased with passing time, but in early neurogenic differentiation of hADSCs, neural expression was higher than late of differentiation. Thus, using of induced cells in early differentiation may be suggested for in vivo application.

Time-Dependent Effect of Encapsulating Alginate Hydrogel on Neurogenic Potential.

OBJECTIVE: Due to the restricted potential of neural stem cells for regeneration of central nervous system (CNS) after injury, providing an alternative source for neural stem cells is essential. Adipose derived stem cells (ADSCs) are multipotent cells with properties suitable for tissue engineering. In addition, alginate hydrogel is a biocompatible polysaccharide polymer that has been used to encapsulate many types of cells. The aim of this study was to assess the proliferation rate and level of expression of neural markers; NESTIN, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) in encapsulated human ADSCs (hADSCs) 10 and14 days after neural induction. MATERIALS AND METHODS: In this experimental study, ADSCs isolated from human were cultured in neural induction media and seeded into alginate hydrogel. The rate of proliferation and differentiation of encapsulated cells were evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay, immunocytoflourescent and realtime reverse transcriptase polymerase chain reaction (RT-PCR) analyzes 10 and 14 days after induction. RESULTS: The rate of proliferation of encapsulated cells was not significantly changed with time passage. The expression of NESTIN and GFAP significantly decreased on day 14 relative to day 10 (P<0.001) but MAP2 expression was increased. CONCLUSION: Alginate hydrogel can promote the neural differentiation of encapsulated hADSCs with time passage.

The beneficial effect of encapsulated human adipose-derived stem cells in alginate hydrogel on neural differentiation.

Tissue engineering employs combination of biomaterials and cell therapy to develop new therapeutic strategies for neurodegenerative diseases, spinal cord, and traumatic brain injuries. Alginate is a biocompatible hydrogel, which has been used broadly to encapsulate many types of cells. Human adipose-derived stem cells (hADSCs) have appropriate property to differentiate into neuron-like cells. Therefore, the aim of this study was to evaluate the effect of alginate hydrogel on the viability and neural differentiation potential of induced hADSCs. After neural induction of isolated hADSCs and encapsulated in alginate hydrogel, the cell viability using MTT assay and their neural differentiation potential by immunocytochemical and real time RT-PCR analysis for neural markers (Nestin, GFAP, and MAP2) were evaluated. Expression of Nestin, GFAP, and MAP2 markers was significantly increased compare to monolayer induced cells (p<0.001), but we did not found any significant effect on viability of induced cells relative to monolayer induced cells. Although neural differentiation of encapsulated cells was increased relative to monolayer induced cells, the viability of these cells was not significantly different in alginate hydrogel as compared with monolayer induced cells.

Transplantation of undifferentiated and induced human exfoliated deciduous teeth-derived stem cells promote functional recovery of rat spinal cord contusion injury model.

Regarding both the neural crest origin and neuronal potential of stem cells from human exfoliated deciduous teeth (SHED), here, we assessed their potential in addition to neural induced SHED (iSHED) for functional recovery when transplanted in a rat model for acute contused spinal cord injury (SCI). Following transplantation, a significant functional recovery was observed in both groups relative to the vehicle and control groups as determined by the open field locomotor functional test. We also observed that animals that received iSHED were in a better state as compared with the SHED group. Immunohistofluorescence evaluation 5 weeks after transplantation showed neuronal and glial differentiation and limited proliferation in both groups. However, myelin basic protein and chondroitin sulfate proteoglycan NG2-oligodendrocyte markers-were increased and glial fibrillary acidic protein-astrocyte marker-was decreased in the iSHED group in comparison with the SHED group. These findings have demonstrated that transplantation of SHED or its derivatives could be a suitable candidate for the treatment of SCI as well as other neuronal degenerative diseases.