Ayça Aksoy

Characterization of mesenchymal stem cells from rat bone marrow: ultrastructural properties, differentiation potential and immunophenotypic markers

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can differentiate into many lineages. Although the growing interest in BM-MSCs has led to a number of characterization studies, some important biochemical and immunohistochemical properties are still lacking. In this study, morphological and immunophenotypic properties of BM-MSCs were examined in detail. Differentiation potential and growth kinetics of adult rat BM-MSCs were also determined. Immunohistochemistry and RT-PCR results indicated that BM-MSCs expressed myogenic (desmin, myogenin, myosin IIa, and α-SMA), neurogenic (γ-enolase, MAP2a,b, c-fos, nestin, GFAP and beta III tubulin), and osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, BMP-2, BMP-4 and type I collagen) markers without stimulation towards differentiation. These expression patterns indicated why these cells can easily differentiate into multiple lineages both in vitro and in vivo. Ultrastructural characteristics of rBM-MSCs showed more developed and metabolically active cells.

Isolation and in vitro characterisation of dental pulp stem cells from natal teeth

Dental pulp stem cells were primarily derived from the pulp tissues of exfoliated deciduous teeth, primary incisors and permanent third molar teeth. The aim of this study was to isolate and extensively characterise SCs derived from human natal dental pulp (hNDP). For characterisation, proliferation capacity, phenotypic properties, ultrastructural and differentiation characteristics and gene expression profiles were utilised. A comparison was done between the properties of NDP-SCs and the properties of mesenchymal stem cells (MSCs) from bone marrow (BM) of the human. Stem cells isolated from hNDP and hBM were analysed by flow cytometry, reverse transcriptase-PCR, Real Time-PCR, and immunocytochemistry. Both cell lines were directionally differentiated towards adipogenic, osteogenic chondrogenic, myogenic and neurogenic lineages. hNDP-SCs and hBM-MSCs expressed CD13, CD44, CD90, CD146 and CD166, but not CD3, CD8, CD11b, CD14, CD15, CD19, CD33, CD34, CD45, CD117, and HLA-DR. Ultrastructural characteristics of hNDP-SCs showed more developed and metabolically active cells. hNDP-SCs and hBM-MSCs expressed some adipogenic (leptin, adipophilin and PPARγ), myogenic (desmin, myogenin, myosinIIa, and α-SMA), neurogenic (γ-enolase, MAP2a,b, c-fos, nestin, NF-H, NF-L, GFAP and betaIII tubulin), osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, and type I collagen) and chondrogenic (type II collagen, SOX9) markers without any stimulation towards differentiation under basal conditions. Embryonic stem cell markers Oct4, Rex-1, FoxD-3, Sox2, and Nanog were also identified. The differentiation potential of hNDP-SCs and hBM-MSCs to adipogenic, osteogenic, chondrogenic, myogenic and neurogenic was shown. This report described the first successful isolation and characterisation of hNDP-SCs.

Human dental pulp stem cells demonstrate better neural and epithelial stem cell properties than bone marrow-derived mesenchymal stem cells.

Dental pulp stem cells (hDP-SCs) were primarily derived from pulp tissues of primary incisors, exfoliated deciduous and permanent third molar teeth. To understand the characteristics of hDP-SCs from impacted third molar, proliferation capacities, gene expression profiles, phenotypic, ultrastructural, and differentiation characteristics were analyzed in comparison with human bone marrow-derived mesenchymal stem cells (hBM-MSCs), extensively. hDP-SCs showed more developed and metabolically active cells. Contrary to hBM-MSCs, hDP-SCs strongly expressed both cytokeratin (CK)-18 and -19, which could involve in odontoblast differentiation and dentine repair. The intrinsic neuro-glia characteristics of hDP-MSCs were demonstrated by the expression of several specific transcripts and proteins of neural stem cell and neurons. These cells not only differentiate into adipogenic, osteogenic, and chondrogenic lineage, but also share some special characteristics of expressing some neural stem cell and epithelial markers. Under defined conditions, hDP-SCs are able to differentiate into both neural and vascular endothelial cells in vitro. Dental pulp might provide an alternative source for human MSCs. hDP-SCs with a promising differentiation capacity could be easily isolated, and possible clinical use could be developed for neurodegenerative and oral diseases in the future.

Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells

The maintenance of viable and functional islets is critical in successful pancreatic islet transplantation from cadaveric sources. During the isolation procedure, islets are exposed to a number of insults including ischemia, oxidative stress and cytokine injury that cause a reduction in the recovered viable islet mass. A novel approach was designed in which streptozotocin (STZ)-damaged rat pancreatic islets (rPIs) were indirectly cocultured with rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) to maintain survival of the cultured rPIs. The results indicated that islets cocultured with rBM-MSCs secreted an increased level of insulin after 14 days, whereas non-cocultured islets gradually deteriorated and cell death occurred. The cocultivation of rBM-MSCs with islets and STZ-damaged islets showed the expression of IL6 and transforming growth factor-β1 in the culture medium, besides the expression of the antiapoptotic genes (Mapkapk2, Tnip1 and Bcl3), implying the cytoprotective, anti-inflammatory and antiapoptotic effects of rBM-SCs through paracrine actions.

Recovery of Fertility in Azoospermia Rats after Injection of Adipose-Tissue-Derived Mesenchymal Stem Cells: The Sperm Generation

The recent reports on the treatment of azoospermia patients, in which spermatozoa could not be traced in their testes, are focused more on the potential use of adult stem cells, like mesenchymal stem cells (MSCs). The aim of this study was to demonstrate the potential use of MSCs derived from adipose tissue in the treatment of azoospermia using rat disease models. After busulfan application, the rats (n = 20) were injected with the GFP+ MSCs into left rete testes. After 12 weeks, the testes with cell injection (right testes) were compared to control (left testes) after dimensional and immunohistochemical analyses. Testes treated with MSCs appeared morphologically normal, but they were atrophic in rats without stem cell treatment, in which the seminiferous tubules were empty. Spermatogenesis was detected, not in every but in some tubules of cell-treated testes. GFP+/VASA+ and GFP+/SCP1+ cells in testes indicated the transdifferentiation of MSCs into spermatogenetic cells in the appropriate microenvironment. Rats with cell treatment were mated to show the full recovery of spermatogenesis, and continuous generations were obtained. The expression of GFP was detected in the mesenchymal stem cells derived from adipose tissue and bone marrow and also in the sperms of offspring. In conclusion, MSCs might be studied for the same purpose in humans in future.

Phenotypic and Proteomic Characteristics of Human Dental Pulp Derived Mesenchymal Stem Cells from a Natal, an Exfoliated Deciduous, and an Impacted Third Molar Tooth

The level of heterogeneity among the isolated stem cells makes them less valuable for clinical use. The purpose of this study was to understand the level of heterogeneity among human dental pulp derived mesenchymal stem cells by using basic cell biology and proteomic approaches. The cells were isolated from a natal (NDPSCs), an exfoliated deciduous (stem cells from human exfoliated deciduous (SHED)), and an impacted third molar (DPSCs) tooth of three different donors. All three stem cells displayed similar features related to morphology, proliferation rates, expression of various cell surface markers, and differentiation potentials into adipocytes, osteocytes, and chondrocytes. Furthermore, using 2DE approach coupled with MALDI-TOF/TOF, we have generated a common 2DE profile for all three stem cells. We found that 62.3 ± 7% of the protein spots were conserved among the three mesenchymal stem cell lines. Sixty-one of these conserved spots were identified by MALDI-TOF/TOF analysis. Classification of the identified proteins based on biological function revealed that structurally important proteins and proteins that are involved in protein folding machinery are predominantly expressed by all three stem cell lines. Some of these proteins may hold importance in understanding specific properties of human dental pulp derived mesenchymal stem cells.

Suppressive effect of compact bone-derived mesenchymal stem cells on chronic airway remodeling in murine model of asthma

New therapeutic strategies are needed in the treatment of asthma besides vaccines and pharmacotherapies. For the development of novel therapies, the use of mesenchymal stem cells (MSCs) is a promising approach in regenerative medicine. Delivery of compact bone (CB) derived MSCs to the injured lungs is an alternative treatment strategy for chronic asthma. In this study, we aimed to isolate highly enriched population of MSCs from mouse CB with regenerative capacity, and to investigate the impact of these cells in airway remodeling and inflammation in experimental ovalbumin-induced mouse model of chronic asthma. mCB-MSCs were isolated, characterized, labeled with GFP and then transferred into mice with chronic asthma developed by ovalbumin (OVA) provocation. Histopathological changes including basement membrane, epithelium, subepithelial smooth thickness and goblet cell hyperplasia, and MSCs migration to lung tissues were evaluated. These histopathological alterations were increased in ovalbumin-treated mice compared to PBS group (P<0.001). Intravenous administration of mCB-MSC significantly reduced these histopathological changes in both distal and proximal airways (P<0.001). We showed that GFP-labeled MSCs were located in the lungs of OVA group 2weeks after intravenous induction. mCB-MSCs also significantly promoted Treg response in ovalbumin-treated mice (OVA+MSC group) (P<0.037). Our studies revealed that mCB-MSCs migrated to lung tissue and suppressed histopathological changes in murine model of asthma. The results reported here provided evidence that mCB-MSCs may be an alternative strategy for the treatment of remodeling and inflammation associated with chronic asthma.

Bone marrow-derived mesenchymal stem cells co-cultured with pancreatic islets display β cell plasticity

The direct co‐culturing effect of rat bone‐marrow‐derived mesenchymal stem cells (rBM‐MSCs) on the pancreatic‐islets (PIs) was studied to obtain functional islet cells. MSCs were isolated from rat bone marrow and cultivated under standard conditions. Following their characterization, the rBM‐MSCs were directly (with cell–islet contact) co‐cultured with recovered PIs together with the single cell cultures of those cell cultures as a control. The effect of direct co‐cultures of rBM‐MSCs with the PIs of normal rats was investigated using immunophenotypical and functional methods. The change in the amount of insulin secretion was evaluated as an indicator for differentiation of rBM‐MSCs. One approache for in vitro differentiation to achieve reprogramming for differentiation into suitable cell types by changing the microenvironment of the cells to provide signals that might activate metabolic pathways is to use co‐cultures with the microenvironment of the specific cells of the desired cell type, tissue/organ extracts, extracellular matrix compounds or biologically absorbable materials. Differentiated rBM‐MSCs were found to be immunopositive for the specific insulin‐producing cell marker, insulin, but not in undifferentiated rBM‐MSCs. The functionality tests by ELISA confirmed that insulin secretion of co‐cultured MSCs with islets was higher than that of islets. These evidences indicated that PIs could be regarded as critical components of the stem cell niche, such that MSCs can be differentiated into insulin‐producing cells (IPCs). Moreover, direct cell‐to‐cell contact might provide additional and independent support. This approach would circumvent the need for PI‐stem cell co‐culture and could potentially facilitate the production of functional IPCs for future clinical applications. Copyright

Isolation and characterization of stem cells from pancreatic islet: pluripotency, differentiation potential and ultrastructural characteristics

BACKGROUND AIMS Stem cells (SC) in different locations have individual characteristics. Important questions to be answered include how these specialties are generated, what the mechanism underlying their generation is, and what their biologic and clinical merits are. A basic approach to answering these questions is to make comparisons between the differences and similarities among the various SC types. They may focus on aspects of biologic marker discovery, capacity of proliferation and differentiation, along with other characteristics. The aim of this study was to characterize in detail the SC isolated from pancreatic islet (PI) and compare their properties with bone marrow (BM)-derived mesenchymal stromal cells (MSC) of the rat. METHODS Immunophenotypic characteristics, proliferation capacities, telomerase activities, pluripotent-related gene expressions, ultrastructure and the potential for multilineage differentiation of PI SC and BM MSC were studied. RESULTS We found that PI SC expressed markers of embryonic SC (Oct-4, Sox-2 and Rex-1) and had a high proliferation capacity, proven also by high telomerase activities. Surprisingly, markers belonging to differentiated cells were expressed by these cells in a constitutive manner. PI SC ultrastructure showed more developed and metabolically active cells. CONCLUSIONS The immunocytochemical identification of both PI SC and BM MSC was demonstrated to be typical MSC. Without stimulation of differentiation markers of adipogenic, chondrogenic, neurogenic, myogenic and osteogenic cells in these SC, the expression of those markers might explain their multilineage differentiation potential. We suggest that, by reason of the respectively high telomerase activity in PI SC, they could be better candidates than BM MSC for cell replacement therapy of type 1 diabetes.

Comparative Analysis of Apoptotic Resistance of Mesenchymal Stem Cells Isolated from Human Bone Marrow and Adipose Tissue

Aim. Mesenchymal stem cells (MSCs) isolated from human bone marrow (hBM) and adipose tissue (hAT) are perceived as attractive sources of stem cells for cell therapy. The aim of this study was to compare MSCs from hBM and hAT for their immunocytochemistry staining and resistance to in vitro apoptosis. Methods. In our study, we investigated the antiapoptotic ability of these MSCs toward oxidative stress induced by hydrogen peroxide (H2O2) and serum deprivation. Results were assessed by MTT and flow cytometry. All experiments were repeated a minimum of three times. Results. Flow cytometry and MTT analysis revealed that hAT-MSCs exhibited a higher resistance toward H2O2-induced apoptosis (n = 3, hBM-hAT viability H2O2  58.43 ± 1.24–73.02 ± 1.44, P < 0.02) and to serum-deprivation-induced apoptosis at days 1 and 4 than the hBM-MSCs (n = 3, hAT-hBM absorbance, resp., day 1: 0.305 ± 0.027–0.234 ± 0.015, P = 0.029, day 4: 0.355 ± 0.003–0.318 ± 0.007, P = 0.001, and day 7: 0.400 ± 0.017–0.356 ± 0.008, P = 0.672). hAT-MSCs showed superior tolerance to oxidative stress triggered by 2 mmol/L H2O2 and also have superior antiapoptosis capacity toward serum-free culture. Conclusion. In this study we found that hAT-MSCs are more resistant to in vitro apoptosis.