Selegiline Differentiates Adult Stem Cells toward Dopaminergic-Like Neurons: A Comparison between Two Cellular Niches of Hippocampal Neurogenesis.

OBJECTIVE: Neural stem cells (NSCs) are suitable therapeutic candidates. Here, we compare the proliferation rate, differentiation potential, and expression levels of specific markers in two groups of cultured NSCs derived from rat subgranular (SGZ) and subventricular (SVZ) zones. MATERIALS AND METHODS: In this experimental study, NSCs isolated from SGZ and SVZ were cultured in α-minimal essential medium (α-MEM) supplemented with 1% penicillin/streptomycin, 10% fetal bovine serum (FBS), 20 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF), and B27 supplement. Glial fibrillary acidic protein (Gfap), p75 neurotrophin receptor (Ngfr), tyrosine kinase receptor A (TrkA), beta-tubulin III (ßTIII), and Nestin gene levels were compared via reverse transcription polymerase chain reaction (RT-PCR) in these NSCs. Nestin and Gfap protein levels were compared by immunoassay. Subsequently, both populations were induced with 10-8 M selegiline for 48 hours, followed by immunohistochemical analysis of tyrosine hydroxylase (TH) levels. One-way ANOVA and Tukey's post-test were used with a significance level of P<0.05. RESULTS: Both groups were successfully expanded in vitro and expressed the neurotrophin receptor genes. The SGZNSCs had a significantly higher proliferation rate and significantly higher numbers of Nestin and Gfap-positive cells. Although the majority of selegiline-induced NSCs were TH-positive, we observed more TH-positive cells in SGZ-derived NSCs and these SGZ-NSCs displayed a shorter differentiation time. CONCLUSION: SGZ-derived NSCs appear to be a more appropriate candidate for therapeutic purposes based on proliferation rate, neurosphere size, and Gfap and Nestin expression levels, as well as differentiation time and TH expression level after dopaminergic induction.

Intravenous Transplantation of Adipose-Derived Mesenchymal Stem Cells Promoted The Production of Dopaminergic Neurons and Improved Spatial Memory in A Rat Model of Parkinson's Disease.

OBJECTIVE: Parkinson's disease (PD) is a neurodegenerative disorder described by the dynamic decline of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Stem cell transplantation is a new therapeutic strategy in the treatment of PD. The objective of the study was to assess the impact of intravenous infusion of adipose-derived mesenchymal stem cells (AD-MSCs) on memory disorder in Parkinsonian rats. MATERIALS AND METHODS: In this experimental study, male Wistar rats were randomly divided to four groups containing sham, cell treatment, control, and lesion. The cell treatment group received intravenous injection of AD-MSCs 12 days after PD induction by bilateral injection of 6-hydroxydopamine. Four weeks after lesion formation, spatial memory was examined using the Morris water maze (MWM) assessment. The rats' brains were removed and assessed by bromodeoxyuridine (BrdU), tyrosine hydroxylase (TH), and glial fibrillary acidic protein (Gfap) immunostaining. RESULTS: Statistical analyses revealed a significant addition and reduction in time spent and escape latency in the target quadrant, respectively, in the cell group as compared to the lesion group. Also, BrdU-labeled cells were present in the substantia nigra (SN). The density of TH-positive cells was significantly increased in the AD-MSCs transplantation group as compared to the lesion group, and the density of astrocytes significantly diminished in the AD-MSCs transplantation group as compared to the lesion group. CONCLUSION: It appears that AD-MSCs treatment for Parkinson's could decrease the density of astrocytes and promote the density of TH-positive neurons. It appears that AD-MSCs could improve spatial memory impairment in PD.

In-vivo oogenesis of oogonial and mesenchymal stem cells seeded in transplanted ovarian extracellular matrix.

OBJECTIVE (S): One way to overcome the recurrence of cancer cells following ovarian tissue transplantation is to use decellularized tissues as a scaffold that does not have any cellular components. These cell-free scaffolds can be seeded with different type of stem cells for ovarian restoration. MATERIALS AND METHODS: OSCs, PMSCs and BMSCs (oogonial, peritoneal and bone marrow mesenchymal stem cells, respectively) were seeded into human decellularized ovarian tissue as 4 groups: Scaffold + OSCs (SO), Scaffold + OSC + PMSCs (SOP), Scaffold + OSC + BMSCs (SOB) and Scaffold + OSC + PMSCs + BMSCs (SOPB). The produced grafts were transplanted into the sub-peritoneal space of ovariectomized NMRI mice as artificial ovary (AO). The expression of Vegf, CD34, Gdf9, Zp3, Ddx4, Amh and Lhr genes in AOs were measured by qRT-PCR. Also, histotechniques were considered to detect the anti GFP, PCNA, VEGF, GDF9, ZP3 and AMH proteins. RESULTS: H & E staining showed follicle-like structures in all groups; the number of these structures, in the SOP and SOB groups, were the highest. In SO group, differentiation ability to oocyte and granulosa cells was observed. Endothelial, oocyte, germ, and granulosa cell-like cells were specially seen in SOP and angiogenesis capability was more in SOB group. However, angiogenesis ability and differentiation to theca cell-like cells were more often in SOPB group. While none of the groups showed a significant difference in AMH level, estradiol levels were significantly higher in SOPB group. CONCLUSION: Integration of OSCs + PMSCs and those OSCs + BMSCs were more conducive to oogenesis.

Co-culture with peritoneum mesothelial stem cells supports the in vitro growth of mouse ovarian follicles.

The important roles played by the ovarian microenvironment and cell interactions in folliculogenesis suggest promising approaches for in vivo growth of ovarian follicles using appropriate scaffolds containing suitable cell sources. In this study, we have investigated the growth of early preantral follicles in the presence of decellularized mesenteric peritoneal membrane (MPM), peritoneum mesothelial stem cells (PMSCs), and conditioned medium (CM) of PMSCs. MPM of mouse was first decellularized; PMSCs were isolated from MPM and cultured and their conditioned medium (CM) was collected. Mouse follicles were separated into four groups: (1) culture in base medium (control), (2) culture in decellularized MPM (DMPM), (3) co-culture with PMSCs (Co-PMSCs), and (4) culture in CM of PMSCs (CM-PMSCs). Qualitative and quantitative assessments were performed to evaluate intact mesenteric peritoneal membrane (IMPM) as well as decellularized ones. After culturing the ovarian follicles, follicular and oocyte diameter, viability, eccentric oocyte percentage, and estradiol hormone amounts were evaluated. Quantitative and qualitative evaluations confirmed removal of cells and retention of the essential fibers in MPM after the decellularization process. Follicular parameters showed that Co-PMSCs better support in vitro growth and development of ovarian follicles than the other groups. The eccentric rate and estradiol production were statistically higher for the Co-PMSCs group than for the CM-PMSCs and control groups. Although the culture of early preantral follicles on DMPM and CM-PMSCs could improve in vitro follicular growth, co-culture of follicles with PMSCs showed even greater improvements in terms of follicular growth and diameter.

Wharton's jelly derived mesenchymal stem cells differentiate into oocyte like cells in vitro by follicular fluid and cumulus cells conditioned medium.

Wharton's jelly derived-mesenchymal stem cells (WJ-MSCs) have a same developmental origin with primordial germ cells. WJ-MSCs perhaps differentiate into oocyte and germ like-cells (OLCs/GLCs) in the presence of appropriate inducers. Human follicular fluid (FF) and cumulus cells conditioned medium (CCM) are naturally rich sources for oocyte development. The aim of this study was to evaluate WJ-MSCs potential for differentiating into OLCs and GLCs exposed to FF and CCM. WJ-MSCs were cultured in two different induction media (10% FF, 10% CCM) for 21 days. Morphological changes and expression of developmental genes were evaluated on days 0, 7, 14 and 21 of culture. Also, on 21st day of culture, the expression of oocyte and germ cell proteins investigated using immunofluorescence staining. Appearance of round shaped cells from 7th day onwards indicated that WJ-MSCs can differentiate into OLCs when exposed to FF and CCM. The size of produced OLCs and expression of oocyte specific genes and proteins were increased more positively in FF group rather than CCM group. Although, WJ-MSCs could differentiate into OLCs by FF and CCM, however, the induction potential of FF for producing OLCs was better than CCM.

Optimizing The Cell Seeding Protocol to Human Decellularized Ovarian Scaffold: Application of Dynamic System for Bio-Engineering.

OBJECTIVE: Decellularized tissue scaffolds provide an extracellular matrix to control stem cells differentiation toward specific lineages. The application of mesenchymal stem cells for artificial ovary production may enhance ex vivo functions of the ovary. On the other hand, the scaffold needs interaction and integration with cells. Thus, the development of ovarian engineered constructs (OVECs) requires the use of efficient methods for seeding of the cells into the ovarian and other types of scaffolds. The main goal of the present study was to develop an optimized culture system for efficient seeding of peritoneum mesenchymal stem cells (PMSCs) into human decellularized ovarian scaffold. MATERIALS AND METHODS: In this experimental study, three methods were used for cellular seeding including rotational (spinner flask) and static (conventional and injection) seeding cultures. OVECs were evaluated with Hematoxylin and Eosin staining and viability analyses for the seeded PMSCs. Then, immunohistochemistry analysis was performed using the best method of cellular seeding for primordial germ cell-like cells, mesenchymal stem cells and proliferation markers. Stereology analysis was also performed for the number of penetrated cells into the OVECs. RESULT: Our results showed that rotational seeding increases the permeability of PMSCs into the scaffold and survival rate of the seeded PMSCs, comparing to the other methods. On the other hand, rotationally seeded PMSCs had a more favorable capability of proliferation with Ki67 expression and differentiation to ovarian specific cells with expression of primordial germ cell line markers without mesenchymal stem cells markers production. Furthermore, stereology showed a more favorable distribution of PMSCs along the outer surfaces of the OVEC with further distribution at the central part of the scaffold. The average total cell values were determined 2142187 cells/mm3 on each OVEC. CONCLUSION: The rotational seeding method is a more favorable approach to cell seeding into ovarian decellularized tissue than static seeding.

Evaluating two ovarian decellularization methods in three species.

Since there is dearth of practical ways to obtain mature follicles from cryopreserved or native ovarian tissues, especially in patients suffering from ovarian dysfunction, tissue engineering may help in restoring ovarian function and/or fertility. In the present study, the effects of sodium dodecyl sulfate (SDS) and sodium hydroxide (NaOH) on the decellularization of ovarian tissues were studied in order to ascertain their suitability in creating suitable bioscaffolds. Cells were removed from the ovarian tissues of mouse, sheep and human. The samples were distributed among three groups, viz., control (not treated), SDS and NaOH treated. Qualitative histological evaluations, quantitative assessments (nuclear contents, collagen and glycosaminoglycan), immunohistochemistry staining (for laminin, fibronectin and Collagen I), cell viability and scanning electron microscopic (SEM) assays were performed for all experimental groups. Finally, suspensions of mouse ovarian cells were injected into human NaOH treated scaffolds and subsequently auto-transplanted to ovariectomized mice. H&E and IHC staining (GDF-9) were performed on human recellularized NaOH treated scaffolds 1 month after auto-transplantation. Although histological studies and quantitative evaluations confirmed the successful decellularization and presence of key factors in ovarian scaffolds under both treatment methods, NaOH showed more interesting outcomes. Cell metabolic activity in sheep and human ovaries treated with NaOH was statistically (p < 0.05) higher than for SDS treated samples after 72 h. Moreover, spherical associations with cuboidal cells in human NaOH treated scaffolds were observed and this follicular reconstruction was also confirmed by GDF-9. NaOH was found to be more suitable than SDS for the decellularization of ovarian tissues and it supports follicular reconstruction better than SDS. This is a valuable finding in tissue engineering research and can help in the creation of appropriate ovarian bioscaffolds.

Induction of Mouse Peritoneum Mesenchymal Stem Cells into Germ Cell-Like Cells Using Follicular Fluid and Cumulus Cells-Conditioned Media.

The peritoneum mesothelium lines body cavities and has the same origin as ovarian surface epithelium with probable existence of peritoneum mesenchymal stem cells (PMSCs). In the present research, PMSCs were isolated from peritoneum and differentiated into ovarian cell-like cells using human follicular fluid (HFF) and human cumulus-conditioned medium (HCCM). Anterior abdominal wall and intestinal peritoneum explants were used for cells isolation and cultured in Dulbecco's modified Eagle's medium. After passage 3, purified PMSCs were assessed for morphology, proliferation rate, and cell viability. Then, isolated PMSCs underwent two characterization procedures: (1) differentiation to mesodermal lineage and (2) expression of mesenchymal (CD90 and CD44) and epithelial cell (CK19) markers. The characterized PMSCs were differentiated into ovarian cell-like cells using 10% HFF and 50% HCCM for 21 days, and the expressions of oocyte (Zp3, Gdf9), germ cell (Ddx4, Dazl), granulosa cell (Amh), and theca cell (Lhr) markers were assessed using real-time polymerase chain reaction and immunocytofluorescence assay. Both anterior abdominal wall and intestinal peritoneum mesenchymal stem cells (AP-MSCs and IP-MSCs) showed mesenchymal characters and differentiated to adipocyte and osteocyte. AP-MSCs expressed mesenchymal- and epithelial cell-specific markers more than IP-MSCs and showed an analytically better proliferation rate. The induced AP-MSCs and IP-MSCs were expressed as germ and oocyte cell-specific markers, and this expression increased in the third week of culture. In both groups of AP-MSCs and IP-MSCs, the expressions of Gdf9, Zp3, Ddx4, Dazl, and Amh genes under just HCCM induction showed upregulation significantly on the 21st day of culture compared with day 0. But in protein synthesis of all mentioned genes, both HFF and HCCM had equal induction effect on the 21st day of culture against the 0th day. In addition, LHR was not expressed in any groups. Finally, in both characterization and differentiation procedures, the AP-MSCs respond to inducers better than IP-MSCs.