Histological Evaluation of Decellularized Skeletal Muscle Tissue Using Two Different Decellularization Agents.

The aim of the present study was to determine effect of two decellularized agents, sodium dodecyl sulphate (SDS) and Triton X-100, to the skeletal muscle tissue. Final scaffold was evaluated by several histological techniques to analyse preservation of essential structures including collagen and elastic fibres, basement membranes, glycosaminoglycans and also to confirm elimination of nuclear and cytoplasmic components which are redundant in effectively prepared decellularized scaffolds. Comparison of tissue scaffolds processed with different detergents proved that SDS is superior to Triton X-100 as it can effectively decellularize muscle tissue.

Characterization of dental pulp stem cells from impacted third molars cultured in low serum-containing medium.

We isolated and expanded stem cells from dental pulp from extracted third molars using an innovative culture method consisting of low serum-containing medium supplemented with epidermal growth factor and platelet-derived growth factor BB. We evaluated the differentiation potential of these cells when they were growing either adherently or as micromass/spheroid cultures in various media. Undifferentiated and differentiated cells were analyzed by flow cytometry, immunocytochemistry and immunoblotting. The flow cytometry results showed that the dental pulp stem cells (DPSCs) were positive for mesenchymal stromal cell markers, but negative for hematopoietic markers. Immunocytochemical and/or immunoblotting analyses revealed the expression of numerous stem cell markers, including nanog, Sox2, nestin, Musashi-1 and nucleostemin, whereas they were negative for markers associated with differentiated neural, vascular and hepatic cells. Surprisingly, the cells were only slightly positive for α-smooth muscle actin, and a heterogeneous expression of CD146 was observed. When cultured in osteogenic media, they expressed osteonectin, osteopontin and procollagen I, and in micromass cultures, they produced collagen I. DPSCs cultured in TGF-ß1/3-supplemented media produced extracellular matrix typical of cartilaginous tissue. The addition of vascular endothelial growth factor to serum-free media resulted in the expression of endothelial markers. Interestingly, when cultured in neurogenic media, DPSCs exhibited de novo or upregulated markers of undifferentiated and differentiated neural cells. Collectively, our data show that DPSCs are self-renewing and able to express markers of bone, cartilage, vascular and neural tissues, suggesting their multipotential capacity. Their easy accessibility makes these cells a suitable source of somatic stem cells for tissue engineering.

Telomere attrition occurs during ex vivo expansion of human dental pulp stem cells.

We provide a detailed characteristic of stem cells isolated and expanded from the human dental pulp. Dental pulp stem cells express mesenchymal cell markers STRO-1, vimentin, CD29, CD44, CD73, CD90, CD166, and stem cell markers Sox2, nestin, and nucleostemin. They are multipotent as shown by their osteogenic and chondrogenic potential. We measured relative telomere length in 11 dental pulp stem cell lines at different passages by quantitative real-time PCR. Despite their large proliferative capacity, stable viability, phenotype, and genotype over prolonged cultivation, human dental pulp stem cells suffer from progressive telomere shortening over time they replicate in vitro. Relative telomere length (T/S) was inversely correlated with cumulative doubling time. Our findings indicate that excessive ex vivo expansion of adult stem cells should be reduced at minimum to avoid detrimental effects on telomere maintenance and measurement of telomere length should become a standard when certificating the status and replicative age of stem cells prior therapeutic applications.

Methotrexate released in vitro from bone cement inhibits human stem cell proliferation in S/G2 phase.

Methotrexate (MTX) released from bone cement showed a useful local effect in animal models of bone tumours. However, local toxic reactions such as impaired wound healing were observed in areas surrounding the MTX-loaded implant. Therefore, we hypothesised that MTX released from bone cement would have harmful effects on human mesenchymal stem cells (MSC)-one of the basic components of bone marrow and tissue reparatory processes. Moreover, elution of MTX was calculated from implants prepared either with liquid or powdered MTX. During the 28-day incubation, the cement compounded with liquid MTX showed the highest elution rate of the drug. MTX released from pellets produced a significant decrease in proliferation of MSC as a consequence of a blockade of their cell cycle in the S/G2 phase. These findings indicate impairment of stem cell function in marginal areas surrounding the MTX-loaded cement and may help to explain problems with regeneration of tissues in these locations.

Osteogenic differentiation of human dental pulp-derived stem cells under various ex-vivo culture conditions.

Dental pulp stem cells (DPSCs) can be easily isolated and cultured in low-serum containing medium supplemented with growth factors PDGF-BB and EGF while exhibiting multipotency and immature phenotypic characteristics. In the present study, we investigated their potential to differentiate towards osteogenic lineages using various culture conditions in order to optimize their therapeutic use. DPSCs were cultured either as a cell monolayer or as three-dimensional (3D) micro-mass structures. Monolayers preincubated with bFGF and valproic acid for one week prior their differentiation were cultured in serum containing standard osteodifferentiation medium for four weeks, which resulted in multilayered nodule formation. Micro-mass structures were cultured for same period either in serum containing medium or under serum-free conditions supplemented with TGF-beta3 with or without BMP-2. Histochemically, we detected massive collagen I and weak calcium phosphate depositions in multilayered nodules. When culture 3D-aggregates in either standard osteodifferentiation medium or serum-free medium containing TGF-beta3, only small amount of collagen I fibres was observed and almost no deposits of calcium phosphate were detected. In contrast, in presence of both TGF-beta3 and BMP-2 in the serum-free medium a significant amount of collagen I fibers/bundles and calcification were detected, which is in line with osteogenic effect of BMP-2. Thus, our data indicate that certain environmental cues can enhance differentiation process of DPSCs into osteogenic lineage, which suggest their possible utilization in tissue engineering.

Stem cells from human exfoliated deciduous teeth--isolation, long term cultivation and phenotypical analysis.

AIMS: Our aims were to isolate stem cells from human exfoliated deciduous teeth (SHED), to cultivate them in vitro and to investigate their basic biological properties, phenotype and to compare our findings with dental pulp stem cells (DPSC) isolated from permanent teeth. METHODS: Dental pulp was gently evacuated from exfoliated teeth. After enzymatic dissociation of dental pulp, SHED were cultivated in modified cultivation media for mesenchymal adult progenitor cells containing 2% FCS and supplemented with growth factors and insulin, transferrin, sodium (ITS) supplement. Cell viability and other biological properties were examined using a Vi-Cell analyzer and a Z2-Counter. DNA analyses and phenotyping were performed with flow cytometry. RESULTS: We were able to cultivate SHED over 45 population doublings. Our results showed that SHED cultivated under same conditions as DPSC had longer average population doubling time (41.3 hrs for SHED vs. 24.5 hrs for DPSC). Phenotypic comparison of cultivated SHED to that of cultivated DPSC showed differential expression CD29, CD44, CD71, CD117, CD 166. During long-term cultivation, SHED did not showed any signs of degeneration or spontaneous differentiation. CONCLUSIONS: We isolated stem cells from exfoliated teeth. In comparison to DPSC, SHED proliferation rate was about 50% slower, and SHED showed slightly different phenotype. These cells may be extremely useful for stem cell tissue banking, further stem cell research and future therapeutic applications.

Directed reprogramming of comprehensively characterized dental pulp stem cells extracted from natal tooth.

The aim of this study was to extensively characterise natal dental pulp stem cells (nDPSC) and assess their efficiency to generate human induced pluripotent stem cells (hiPSC). A number of distinguishing features prompted us to choose nDPSC over normal adult DPSC, in that they differed in cell surface marker expression and initial doubling time. In addition, nDPSC expressed 17 out of 52 pluripotency genes we analysed, and the level of expression was comparable to human embryonic stem cells (hESC). Ours is the first group to report comprehensive characterization of nDPSC followed by directed reprogramming to a pluripotent stem cell state. nDPSC yielded hiPSC colonies upon transduction with Sendai virus expressing the pluripotency transcription factors POU5F1, SOX2, c-MYC and KLF4. nDPSC had higher reprogramming efficiency compared to human fibroblasts. nDPSC derived hiPSCs closely resembled hESC in terms of their morphology, expression of pluripotency markers and gene expression profiles. Furthermore, nDPSC derived hiPSCs differentiated into the three germ layers when cultured as embryoid bodies (EB) and by directed differentiation. Based on our findings, nDPSC present a unique marker expression profile compared with adult DPSC and possess higher reprogramming efficiency as compared with dermal fibroblasts thus proving to be more amenable for reprogramming.

Human prominin-1 (CD133) is detected in both neoplastic and non-neoplastic salivary gland diseases and released into saliva in a ubiquitinated form.

Prominin-1 (CD133) is physiologically expressed at the apical membranes of secretory (serous and mucous) and duct cells of major salivary glands. We investigated its expression in various human salivary gland lesions using two distinct anti-prominin-1 monoclonal antibodies (80B258 and AC133) applied on paraffin-embedded sections and characterized its occurrence in saliva. The 80B258 epitope was extensively expressed in adenoid cystic carcinoma, in lesser extent in acinic cell carcinoma and pleomorphic adenoma, and rarely in mucoepidermoid carcinoma. The 80B258 immunoreactivity was predominately detected at the apical membrane of tumor cells showing acinar or intercalated duct cell differentiation, which lined duct- or cyst-like structures, and in luminal secretions. It was observed on the whole cell membrane in non-luminal structures present in the vicinity of thin-walled blood vessels and hemorrhagic areas in adenoid cystic carcinoma. Of note, AC133 labeled only a subset of 80B258-positive structures. In peritumoral salivary gland tissues as well as in obstructive sialadenitis, an up-regulation of prominin-1 (both 80B258 and AC133 immunoreactivities) was observed in intercalated duct cells. In most tissues, prominin-1 was partially co-expressed with two cancer markers: carcinoembryonic antigen (CEA) and mucin-1 (MUC1). Differential centrifugation of saliva followed by immunoblotting indicated that all three markers were released in association with small membrane vesicles. Immuno-isolated prominin-1-positive vesicles contained CEA and MUC1, but also exosome-related proteins CD63, flotillin-1, flotillin-2 and the adaptor protein syntenin-1. The latter protein was shown to interact with prominin-1 as demonstrated by its co-immunoisolation. A fraction of saliva-associated prominin-1 appeared to be ubiquitinated. Collectively, our findings bring new insights into the biochemistry and trafficking of prominin-1 as well as its immunohistochemical profile in certain types of salivary gland tumors and inflammatory diseases.

The effect of ATM kinase inhibition on the initial response of human dental pulp and periodontal ligament mesenchymal stem cells to ionizing radiation.

PURPOSE: This study evaluates early changes in human mesenchymal stem cells (MSC) isolated from dental pulp and periodontal ligament after γ-irradiation and the effect of ataxia-telangiectasia mutated (ATM) inhibition. METHODS: MSC were irradiated with 2 and 20 Gy by (60)Co. For ATM inhibition, specific inhibitor KU55933 was used. DNA damage was measured by Comet assay and γH2AX detection. Cell cycle distribution and proteins responding to DNA damage were analyzed 2-72 h after the irradiation. RESULTS: The irradiation of MSC causes an increase in γH2AX; the phosphorylation was ATM-dependent. Irradiation activates ATM kinase, and the level of p53 protein is increased due to its phosphorylation on serine15. While this phosphorylation of p53 is ATM-dependent in MSC, the increase in p53 was not prevented by ATM inhibition. A similar trend was observed for Chk1 and Chk2. The increase in p21 is greater without ATM inhibition. ATM inhibition also does not fully abrogate the accumulation of irradiated MSC in the G2-phase of the cell-cycle. CONCLUSIONS: In irradiated MSC, double-strand breaks are tagged quickly by γH2AX in an ATM-dependent manner. Although phosphorylations of p53(ser15), Chk1(ser345) and Chk2(thr68) are ATM-dependent, the overall amount of these proteins increases when ATM is inhibited. In both types of MSC, ATM-independent mechanisms for cell-cycle arrest in the G2-phase are triggered.

Irradiation of adult human dental pulp stem cells provokes activation of p53, cell cycle arrest, and senescence but not apoptosis.

Adult human dental pulp contains stem cells (DPSCs) that are capable of differentiation into osteoblasts, odontoblasts, adipocytes, and neuronal-like cells. Because these cells have potential use in tissue regeneration, herein we characterized the response of DPSC lines to ionizing radiation (IR). These DPSC lines have been developed from the extracted molars of healthy donors. DPSCs were cultivated in a unique media supplemented with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Since tissue homeostasis depends on a precise balance among cell proliferation, senescence, and cell death, we explored the effects of IR (2-20 Gy) on the proliferative activity of DPSCs and the molecular pathways involved. Even the highest dose used (20 Gy) did not induce DPSC apoptosis. After irradiation with doses of 6 and 20 Gy, DPSCs accumulated in the G2 phase of the cell cycle. DPSCs responded to IR (20 Gy) with senescence detected as SA-ß-galactosidase positivity, beginning on the third day after irradiation. Twenty-four hours after irradiation, p53 and its serine 15 and 392 phosphorylated forms were detected. At this time, p21 (WAF1) was induced. Increases in protein p16 were observed from the third day following irradiation and continued till the end of the examination (Day 13). We conclude that DPSCs respond to IR-induced damage by permanent cell cycle arrest in the G2 phase and by stress-induced premature senescence.

Dental pulp stem cells and their characterization.

AIMS: Our aims were to isolate dental pulp stem cells, to cultivate them in various media and to investigate their basic biological properties and phenotype. METHODS: 16 lines of dental pulp stem cells (DPSCs) were isolated from an impacted third molar. After enzymatic dissociation of dental pulp, DPSCs were cultivated in modified cultivation media for mesenchymal adult progenitor cells containing 2 % or 10 % fetal calf serum (FCS), or in modified 2 % FCS cultivation media supplemented with ITS. Cell viability and other biological properties were examined periodically using a Vi-Cell analyzer and Z2-Counter. DNA analysis and phenotyping were done using flow cytometry. RESULTS: We were able to cultivate DPSCs in all tested cultivation media over 40 population doublings. Our results showed that DPSCs cultivated in medium supplemented with ITS had shorter average population doubling time (24.5, 15.55-35.12 hours) than DPSCs cultivated in 2 % FCS (55.43, 21.57-187.14 hours) or 10 % FCS (42.56, 11.86 - 101.3 hours). Cell diameter was not affected and varied from 15 to 16 microm. DPSCs viability in the 9(th) passage was over 90 %. Our phenotypical analysis was highly positivity for CD29, CD44, CD90 and HLA I, and negative for CD34, CD45, CD71, HLA II. DPSC lines cultivated in all media showed no signs of degeneration or spontaneous differentiation during the expansion process. CONCLUSIONS: We showed that ITS supplement in the cultivation media greatly increased the proliferative activity of DPSCs. Other DPSC biological properties and phenotype were not affected.