Adipose-derived stem cells: a review of osteogenesis differentiation

Second part of manuscript based on osteogenesis differentiation of stem cells. Bones are highly regenerative organs but there are still many problems with therapy of large bone defects. Sometimes there is necessary to make a replacement or expansion new bone tissue. Stem cells might be a good solution for this especially ADSCs which manage differentiate into osteoblast in in vitro and in vivo conditions

Impact of antibiotics on the proliferation and differentiation of human adipose-derived mesenchymal stem cells

Adipose tissue is a promising source of mesenchymal stem cells. Their potential to differentiate and regenerate other types of tissues may be affected by several factors. This may be due to in vitro cell-culture conditions, especially the supplementation with antibiotics. The aim of our study was to evaluate the effects of a penicillin-streptomycin mixture (PS), amphotericin B (AmB), a complex of AmB with copper (II) ions (AmB-Cu2+) and various combinations of these antibiotics on the proliferation and differentiation of adipose-derived stem cells in vitro. Normal human adipose-derived stem cells (ADSC, Lonza) were routinely maintained in a Dulbecco’s Modified Eagle Medium (DMEM) that was either supplemented with selected antibiotics or without antibiotics. The ADSC that were used for the experiment were at the second passage. The effect of antibiotics on proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine-B (SRB) tests. Differentiation was evaluated based on Alizarin Red staining, Oil Red O staining and determination of the expression of ADSC, osteoblast and adipocyte markers by real-time RT-qPCR. The obtained results indicate that the influence of antibiotics on adipose-derived stem cells depends on the duration of exposure and on the combination of applied compounds. We show that antibiotics alter the proliferation of cells and also promote natural osteogenesis, and adipogenesis, and that this effect is also noticeable in stimulated osteogenesis

Adipose-Derived Stem Cells undergo differentiation after co-culture with porcine Limbal Epithelial Stem Cells

Mesenchymal stem cells (MSCs) are objects of interest in regenerative medicine. They are used for various therapies such as for the regeneration of bone, chondrocytes and other tissues. Adipose derived stem cells (ADSCs) inter alia are particularly easy to access, they are relatively abundant in fat tissue. ADSCs could be differentiated into many types of cells. To date, it has been proven that ADSCs only differentiate into mesodermal cell lineages. In this study, we present the differentiation of ADSCs into the corneal epithelium. Human ADSCs were placed in a co-culture with porcine limbal epithelial stem cells (LESCs). After 14 days of cultivation, total RNA was extracted for the analysis of the molecular markers (expression of genes of interest). The gene expression was assessed by real-time RT-qPCR. The expression of the surface molecular markers of ADSCs is modulated after co-culturing. We have observed the decrease in CD73, CD90 and CD105 mRNA expression, while the expression of mRNA coding for CK3 and CK12 mRNA was increased in ADSCs co-cultured with porcine limbal epithelial stem cells as compared to the control. We conclude that the coculture of LESCs and ADSCs changed ADSCs’ molecular markers gene expression indicating initiation of differentiation towards limbal cells

Ekspresja genów receptorów melatoninowych i genów związanych z regulacją ich aktywności w gruczolakoraku endometrium

Objectives: The aim of the study was to evaluate transcription activity of melatonin receptors and genes associated with regulation of their activity in endometrial adenocarcinoma to identify probable diagnostic and prognostic molecular markers. Material and methods: The material included endometrial adenocarcinoma tissue samples of histopathological grades G1, G2, G3, and normal endometrium. The molecular analysis was performed on 37 patient samples. Total RNA was extracted and used for the microarray HG-U133A analysis. Among 22 283 ID mRNA, only entities of genes associated with regulation of melatonin receptors activity were selected. qRT-PCR was employed for validation, what allowed to compare melatonin receptor genes activation in endometrial cancer tissues to the normal endometrium. Results: The results of the microarray experiments showed that only 18 ID mRNA were differential in endometrial cancer samples as compared to the control at p-value1.5. These genes were identified as differentially expressed in grade G2- ASMTL, GNA11, PER2, PTGDS and in grade G3- GNAI2, GNA11. Silencing of RGS4 encoding RGP4, which regulates signal transmission by G protein, was observed in all cancer groups, independently of the histopathological grade. Conclusions: The profile expression of genes associated with regulation of melatonin receptors activity was different and dependent on the histopathological grade of endometrial cancer and can be an additional diagnostic and prognostic marker. Statistically significant was the down-regulation of melatonin biosynthesis genes (ASMTL) and melatonin signal transmitters (GNA11, GNA12, RTGSCel pracy: Celem pracy była ocena aktywności transkrypcyjnej genów kodujących receptory melatoninowe oraz genów zaangażowanych w ich regulację w gruczolakoraku endometrium pod kątem poszukiwania molekularnych markerów diagnostycznych i prognostycznych tego nowotworu. Materiał i metody: Materiał do badań stanowiły wycinki gruczolakoraka endometrium typu endometrioidalnego w stopniu histologicznego zróżnicowania G1, G2, G3 oraz endometrium prawidłowego. Badania molekularne wykonano u 37 kobiet. Po izolacji RNA z tkanek techniką mikromacierzy oligonukleotydowych HG-U133A (Affymetrix) spośród 22 283 ID mRNA wyznaczono profil stężenia mRNA genów związanych z aktywnością receptorów melatoninowych. Metodą qRT-PCR oceniono zmiany aktywności transkrypcyjnej genów receptorów melatoninowych w wycinkach gruczolakoraka w porównaniu do endometrium prawidłowego. Wyniki: Analiza transkryptomów metodą mikromacierzy ekspresyjnych (Affymetrix) pozwoliła na wytypowanie 18 ID mRNA dla genów związanych z aktywnością receptorów melatoninowych różnicujących raka endometrium od kontroli, przy założeniu, że wartość p1,5. Wśród genów specyficznie różnicujących raka są: w stopniu G2- ASMTL, GNA11, PER2, PTGDS oraz w stopnia G3- GNA12, GNA11. Wyciszenie genu kodującego białko RGS4, regulujące czas i nasilenie przekazywania sygnału przy udziale białka G (negatywnego regulatora) obserwowano we wszystkich wycinkach raka, niezależnie od stopnia zróżnicowania histologicznego. Wnioski: Profil stężenia mRNA genów uczestniczących w regulacji aktywności biologicznej receptorów melatoninowych zależy od stopnia zróżnicowania histologicznego gruczolakoraka endometrium i może stanowić uzupełniający marker diagnostyczny i prognostyczny raka endometrium. Istotne statystycznie obniżenie ekspresji genów zaangażowanych w proces biosyntezy melatoniny (ASMTL) oraz genów kodujących białka związane z transdukcją sygnału receptorów melatoninowych (GNA11, GNA12, RTGS4, HTR2B i GNRH2), mogą stanowić obiecujące ogniwo w podjęciu nowych terapeutycznych rozwiązań w leczeniu gruczolakoraka endometrium

DNA methylation: gene expression regulation

Epigenetic modifications are responsible for the modulation of gene expression without affecting the nucleotide sequence. The observed changes in transcriptional activity of genes in tumor tissue compared to normal tissue, are often the result of DNA methylation within the promoter sequences of these genes. This modification by attaching methyl groups to cytosines within CpG islands results in silencing of transcriptional activity of the gene, which in the case of tumor suppressor genes is manifested by abnormal cell cycle, proliferation and excessive destabilization of the repair processes. Further studies of epigenetic modifications will allow a better understanding of mechanisms of their action, including the interdependence between DNA methylation and activity of proteins crucial to the structure of chromatin and gene activity. Wider knowledge of epigenetic mechanisms involved in the process of malignant transformation and pharmacological regulation of the degree of DNA methylation provides an opportunity to improve the therapeutic actions in the fight against cancer

Regulation of Adipose-Derived Stem Cell Activity by Melatonin Receptors in Terms of Viability and Osteogenic Differentiation

Melatonin is a hormone secreted mainly by the pineal gland and acts through the Mel1A and Mel1B receptors. Among other actions, melatonin significantly increases osteogenesis during bone regeneration. Human adipose-derived mesenchymal stem cells (ADSCs) are also known to have the potential to differentiate into osteoblast-like cells; however, inefficient culturing due to the loss of properties over time or low cell survival rates on scaffolds is a limitation. Improving the process of ADSC expansion in vitro is crucial for its further successful use in bone regeneration. This study aimed to assess the effect of melatonin on ADSC characteristics, including osteogenicity. We assessed ADSC viability at different melatonin concentrations as well as the effect on its receptor inhibitors (luzindole or 4-P-PDOT). Moreover, we analyzed the ADSC phenotype, apoptosis, cell cycle, and expression of MTNR1A and MTNR1B receptors, and its potential for osteogenic differentiation. We found that ADSCs treated with melatonin at a concentration of 100 µM had a higher viability compared to those treated at higher melatonin concentrations. Melatonin did not change the phenotype of ADSCs or induce apoptosis and it promoted the activity of some osteogenesis-related genes. We concluded that melatonin is safe, non-toxic to normal ADSCs in vitro, and can be used in regenerative medicine at low doses (100 μM) to improve cell viability without negatively affecting the osteogenic potential of these cells

Limbal epithelial stem cells in regeneration of corneal epithelium – clinical and molecular aspects

Limbal epithelial stem cells (LESC) are located at the junction between the cornea and sclera. Their function is to reconstruc-ting and replace damaged or dysfunctional cells. In this paper information on the characteristics of LESC, describes the methods of their identification using surface molecular markers and also morphological characteristics are included. The methods of LESC cultivation in vitro as a potential source of stem cells for therapy are also presented. The issues of corneal disorders due to limbal stem cell deficiency (LSCD) caused by various factors were described and discussed as well as the treatment methods currently applied in medicine. The standard treatment of LSCD is based on pharmacotherapy and autologous transplantation of the limbus which is rich in stem cells. However, in the case of total stem cell deficiency (TLSCD) allogenic transplantation is necessary.Komórki macierzyste rąbka rogówki (limbal epithelial stem cells – LESC) zlokalizowane są na granicy rogówki i spojówki. Ich funkcja polega na odbudowie rogówki poprzez zastępowanie uszkodzonych lub niefunkcjonalnych komórek. W niniejszej pracy zawarto informacje dotyczące charakterystyki LESC, opisano sposoby ich identyfikacji z wykorzystaniem markerów powierzchniowych oraz cech morfologicznych. W pracy zawarto również informacje dotyczące hodowli komórek w warunkach in vitro jako potencjalnego źródła komórek wykorzystywanych w terapii oraz poruszono zagadnienia dysfunkcji LESC spowodowanych niedoborem komórek macierzystych wywołanych różnymi czynnikami, a także przedstawiono stosowane obecnie metody leczenia LSCD (limbal stem cell deficiency). Standardowe leczenie niedoboru komórek macierzystych rąbka rogówki polega na farmakoterapii oraz transplantacji autologicznego rąbka rogówki bogatego w komórki macierzyste. W przypadku całkowitego niedoboru LESC konieczne jest wykonanie przeszczepów allogenicznych

Influence of Adalimumab on the Expression Profile of Genes Associated with the Histaminergic System in the Skin Fibroblasts In Vitro

Objective. The aim of this study was to evaluate the influence of adalimumab on expression profile of genes associated with the histaminergic system in Normal Human Dermal Fibroblast (NHDF) cells stimulated with 8.00 μg/ml of adalimumab and the identification of miRNAs regulating these genes’ expression. Methods. NHDFs were cultured with or without the presence of adalimumab for 2, 8, and 24 hours. The expression profile of genes and miRNA were determined with the use of microarray technology. Results. Among 22283 ID mRNA, 65 are associated with the histaminergic system. It can be observed that 15 mRNAs differentiate NHDFs cultures with adalimumab form control. The analysis of miRNAs showed that, among 1105 ID miRNA, 20 miRNAs are differentiating in cells treated with adalimumab for 2 hours, 9 miRNA after 8 hours, and only 3 miRNAs after 24 hours. Conclusion. It was also determined that miRNAs play certain role in the regulation of the expression of genes associated with the histaminergic system. The results of this study confirmed the possibility of using both genes associated with this system as well as miRNAs regulating their expression, as complementary molecular markers of sensitivity to the adalimumab treatment

From Alpha to Delta—Genetic Epidemiology of SARS-CoV-2 (hCoV-19) in Southern Poland

In Poland, the first case of SARS-CoV-2 infection was confirmed in March 2020. Since then, many circulating virus lineages fueled rapid pandemic waves which inflicted a severe burden on the Polish healthcare system. Some of these lineages were associated with increased transmissibility and immune escape. Mutations in the viral spike protein, which is responsible for host cell recognition and serves as the primary target for neutralizing antibodies, are of particular importance. We investigated the molecular epidemiology of the SARS-CoV-2 clades circulating in Southern Poland from February 2021 to August 2021. The 921 whole-genome sequences were used for variant identification, spike mutation, and phylogenetic analyses. The Pango B.1.1.7 was the dominant variant (n = 730, 89.68%) from March 2021 to July 2021. In July 2021, the B.1.1.7 was displaced by the B.1.617.2 lineage with 66.66% in July 2021 and 92.3% in August 2021 frequencies, respectively. Moreover, our results were compared with the sequencing available on the GISAID platform for other regions of Poland, the Czech Republic, and Slovakia. The analysis showed that the dominant variant in the analyzed period was B.1.1.7 in all countries and Southern Poland (Silesia). Interestingly, B.1.1.7 was replaced by B.1.617.2 earlier in Southern Poland than in the rest of the country. Moreover, in the Czech Republic and Slovakia, AY lineages were predominant at that time, contrary to the Silesia region