Despite several scientific or ethical issues, fetal bovine serum (FBS) remains the standard nutrient supplement in the mesenchymal stem cell cultivation medium. Cell amplification plays an important role in human stem cell therapies. Increasing interest in this field has supported attempts to find suitable human alternatives to FBS for in vitro cell propagation. Human platelet lysate (hPL) has recently been determined as one of them. Our study aimed to evaluate the influence of 2% hPL in the growth medium for in vitro expansion of human natal dental pulp stem cells (hNDP-SCs). The effect was determined on proliferation rate, viability, phenotype profile, expression of several markers, relative telomere length change, and differentiation potential of four lineages of hNDP-SCs. As a control, hNDP-SCs were simultaneously cultivated in 2% FBS. hNDP-SCs cultivated in hPL showed a statistically significantly higher proliferation rate in initial passages. We did not observe a statistically significant effect on mesenchymal stem cell marker (CD29, CD44, CD73, CD90) or stromal-associated marker (CD13, CD166) expression. The cell viability, relative telomere length, or multipotency remained unaffected in hNDP-SCs cultivated in hPL-medium. In conclusion, hPL produced under controlled and standardized conditions is an efficient serum supplement for in vitro expansion of hNDP-SCs.

• Klíčová slova
• culture medium nutrient supplement, fetal bovine serum, human natal stem cells, human platelet lysate, mesenchymal stem cells, regenerative medicine, stem cell cultivation,
• MeSH
• buněčná diferenciace MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky * MeSH
• proliferace buněk MeSH
• živiny MeSH
• zubní dřeň * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

It is primarily important to define the standard features and factors that affect dental pulp stem cells (DPSCs) for their broader use in tissue engineering. This study aimed to verify whether DPSCs isolated from various teeth extracted from the same donor exhibit intra-individual variability and what the consequences are for their differentiation potential. The heterogeneity determination was based on studying the proliferative capacity, viability, expression of phenotypic markers, and relative length of telomere chromosomes. The study included 14 teeth (6 molars and 8 premolars) from six different individuals ages 12 to 16. We did not observe any significant intra-individual variability in DPSC size, proliferation rate, viability, or relative telomere length change within lineages isolated from different teeth but the same donor. The minor non-significant variances in phenotype were probably mainly because DPSC cell lines comprised heterogeneous groups of undifferentiated cells independent of the donor. The other variances were seen in DPSC lineages isolated from the same donor, but the teeth were in different stages of root development. We also did not observe any changes in the ability of cells to differentiate into mature cell lines-chondrocytes, osteocytes, and adipocytes. This study is the first to analyze the heterogeneity of DPSC dependent on a donor.

• Klíčová slova
• dental stem cells, intra-individual variability, mesenchymal stem cells, regenerative medicine, same donor isolation, stem cell characterization,
• MeSH
• buněčná diferenciace fyziologie   MeSH
• buněčné linie MeSH
• buněčný rodokmen fyziologie   MeSH
• chondrocyty fyziologie   MeSH
• dárci tkání MeSH
• individuální biologická variabilita MeSH
• kmenové buňky fyziologie   MeSH
• lidé MeSH
• mladiství MeSH
• osteocyty fyziologie   MeSH
• proliferace buněk fyziologie   MeSH
• tukové buňky fyziologie   MeSH
• zubní dřeň fyziologie   MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Dental pulp stem cells (DPSCs) are a type of easily accessible adult mesenchymal stem cell. Due to their ease of access, DPSCs show great promise in regenerative medicine. However, the tooth extractions from which DPSCs can be obtained are usually performed at a period of life when donors would have no therapeutic need of them. For this reason, it is imperative that successful stem cell storage techniques are employed so that these cells remain viable for future use. Any such techniques must result in high post-thaw stem cell recovery without compromising stemness, proliferation, or multipotency. Uncontrolled-rate freezing is not a technically or financially demanding technique compared to expensive and laborious controlled-rate freezing techniques. This study was aimed at observing the effect of uncontrolled-rate freezing on DPSCs stored for 6 and 12 months. Dimethyl sulfoxide at a concentration of 10% was used as a cryoprotective agent. Various features such as shape, proliferation capacity, phenotype, and multipotency were studied after DPSC thawing. The DPSCs did not compromise their stemness, viability, proliferation, or differentiating capabilities, even after one year of cryopreservation at -80 °C. After thawing, they retained their stemness markers and low-level expression of hematopoietic markers. We observed a size reduction in recovery DPSCs after one year of storage. This observation indicates that DPSCs can be successfully used in potential clinical applications, even after a year of uncontrolled cryopreservation.

• Klíčová slova
• cryopreservation, dental stem cells, regenerative medicine, stem cell storage, uncontrolled-rate freezing,
• MeSH
• buněčná diferenciace * MeSH
• kmenové buňky cytologie   účinky léků   MeSH
• kryoprezervace metody   MeSH
• kryoprotektivní látky farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mladiství MeSH
• proliferace buněk MeSH
• zubní dřeň cytologie   účinky léků   MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kryoprotektivní látky MeSH

Telomeres are repetitive nucleoprotein DNA sequences that shorten with each cell division. The stem cells activate telomerase to compensate for the telomere loss. This study aimed to evaluate the effect of cultivation passaging on the relative telomere length and proliferation capacity of dental pulp stem cells. We used ten dental pulp stem cell (DPSC) lineages stored for 12 months using uncontrolled-rate freezing to reach the study's goal. We analyzed their proliferation rate, phenotype using flow cytometry, multipotency, and relative telomere length using a qPCR analysis. We determined the relative telomere length in the added study by performing analysis after one, two, and three weeks of cultivation with no passaging. We documented the telomere attrition with increasing passaging. The shorter the relative telomere length, the lower reached population doublings, and longer population doubling time were observed at the end of the cultivation. We observed the telomere prolongation in DPSCs cultivated for two weeks with no passaging in the added subsequent study. We concluded that excessive proliferation demands on DPSCs during in vitro cultivation result in telomere attrition. We opened the theory that the telomerase might be more efficient during cell cultivation with no passaging. This observation could help in preserving the telomere length during ex vivo DPSC expansion.

• Klíčová slova
• dental pulp stem cells, qPCR, relative telomere length measurement, telomerase, telomere,
• MeSH
• fenotyp MeSH
• imunohistochemie MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• proliferace buněk genetika   fyziologie   MeSH
• průtoková cytometrie MeSH
• telomerasa metabolismus   MeSH
• telomery genetika   MeSH
• zubní dřeň cytologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• telomerasa MeSH

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.

• MeSH
• biologické markery MeSH
• buněčná diferenciace genetika   MeSH
• embryoidní tělíska cytologie   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• karyotyp MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• Krüppel-like faktor 4 MeSH
• kultivované buňky MeSH
• lidé MeSH
• předmléčné zuby cytologie   MeSH
• přeprogramování buněk * MeSH
• transkriptom MeSH
• vývojová regulace genové exprese MeSH
• zubní dřeň cytologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• KLF4 protein, human MeSH Prohlížeč
• Krüppel-like faktor 4 MeSH

Asporin has been reported as a tumor suppressor in breast cancer, while asporin-activated invasion has been described in gastric cancer. According to our in silico search, high asporin expresion associates with significantly better relapse free survival (RFS) in patients with low-grade tumors but RFS is significantly worse in patients with grade 3 tumors. In line with other studies, we have confirmed asporin expression by RNA scope in situ hybridization in cancer associated fibroblasts. We have also found asporin expression in the Hs578T breast cancer cell line which we confirmed by quantitative RT-PCR and western blotting. From multiple testing, we found that asporin can be downregulated by bone morphogenetic protein 4 while upregulation may be facilited by serum-free cultivation or by three dimensional growth in stiff Alvetex scaffold. Downregulation by shRNA inhibited invasion of Hs578T as well as of CAFs and T47D cells. Invasion of asporin-negative MDA-MB-231 and BT549 breast cancer cells through collagen type I was enhanced by recombinant asporin. Besides other investigations, large scale analysis of aspartic acid repeat polymorphism will be needed for clarification of the asporin dual role in progression of breast cancer.

• Klíčová slova
• 3D cultivation, asporin, breast cancer, grade, stiffness,
• MeSH
• extracelulární matrix - proteiny metabolismus   MeSH
• fibroblasty metabolismus   patologie   MeSH
• Kaplanův-Meierův odhad MeSH
• lidé MeSH
• nádorové mikroprostředí fyziologie   MeSH
• nádory prsu metabolismus   mortalita   patologie   MeSH
• přežití bez známek nemoci MeSH
• prognóza MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ASPN protein, human MeSH Prohlížeč
• extracelulární matrix - proteiny MeSH