Differentiation Of Endometrial Stem Cells Research Articles

Tissue Engineering Using Differentiation of Endometrial Stem Cells into Ameloblast Cells and Implantation on Hydroxyapatite and Collagen Nanocomposite Scaffold

Abstract Background: Dental tissue engineering (DTE) is an inspiring biological approach to replacing lost teeth. The strength of the differentiation of basic endometrial cells to ameloblasts is yet to be fully understood. Moreover, the construction of a scaffold, upon which ameloblasts can both grow and replicate, has been a challenge in DTE. Methods: This is an experimental study. Basic endometrial cells were first isolated and cultured before induction of differentiation to ameloblast cells by adjacency to mesenchymal rat cells and application of fibroblast growth factor 8. Hydroxyapatite nanoparticles were synthesized by freeze-drying method. The nanocomposite collagen and hydroxyapatite scaffold were synthesized by the solvent casting method. Finally, the amyloblast cells were implanted into the hydroxyapatite and collagen nanocomposite scaffold. Results: The success of cell differentiation was confirmed through staining using specific antibodies against ameloblastin and amelogenin markers. Expression of ameloblast-specific mRNAs, such as ameloblastins, amelogenins, and cytokeratin 14 was detected in the differentiated cells. Alizarin red staining clearly illustrates mineralization nodules in the differentiated cells. Staining against specific ameloblast markers showed that the differentiated ameloblast cells are expressed on the scaffold, unlike the control group. Conclusion: Based on the findings of this study, basic endometrial cells are capable of expressing the traits of ameloblast cells. The scaffold used in the present study conforms to most criteria used for dental tissue reconstruction and can be applied in DTE.

Defining the role of 17β-estradiol in human endometrial stem cells differentiation into neuron-like cells.

Human endometrial stem cells (hEnSCs) that can be differentiated into various neural cell types have been regarded as a suitable cell population for neural tissue engineering and regenerative medicine. Considering different interactions between hormones, growth factors, and other factors in the neural system, several differentiation protocols have been proposed to direct hEnSCs towards specific neural cells. The 17β-estradiol plays important roles in the processes of development, maturation, and function of nervous system. In the present research, the impact of 17β-estradiol (estrogen, E2) on the neural differentiation of hEnSCs was examined for the first time, based on the expression levels of neural genes and proteins. In this regard, hEnSCs were differentiated into neuron-like cells after exposure to retinoic acid (RA), epidermal growth factor (EGF), and also fibroblast growth factor-2 (FGF2) in the absence or presence of 17β-estradiol. The majority of cells showed a multipolar morphology. In all groups, the expression levels of nestin, Tuj-1 and NF-H (neurofilament heavy polypeptide) (as neural-specific markers) increased during 14 days. According to the outcomes of immunofluorescence (IF) and real-time PCR analyses, the neuron-specific markers were more expressed in the estrogen-treated groups, in comparison with the estrogen-free ones. These findings suggest that 17β-estradiol along with other growth factors can stimulate and upregulate the expression of neural markers during the neuronal differentiation of hEnSCs. Moreover, our findings confirm that hEnSCs can be an appropriate cell source for cell therapy of neurodegenerative diseases and neural tissue engineering.

The effect of female sex steroid hormones on osteogenic differentiation of endometrial stem cells.

Bone regeneration is a significant and crucial health issue worldwide. Tissue bioengineering has shown itself to be the best substitute for common clinical treatment of bone loss. The suitable cell source is human endometrial stem cells (hEnSCs) which have several suitable characteristics for this approach. Since sex steroid hormones are involved in expansion and conservation of the skeleton, the effect of two sex steroid hormones known as estrogen (17-β estradiol) and progesterone on osteogenic differentiation of hEnSCs were examined. For this purpose, hEnSCs were treated with 17-β estradiol and progesterone separately (1 × 10-6 M) and simultaneously (1 × 10-7 M). Osteogenic differentiation tests including measurement of total mineral calcium content, Alizarin Red staining, the quantitative expression levels of some osteogenic markers by Real-time RT-PCR, and immunofluorescence staining were performed at 7 and 14days of differentiation. To exhibit the morphology of the cells in osteogenic and culture medium, the hEnSCs were stained with Acridine Orange (AO) solution. In this research, MTT assay and AO staining revealed progesterone and 17-β estradiol increase the proliferation of hEnSCs in a dose-dependent manner. Furthermore, the results of calcium content analysis, Real-time RT-PCR assay, and all tests of differentiation staining have shown that 17-β estradiol and progesterone cannot induce hEnSCs' osteogenic differentiation. In conclusion, it is indicated that 17-β estradiol and progesterone do not have positive effects on hEnSCs' osteogenic differentiation in vitro.

Differentiation of endometrial stem cells into insulin-producing cells using signaling molecules and zinc oxide nanoparticles, and three-dimensional culture on nanofibrous scaffolds.

Diabetes mellitus is the most common metabolic disorder with a high mortality and morbidity rate. A new promising strategy to treat DM is pancreatic tissue engineering. We described a 3D culture system accompanied by signaling factors to differentiate hEnSCs into IPCs in the presence of nZnO. We isolated EnSCs and cultured it in DMEM/F12 medium. Nanofibrous PLA/Cs scaffold was prepared through the electrospinning method. The morphological properties of the scaffolds and cells were evaluated by SEM. MTT assay was used to investigate the metabolic activity of the hEnSCs cultured on the scaffolds and a four-stage protocol was applied to differentiate hEnSCs. The differentiated cells were tested for pancreatic markers by immunocytochemistry, qRT-PCR and DTZ staining. The results of this study revealed that hEnSCs cultured on PLA/Cs scaffold and treated with nZnO can efficiently differentiate into IPCs. The examination of differentiated cell morphology showed their near similarity with pancreatic islet cells, and DTZ staining emphasized the presence of insulin granules inside their cytoplasm. Moreover, qRT-PCR and immunofluorescent staining results showed the efficient expression of specific gene markers of IPCs in resultant differentiated cells. Moreover, PLA/CS and nZnO were able to provide a good nanoenvironment for the differentiation of hEnSCs into IPCS the in presence of other molecules.

In vitro proliferation and osteogenic differentiation of endometrial stem cells and dental pulp stem cells.

Stem-cell-based therapies were introduced aiming to overcome the limitations of the existing procedures for regeneration of mineralized tissues. Stem cells isolated from the endometrial tissue and dental pulp have the capacity to differentiate into various functional cells including osteoblasts. However, studies comparing their ability to regenerate mineralized tissue are lacking. The purpose of this study was to compare the proliferation and osteogenic differentiation potential of endometrial stem cells (EnSCs) and dental pulp stem cells (DPSCs) using in vitro cell culture technique. The DPSCs and EnSCs were isolated from human dental pulp and endometrium, respectively. Their proliferation and osteogenic potential were compared in the same osteogenic medium (OM) after 3, 5, 7 and 10days using the methyl thiazol tetrazolium assay, alizarin red staining, and real-time quantitative reverse transcription polymerase chain reaction (Real-Time qRT-PCR). The EnSCs showed higher proliferation rate compared to DPSCs. Regarding osteogenesis, alizarin red-positive colonies appeared earlier and in greater amounts in DPSCs group. The real-time qRT-PCR demonstrated significantly greater osteogenic potential of DPSCs compared to EnSCs. Our findings revealed significant differences in stem cell properties based on the tissue source. The EnSCs had greater proliferation rate than DPSCs, while DPSCs showed greater osteogenic potential compared to EnSCs in the same OM.

Indirect Co-Culture with Schwann Cells as a New Approach for Human Endometrial Stem Cells Neural Transdifferentiation

Neurodegenerative diseases such as Spinal Cord Injury (SCI) refer to the loss of neuronal cells in the central nervous system. Cell replacement therapy is currently the most propitious approach for the treatment of such diseases. Endometrial Stem Cells (EnSC) due to their unique characteristics have recently gained extensive attention as a new source of mesenchymal-like stem cells for tissue engineering purposes. Research has shown the potency of these cells to differentiate into several cell lineages including osteocytes, adipocytes and neural cells using chemical induction. In this present study we describe how human EnSCs can be induced to differentiate into neuron-like cells when co-cultured with human Schwann cells via insert wells which inhibit intercellular contact of the two cell types. Quantitative gene expression analysis and immunocytochemistry was performed after one week of co-culture. The results confirmed neuronal differentiation of EnSCs which support the notion that Schwann cells produce and secrete neurotrophic factors that can act upon cells without physical contact. Our present findings support the differentiation of EnSCs and the potency of these cells for the treatment of degenerative and acquired disorders of the nervous system.

The effect of purmorphamine on differentiation of endometrial stem cells into osteoblast-like cells on collagen/hydroxyapatite scaffolds

We assessed the effect of purmorphamine along with collagen/hydroxyapatite scaffold in inducing osteogenesis of human endometrial stem cells (hEnSCs). The adhesion, viability, proliferation, and differentiation of cells on scaffold were assayed with SEM, MTT, real time-PCR, and ALP assay, respectively. The results were shown good integration of cells with scaffold. Also, qRT-PCR of differentiated cells shows that osteoblast cell markers are expressed after 21d in 2D and scaffold groups while in the scaffold group the expression of these markers were higher than the 2D group. Based on our findings, collagen/hydroxyapatite scaffold with PMA has the potential role in osteogenic differentiation of hEnSCs.

Improved human endometrial stem cells differentiation into functional hepatocyte-like cells on a glycosaminoglycan/collagen-grafted polyethersulfone nanofibrous scaffold.

Liver tissue engineering (TE) is rapidly emerging as an effective technique which combines engineering and biological processes to compensate for the shortage of damaged or destroyed liver tissues. We examined the viability, differentiation, and integration of hepatocyte-like cells on an electrospun polyethersulfone (PES) scaffold, derived from human endometrial stem cells (hEnSCs). Natural polymers were separately grafted on plasma-treated PES nanofibers, that is, collagen, heparan sulfate (HS) and collagen-HS. Galactosilated PES (PES-Gal) nanofibrous were created. The engineering and cell growth parameters were considered and compared with each sample. The cellular studies revealed increased cell survival, attachment, and normal morphology on the bioactive natural polymer-grafted scaffolds after 30 days of hepatic differentiation. The chemical and molecular assays displayed hepatocyte differentiation. These cells were also functional, showing glycogen storage, α-fetoprotein, and albumin secretion. The HS nanoparticle-grafted PES nanofibers demonstrated a high rate of cell proliferation, differentiation, and integration. Based on the observations mentioned above, engineered tissue is a good option in the future, for the commercial production of three-dimensional liver tissues for clinical purposes. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2516-2529, 2017.

Assessment of the Neural-Like Cells Differentiation from Endometrial Stem Cells following Fluoxetine Treatment

Fluoxetine is a common antidepressant which selectively inhibits serotonin reuptake at synaptic level. Some research findings have proposed the effect this drug on neurogenesis, neuronal survival as well as proliferation of the neural progenitor cells. Endometrium is a part of uterus which harbors mesenchymal stem cells. This source of stem cells can be differentiated into neural cells which may potentially be used in treating many diseases. Given the above, this study was designed to assess the effect of fluoxetine on neural cells differentiation from endometrial stem cells. Endometrial stem cells obtained from stem cell banking were cultured in Dulbecco’s modified eagle’s medium (DMED) containing fetal bovine serum (FBS) in the presence of Retinoic Acid, fluoxetine and Retinoic Acid+fluoxetine for 10 days. To assess the differentiation of endometrial stem cells into neural-like cells, we used immunocytochemistry and RT-PCR. The viability of cells was assessed using the trypan blue test. Data analysis revealed that 61% of endometrial stem cells differentiated to neural-like cells. Moreover, the biopotency of neural-like cells on fluoxetine treatment was more pronounced across differentiation days. Based on our findings, fluoxetine was shown to be a suitable inducer for the differentiation of neural-like cells from endometrial stem cells.

Human endometrial stem cells differentiation into functional hepatocyte‐like cells

Human endometrial stem cells differentiation into functional hepatocyte‐like cells

Human endometrial stem cells differentiation into functional hepatocyte‐like cells

In spite of certain clinical limitations, such as teratoma formation, the use of stem cells is considered as an appropriate source in cell therapy and tissue engineering. This study shows human endometrial stem cells (hEnSCs) has exceptional differentiation ability in hepatocyte formation. hEnSCs have high purification rate and immune-tolerance, and can be used as an appropriate substitute for hepatocytes in liver disorders. Differentiation required hepatogenic medium. Quantitative reverse transcription-polymerase chain reaction and immunofluorescent staining of hepatic genes and proteins including cytokeratin 18 (ck18), alpha-fetoprotein (afp), and albumin (alb) were used to assess differentiation. Cells differentiated with a hepatocyte-like morphology and expressed hepatic markers on 30 days of differentiation. The Periodic Acid-Schiff (PAS) reaction showed storage of glycogen, and albumin and afp secretions were also detected. In vitro hEnSCs behave like hepatocyte after differentiation and may be a suitable source of cells in liver regeneration.

Endometrial stem cell differentiation into smooth muscle cell: a novel approach for bladder tissue engineering in women

To investigate manufacturing smooth muscle cells (SMCs) for regenerative bladder reconstruction from differentiation of endometrial stem cells (EnSCs), as the recent discovery of EnSCs from the lining of women's uteri, opens up the possibility of using these cells for tissue engineering applications, such as building up natural tissue to repair prolapsed pelvic floors as well as building urinary bladder wall. Human EnSCs that were positive for cluster of differentiation 146 (CD146), CD105 and CD90 were isolated and cultured in Dulbecco's modified Eagle/F12 medium supplemented with myogenic growth factors. The myogenic factors included: transforming growth factor β, platelet-derived growth factor, hepatocyte growth factor and vascular endothelial growth factor. Differentiated SMCs on bioabsorbable polyethylene-glycol and collagen hydrogels were checked for SMC markers by real-time reverse-transcriptase polymerase chain reaction (RT-PCR), western blot (WB) and immunocytochemistry (ICC) analyses. Histology confirmed the growth of SMCs in the hydrogel matrices. The myogenic growth factors decreased the proliferation rate of EnSCs, but they differentiated the human EnSCs into SMCs more efficiently on hydrogel matrices and expressed specific SMC markers including α-smooth muscle actin, desmin, vinculin and calponin in RT-PCR, WB and ICC experiments. The survival rate of cultures on the hydrogel-coated matrices was significantly higher than uncoated cultures. Human EnSCs were successfully differentiated into SMCs, using hydrogels as scaffold. EnSCs may be used for autologous bladder wall regeneration without any immunological complications in women. Currently work is in progress using bioabsorbable nanocomposite materials as EnSC scaffolds for developing urinary bladder wall tissue.

Effect of dentine matrix proteins on human endometrial adult stem-like cells: In vitro regeneration of odontoblasts cells

ObjectiveHuman endometrium has enormous regenerative capacity due to the presence of endometrial stem cells. The present study sought to assess the possibility of differentiation of these cells into odontoblast-like cells by in vitro induction. MethodsEndometrial stem-like cells were obtained using enzymatic digestion of the biopsy samples of the endometrium after hysterectomy and cultured in Dulbecco's Modified Eagle's Medium (DMEM) which contained dentine non-collagenous proteins (dNCPs). The results were evaluated using the following assays: analysis of morphology, enzymatic assay for measuring alkaline phosphatase activity, Alizarin Red staining to detect in vitro formation of mineralized nodules, immunofluorescence for detection of dentine sialoprotein (DSP) and dentine-matrix protein (DMP1), and Western blotting for DMP1 expression. ResultsFollowing induction, endometrial stem-like cells demonstrated high alkaline phosphatase activity, and expression of DSP and DMP1 confirmed the odontoblast phenotype. DMP1 level increased in samples treated with dNCPs. ConclusionsStudy results indicated that odontoblastic differentiation of endometrial stem cells can be induced by extracellular matrix proteins (e.g. dNCPs). The capacity of endometrial stem-like cells to differentiate into odontoblast-like cells under specific conditions gives new insights into the mechanism of odontogenesis and highlights the potential of such approaches for further research on dental tissue regeneration.

Comparison of osteogenic medium and uniaxial strain on differentiation of endometrial stem cells

Mechanotransduction plays a pivotal role in remodeling and repair of skeletal tissues. This mechanism has been widely used in bone tissue engineering especially under in vitro conditions. To date, various stem cells have been used for this purpose. The present study was the first to evaluate the effect of mechanical loading on differentiation of human endometrial stem cells (hESCs) to osteoblasts. Adhesion of endometrial stem cells after isolation and culture on a silicone membrane covered with collagen was evaluated under scanning electron microscope (SEM). Twenty-four hours after cell culture on the membrane and ensuring appropriate cell adhesion, a group of cells in a conventional culture medium received 3% static uniaxial strain. In the positive control group, cells cultured on the membrane were placed in an osteogenic medium without receiving any mechanical strain. The negative control group was placed in a regular medium and received no strain either. Two weeks later, cultured cells were evaluated for expression of osteogenic markers using immunofluorescence staining and real-time polymerase chain reaction (PCR). Data of real-time PCR was analyzed by ANOVA. P < 0.05 was considered statistically significant. SEM analysis revealed adequate cell adhesion to the membrane after 24 h. Two weeks after loading, expression of markers in the positive control group was significantly higher compared to test group. We can conclude that static uniaxial strain exerted on hESCs results in their differentiation to osteoblasts. However, this magnitude of static strain in the tested time period cannot yield excellent differentiation when compared to the osteogenic medium.

Demonstration of Multipotent Stem Cells in the Adult Human Endometrium by In Vitro Chondrogenesis

Stem cells are defined by their unique capacity for self-renewal and multilineage differentiation. Stem cells have been obtained from multiple extramedullary tissues. Recently, a population of progenitor cells have been identified in the endometrium. However, multilineage differentiation of endometrial stem cells has not been reported. Endometrial tissue was obtained from reproductive-aged women undergoing surgery for benign disease, from which monolayer endometrial stromal cell (ESC), myometrial, fibroid, fallopian tube, and uterosacral ligament tissue cultures were generated. Once confluent, cells were trypsinized and centrifuged in conical tubes to form a cell pellet. Cell pellets were cultured in a defined chondrogenic media (CM) containing dexamethasone and transforming growth factor (TGF)-beta2 or TGF-beta 3 for 3 to 21 days. Samples were analyzed for markers of human articular cartilage, including sulfated glycosaminoglycans and expression of type II collagen. ESC pellets cultured in CM were found to contain cells that resemble chondrocytes. These cells expressed sulfated glycosaminoglycans and type II collagen typical of human articular cartilage. Myometrial, fibroid, fallopian tube, and uterosacral ligament cells were unable to undergo chondrogenic differentiation using the pellet culture method. Cells derived from the endometrium were able to differentiate into a heterologous cell type: chondrocytes, thus demonstrating the presence of multipotent stem cells. Endometrium is a potential source of multipotent stem cells.