Characteristics of Human Endometrial Stem Cells in Tissue and Isolated Cultured Cells: An Immunohistochemical Aspect

Background:

The aim of this study was to investigate the percentage of the stem cells population in human endometrial tissue sections and cultured cells at fourth passage.

Methods:

Human endometrial specimens were divided into two parts, one part for morphological studies and the other part for in vitro culture. Full thickness of human normal endometrial sections and cultured endometrial cells at fourth passage were analyzed via immunohistochemistry for CD146 and some stemness markers such as Oct4, Nanog, Sox2, and Klf4 and the expression of typical mesenchymal stem cell markers CD90, CD105.

Results:

11.88±1.29% of human endometrial cells within tissue sections expressed CD146 marker vs. 28±2.3% of cultured cells, CD90 and CD105 were expressed by functionalis stroma (85±2.4 and 89±3.2%) than basalis stroma (16±1.4 and 17±1.9%), respectively (P<0.05). Oct4 and Nanog-expressing cells comprise 1.43±0.08 and 0.54±0.01% of endometrial stromal cells in endometrial sections vs. 12±3.1% and 8±2.9% of cultured cells, respectively. They reside near the glands in the basal layer of endometrium. Sox2 and Klf4 were not commonly expressed in tissue samples and cultured cells. CD9 and EpCAM were expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells.

Conclusion:

The human endometrial stem cells and pluripotency markers may be localized more in basalis layer of endometrium. The immunostaining observations of endometrial cells at fourth passage were correlated with the immunohistochemistry data.Key Words: Endometrium, Immunohistochemistry, Mesenchymal stem cells     

Differentiation of Umbilical Cord Lining Membrane-Derived Mesenchymal Stem Cells into Endothelial-Like Cells

Background: Stem cell therapy for the treatment of vascular-related diseases through functional revascularization is one of the most important research areas in tissue engineering. The aim of this study was to investigate the in vitro differentiation of umbilical CL-MSC into endothelial lineage cells. Methods: In this study, isolated cells were characterized for expression of MSC-specific markers and osteogenic and adipogenic differentiation. They were induced to differentiate into endothelial-like cells and then examined for expression of the endothelial-specific markers, karyotype, and functional behavior of cells. Results: Isolated cells expressed MSC-specific markers and differentiated into adipocytes and osteoblasts. After endothelial differentiation, they expressed CD31, vWF, VE-cadherin, VEGFR1, and VEGFR2 at both mRNA and protein level, but their morphological changes were not apparent when compared with those of undifferentiated cells. There were no significant changes in karyotype of differentiated cells. Furthermore, angiogenesis assay and LDL uptake assay showed that differentiated cells were able to form the capillary-like structures and uptake LDL, respectively. Conclusion: The results indicated that umbilical CL-MSC could differentiate into functional endothelial-like cells. Also, they are suitable for basic and clinical studies to cure several vascular-related diseases. Key Words: Endothelial differentiation, Endothelial-like cells, Mesenchymal stem cells, Umbilical cord lining membrane     

Interaction between LINC-ROR and Stemness State in Gastric Cancer Cells with Helicobacter pylori Infection

Background:LINC-ROR, as a cancer-related lncRNA, has vital roles in stem cell survival, pluripotency, differentiation, and self-renewal in hESCs. However, cancer-related molecular mechanisms, its functional roles, and clinical value of LINC-ROR in GC remain unclear. In this study, we aimed to investigate probable interplay between LINC-ROR with SALL4 stemness regulator and their role with the development of the disease. Methods:The mRNA expression profile of LINC-ROR and SALL4 was assessed in tumoral and adjacent non-cancerous tissues of GC patients, using quantitative real-time PCR. Results:Significant LINC-ROR underexpression and SALL4 overexpression were observed in 55.81% and 75.58% (p < 0.0001) of samples, respectively. The expression of LINC-ROR and SALL4 were significantly correlated with each other (p = 0.044). There was an association between the underexpression of LINC-ROR and sex, stage of tumor progression, tumor type, and location of tumor (p < 0.05), and H. pylori infection with SALL4 expression (p = 0.036). There were also significant correlations between concomitant mRNA expression of SALL4 and LINC-ROR in tumors located at distal noncardiac, positive for H. pylori infection, tumors with invasion into the muscle layer of the stomach, and grade II tumor (p < 0.05). Conclusion:The clinical results of the SALL4-LINC-ROR association propose a probable functional interaction between these markers in tumor maintenance and aggressiveness. Our study can help to understand one of the mechanisms involved in the progression of GC through the function of these regulators. Key Words: LINC-ROR, Non-coding RNA, SALL4     

Sol-Gel Synthesis,in vitro Behavior,and Human Bone Marrow-Derived Mesenchymal Stem Cell Differentiation and Proliferation of Bioactive Glass 58S

Background:Bioactive glasses 58S, are silicate-based materials containing calcium and phosphate, which dissolved in body fluid and bond to the bone tissue. This type of bioactive glass is highly biocompatible and has a wide range of clinical applications. Methods:The 58S glass powders were synthesized via sol-gel methods, using tetraethyl orthosilicate, triethyl phosphate, and calcium nitrate, as precursors. Upon the analyses of phase and chemical structures of bioactive glass in different gelation times (12, 48, and 100 h), the appropriate heat treatment (at 525, 575, and 625 °C) was performed to eliminate nitrate compounds and stabilize the glass powder samples. The in vitro assay in SBF solution revealed the bioactivity of the synthesized 58S glass through the morphological (SEM), chemical structure (FTIR), release of calcium, phosphorous and silicon elements, pH variations, and weight loss measurements. The behavior of MSCs in the presence of bioactive glass powders was studied by MTT cytotoxicity, cell staining, ALP activity and biomineralization tests, as well as by the evaluation of ALP, osteocalcin, osteonectin, collagen I, and RUNX2 gene expression. Results: The results confirmed a gelation time of 100 h and a calcination temperature of 575 °C at optimal conditions for the synthesis of nitrate-free bioactive glass powders. Conclusion:The glass spherical nanoparticles in the range of 20-30 nm possess the improved bioactivity and osteogenic properties as demanded for bone tissue engineering. Key Words: Bioactive glass 58S, Gene expression, Mesenchymal stem cells     

Evaluation of Neurogenic Potential of Human Umbilical Cord Mesenchymal Cells; a Time- and Concentration-Dependent Manner

Background:

Retinoic acid as one of the most important regulators for cell differentiation was examined in this study for differentiation of human umbilical mesenchymal cells (hUCM).

Methods:

After isolation, hUCM were evaluated for mesenchymal stem cell properties by flow cytometry and alkaline phosphatase assay. Also, doubling time of the cells and their differentiation potential into adipogenic and osteogenic cells were tested. hUCM were then cultured with different concentrations of retinoic acid, and on days 1, 7, and 12, the percentage of differentiated cells was determined by immunostaining for nestin, anti-microtubule associated protein 2 (MAP₂), glutamic acid decarboxylase (GAD), and gamma-aminobutyric acid (GABA) markers.

Results:

The isolated cells were negative for the hematopoietic markers and positive for the mesenchymal markers. They showed the population doubling time 60 ± 3 hours and differentiated into osteogenic and adipogenic cells. A descending trend in nestin and an ascending trend in MAP₂, GAD, and GABA expression were observed from the first day until the last day between different concentrations of retinoic acid.

Conclusion:

hUCM cells may have the potential to differentiate into neural cells in the presence of different incubation period and concentration of retinoic acid.Key Words: Cell differentiation, Neural stem cells, Retinoic acid     

Woodchuck Hepatitis Virus Post-Transcriptional Regulation Element (WPRE) Promotes Anti-CD19 BiTE Expression in Expi293 Cells

Background:Bispecific antibodies represent an important class of mAbs, with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti- CD3 × CD19 bsAb is a bispecific T-cell engager (BiTE) currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts. Methods:WPRE sequence was incorporated at the 3’ end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & Conclusion:Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in HEK-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody. Key Words: Acute lymphoblastic leukemia, Bispecific antibodies, Monoclonal antibody     

Mesenchymal Stem Cells as an Alternative for Schwann Cells in Rat Spinal Cord Injury

Background: Spinal cord has a limited capacity to repair; therefore, medical interventions are necessary for treatment of injuries. Transplantation of Schwann cells has shown a great promising result for spinal cord injury (SCI). However, harvesting Schwann cell has been limited due to donor morbidity and limited expansion capacity. Furthermore, accessible sources such as bone marrow stem cells have drawn attentions to themselves. Therefore, this study was designed to evaluate the effect of bone marrow-derived Schwann cell on functional recovery in adult rats after injury. Methods: Mesenchymal stem cells were cultured from adult rats’ bone marrow and induced into Schwann cells in vitro. Differentiation was confirmed by immunocytochemistry and RT-PCR. Next, Schwann cells were seeded into collagen scaffolds and engrafted in 3 mm lateral hemisection defects. For 8 weeks, motor and sensory improvements were assessed by open field locomotor scale, narrow beam, and tail flick tests. Afterwards, lesioned spinal cord was evaluated by conventional histology and immunohistochemistry. Results: In vitro observations showed that differentiated cells had Schwann cell morphology and markers. In this study, we had four groups (n = 10 each): laminectomy, control, scaffold and scaffold + Schwann cells. Locomotor and sensory scores of cell grafted group were significantly better than control and scaffold groups. In histology, axonal regeneration and remyelination were better than control and scaffold groups. Conclusion: This study demonstrates that bone marrow-derived Schwann cells can be considered as a cell source for Schwann cells in SCI treatment. Key Words: Rats, Spinal cord injuries (SCI), Bone marrow, Schwann cells, Cell transdifferentiation     

Bone Marrow Stromal Cell Transdifferentiation into Oligodendrocyte-Like Cells Using Triiodothyronine as a Inducer with Expression of Platelet-Derived Growth Factor α as a Maturity Marker

Background: The present study investigated the functional maturity of oligodendrocyte derived from rat bone marrow stromal cells (BMSC). Methods: The BMSC were isolated from female Sprague-Dawley rats and evaluated for different markers, such as fibronectin, CD106, CD90, Oct-4 and CD45. Transdifferentiation of OLC from BMSC was obtained by exposing the BMSC to DMSO and 1 µM all-trans-retinoic acid during the pre-induction stage and then induced by heregulin (HRG), platelet-derived growth factor AA (PDGFR-α), fibroblast growth factor and T3. The neuroprogenitor cells (NPC) were evaluated for nestin, neurofilament 68, neurofilament 160 and glial fibrillary acidic protein gene expression using immunocytochemistry. The OLC were assessed by immunocytochemistry for O4, oligo2, O1 and MBP marker and gene expression of PDGFR-α was examined by RT-PCR. Results: Our results showed that the fibronectin, CD106, CD90, CD45 and Oct-4 were expressed after the fourth passage. Also, the yield of OLC differentiation was about 71% when using the O1, O4 and oligo2 markers. Likewise, the expression of PDGFR-α in pre-oligodendrocytes was noticed, while MBP expression was detected in oligodendrocyte after 6 days of the induction. Conclusion: The conclusion of the study showed that BMSC can be induced to transdifferentiate into mature OLC. Key Words: Bone marrow stromal cell, Triiodothyronine, Platelet-derived growth factor α     

The Effect of Human Platelet-Rich Plasma on Adipose-Derived Stem Cell Proliferation and Osteogenic Differentiation

Background: The cultured mesenchymal stem cells (MSC) have been used in many clinical trials; however, there are still some concerns about the cultural conditions. One concern is related to the use of FBS as a widely used xenogeneic supplement in the culture system. Human platelet-rich plasma (hPRP) is a candidate replacement for FBS. In this study, the effect of hPRP on MSC proliferation and osteogenic differentiation has been evaluated. Methods: Human adipose-derived stem cells (hADSC) were expanded. Cells from the third passage were characterized by flow cytometric analysis and used for in vitro experiments. Resazurin and alizarin red stains were used for cell proliferation and osteogenic differentiation assays, respectively. Results: Treatment with hPRP resulted in a statistically significant increase in cell proliferation compare to the negative control group (P<0.001). Cell proliferation in the 15% hPRP group was also significantly higher than that in the 10% hPRP group (P<0.05). Additionally, it caused less osteogenic differentiation of the hADSC compared to the FBS (P<0.001), but in comparison to negative control, it caused acceptable mineralization (P<0.001). Conclusion: These findings indicate that hPRP not only improves the proliferation but also it can be a suitable substitution in osteogenic differentiation for clinical purposes. However, the clinical application value of hPRP still needs more investigation. Key Words: Platelet-Rich Plasma, Adipose tissue, Stem Cells, Cell differentiation, Cell proliferation     

Soft rot and blackleg reactions in diploid potato hybrids inoculated withErwinia spp.

From 1993 to 1996 three groups of potato genotypes were evaluated for resistance toErwinia spp.: (1) 31 interspecific diploid hybrids (28 resistant and three susceptible), (2) five hexaploid or pentaploid somatic hybrids ofSolanum tuberosum (tbr) ×S. brevidens (brd), and (3) eight cultivars. Two evaluation methods were applied: tuber point inoculation with eitherErwinia carotovora ssp.atroseptica (Eca) orE. chrysanthemi (Ech) to test tuber soft rot resistance and stem base inoculation with Eca to test blackleg resistance. Some resistant diploid hybrids and somatic hybrids oftbr × brd were superior to cultivars for both tuber and stem resistance. Tuber and stem resistance toErwinia spp. in the most resistant diploid hybrids were equal to the highly resistant somatic hybrids oftbr xbrd. Tuber resistance to Eca was highly correlated to tuber resistance to Ech (r=0.815***). In two years of evaluation for stem resistance, three diploid hybrids and a derivative of one of the somatic hybrids (USA M 264) failed to develop symptoms of blackleg following inoculation with Eca, Analyses of variance for tuber and stem resistance indicated significant effects of genotype, year and genotype × year interaction. A positive relationship between tuber and stem resistance toErwinia spp. has been found, however the genetic control of resistance in tuber and stem is partially independent. In the case of Eca the correlation coefficient was r = 0.725***. Therefore it should be possible to obtain resistant genotypes to both blackleg and tuber soft rot. Several resistant diploid hybrids were selected from among those tested, which also have several other characters desirable for potato breeders.     

Contribution of synthetic tetraploids (AAAA) and diploids (AA) to black Sigatoka resistance and bunch weight to their triploid progenies

The generation of banana triploids from tetraploid-diploid crosses requires knowledge on the influence of the parents on black Sigatoka resistance and agronomic traits to the triploid progenies. The objective of this investigation was to determine the influence of tetraploid and diploid parents on black Sigatoka resistance and agronomic traits in the triploid progenies generated from tetraploid-diploid crosses. The mating scheme was designed as a 4 × 5 North Carolina II mating design. Due to problems in seed set and germination, progenies from 2 male parents with 4 female parents were evaluated at two sites in Uganda. The results showed that the male-parent triploid progeny heritability estimate for the number of leaves at harvest was greater than the female parent estimate. The diploid parents had higher correlation coefficients for the total leaves at harvest with the triploid progenies than tetraploid parents with triploid progenies. Disease development over time took more days in diploid parents than in the tetraploid parents with the triploid progenies as intermediates. These results suggested that diploids transferred black Sigatoka resistance to the triploid progenies as measured by the number of standing leaves and disease development overtime. There was a positive correlation (P < 0.05) between tetraploid female parents and triploid progenies for plant height and bunch weight. The triploid progeny-tetraploid female parent heritability estimates for plant height (0.92) and bunch weight (0.72) were highly significant. These results indicated that the female synthetic tetraploids influenced plant height and bunch weight in the triploid progenies. Therefore, it is important to select the tetraploids with heavy bunches to effectively improve yield in triploid progenies generated by tetraploid-diploid crosses. The tetraploid-diploid progenies had a significant (P < 0.05) family-by-site interaction for bunch weight indicating that new banana genotypes need to be tested across different environments to select stable genotypes to promote to end-users.     

Neuronal Differentiation of Rat Hair Follicle Stem Cells: the Involvement of the Neuroprotective Factor Seladin-1 (DHCR24)

Background: The seladin-1 (selective Alzheimer disease indicator-1), also known as DHCR24, is a gene found to be down-regulated in brain region affected by Alzheimer disease (AD). Whereas, hair follicle stem cells (HFSC), which are affected in with neurogenic potential, it might to hypothesize that this multipotent cell compartment is the predominant source of seladin-1. Our aim was to evaluate seladin-1 gene expression in hair follicle stem cells. Methods: In this study, bulge area of male Wistar rat HFSC were cultured and then characterized with Seladin-1 immunocytochemistry and flow cytometry on days 8 to 14. Next, 9-11-day cells were evaluated for seladin-1 gene expression by real-time PCR. Results: Our results indicated that expression of the seladin-1 gene (DHCR24) on days 9, 10, and 11 may contribute to the development of HFSC. However, the expression of this gene on day 11 was more than day 10 and on 10^th day was more than day 9. Also, we assessed HFSC on day 14 and demonstrated these cells were positive for β-ш tubulin, and seladin-1 was not expressed in this day. Conclusion: HFSC express seladin-1 and this result demonstrates that these cells might be used to cell therapy for AD in future. Key Words: Seladin-1 (selective Alzheimer disease indicator-1), Alzheimer disease (AD), Hair follicle stem cells     

The Role of Biodegradable Engineered Nanofiber Scaffolds Seeded with Hair Follicle Stem Cells for Tissue Engineering

Background

The aim of this study was to fabricate the poly caprolactone (PCL) aligned nanofiber scaffold and to evaluate the survival, adhesion, proliferation, and differentiation of rat hair follicle stem cells (HFSC) in the graft material using electrospun PCL nanofiber scaffold for tissue engineering applications.

Methods

The bulge region of rat whisker was isolated and cultured in DMEM: nutrient mixture F-12 supplemented with epidermal growth factor. The morphological and biological features of cultured bulge cells were observed by light microscopy using immunocytochemistry methods. Electrospinning was used for production of PCL nanofiber scaffolds. Scanning electron microscopy (SEM), 3-(4, 5-di-methylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and histology analysis were used to investigate the cell morphology, viability, attachment and infiltration of the HFSC on the PCL nanofiber scaffolds.

Results

The results of the MTT assay showed cell viability and cell proliferation of the HFSC on PCL nanofiber scaffolds. SEM microscopy images indicated that HFSC are attached, proliferated and spread on PCL nanofiber scaffolds. Also, immunocytochemical analysis showed cell infiltration and cell differentiation on the scaffolds.

Conclusion

The results of this study reveal that PCL nanofiber scaffolds are suitable for cell culture, proliferation, differentiation and attachment. Furthermore, HFSC are attached and proliferated on PCL nanofiber scaffolds.Key Words: Nanofiber, Electrospinning, Stem cells, Tissue engineering     

HOTAIR Induces the Downregulation of miR-200 Family Members in Gastric Cancer Cell Lines

Background:Gastric cancer is the fourth most common human malignancy and the second reason for cancer morbidity worldwide. LncRNA HOTAIR has recently emerged as a promoter of metastasis in various cancer types, including GC, through the EMT process. However, the exact mechanism of HOTAIR in promoting EMT is unknown. Aberrant expression of the miR-200 family has been linked to the occurrence and development of various types of malignant tumors. This study investigates the correlation between the HOTAIR and miR-200 family gene expression patterns in GC cell lines. We investigated the miR-200 and HOTAIR due to their common molecular features in the EMT process. Methods:AGS and MKN45 cell lines were transfected with si-HOTAIR, along with a negative control. The effect of HOTAIR knockdown was also analyzed on cell viability and also on the expression of miR-200 family members, including miR-200a, -200b, and -200c, in cell lines using qRT-PCR. Statistical analysis was performed to find the potential correlation between the expression level of HOTAIR and miRs. Results:Our results showed significant increased miR-200 family expression level in transfected AGS and MKN45 GC cells (fold changes > 2; p < 0.001). Moreover, a negative correlation was observed between HOTAIR and miR-200 expression levels in GC cell lines (p < 0.05). Conclusion:Our findings showed a significant association between miR-200 family and HOTAIR expression levels in GC cell lines. Taken together, the HOTAIR-miR-200 axis seems to play a vital role in human GC, suggesting a potential therapeutic target in future GC treatment. Key Words: Gene expression, Long noncoding RNA, HOTAIR, MicroRNAs     

Valproic Acid Ameliorates Locomotor Function in the Rat Model of Contusion via Alteration of Mst1, Bcl-2, and Nrf2 Gene Expression

Background:In animal models of inflammatory diseases, Mst1 facilitates the programmed cell death as a novel pro-apoptotic kinase. This research aimed to determine the expression level of Mst1 gene in a rat model of SCI treated with VPA. Methods:Severe rat model contusion was used for evaluation of the neuroprotective effect of valproic acid. The BBB test, was performed to determine locomotor functions. H&E staining and TUNEL assay were performed to detect cavity formation and apoptosis, respectively. The mRNA levels of the genes Mst1, Nrf2, and Bcl-2 were evaluated, using quantitative RT-PCR. Results:The results revealed that Mst1 gene expression and TUNEL-positive cells in the VPA-treated group were significantly reduced as compared to the untreated group (p ≤ 0.05). Conclusion:Our findings indicate that VPA has therapeutic potential and can be a candidate for the treatment of neurodegenerative disorders and traumatic injury as a promising drug. Key Words: Bcl-2, Contusion, Mst1, Nrf2, Valproic acid