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Background: Embryonic pluripotency markers are recognized for their role in ER− BC aggressiveness, but their significance in ER+ BC remains unclear. This study aims to investigate the prevalence of expression of pluripotency markers in ER+ BC and their effect on survival and prognostic indicators. Methods: We analyzed data of ER+ BC patients from three large cancer datasets to assess the expression of three pluripotency markers (NANOG, SOX‐2, and OCT4), and the stem cell marker ALDH1A1. Additionally, we investigated associations between gene expression, through mRNA‐Seq analysis, and overall survival (OS). The prevalence of mutational variants within these genes was explored. Using immunohistochemistry (IHC), we examined the expression and associations with clinicopathologic prognostic indicators of the four markers in 81 ER+ BC patients. Results: Through computational analysis, NANOG and ALDH1A1 genes were significantly upregulated in ER+ BC compared to ER‐ BC patients (p < 0.001), while POU5F1 (OCT4) was downregulated (p < 0.001). NANOG showed an adverse impact on OS whereas ALDH1A1 was associated with a highly significant improved survival in ER+ BC (p = 4.7e‐6), except for the PR− and HER2+ subgroups. Copy number alterations (CNAs) ranged from 0.4% to 1.6% in these genes, with the highest rate detected in SOX2. In the IHC study, approximately one‐third of tumors showed moderate to strong expression of each of the four markers, with 2–4 markers strongly co‐expressed in 56.8% of cases. OCT‐4 and ALDH1A1 showed a significant association with a high KI‐67 index (p = 0.009 and 0.008, respectively), while SOX2 showed a significant association with perinodal fat invasion (p = 0.017). Conclusion: Pluripotency markers and ALDH1A1 are substantially expressed in ER+ BC tumors with different, yet significant, associations with prognostic and survival outcomes. This study suggests these markers as targets for prospective clinical validation studies of their prognostic value and their possible therapeutic roles. [ABSTRACT FROM AUTHOR]

Abstract Background Embryonic pluripotency markers are recognized for their role in ER− BC aggressiveness, but their significance in ER+ BC remains unclear. This study aims to investigate the prevalence of expression of pluripotency markers in ER+ BC and their effect on survival and prognostic indicators. Methods We analyzed data of ER+ BC patients from three large cancer datasets to assess the expression of three pluripotency markers (NANOG, SOX‐2, and OCT4), and the stem cell marker ALDH1A1. Additionally, we investigated associations between gene expression, through mRNA‐Seq analysis, and overall survival (OS). The prevalence of mutational variants within these genes was explored. Using immunohistochemistry (IHC), we examined the expression and associations with clinicopathologic prognostic indicators of the four markers in 81 ER+ BC patients. Results Through computational analysis, NANOG and ALDH1A1 genes were significantly upregulated in ER+ BC compared to ER‐ BC patients (p

The capacity to regenerate entire body parts, tissues, and organs had generally been thought to be lost in evolution with very few exceptions (e.g. the liver) surviving in mammals. The discovery of the MRL mouse and the elucidation of the underlying molecular pathway centering around hypoxia inducible factor, HIF-1α, has allowed a drug and materials approach to regeneration in mice and hopefully humans. The HIF-1α pathway is ancient and permitted the transition from unicellular to multicellular organisms. Furthermore, HIF-1α and its regulation by PHDs, important oxygen sensors in the cell, provides a perfect drug target. We review the historical background of regeneration biology, the discovery of the MRL mouse, and its underlying biology, and novel approaches to drugs, targets, and delivery systems (see Fig. 1).

The ability of human pluripotent stem cells for unlimited proliferation and self-renewal promotes their application in the fields of regenerative medicine. The morphological assessment of growing colonies and cells, as a non-invasive method, allows the best clones for further clinical applications to be safely selected. For this purpose, we analyzed seven morphological parameters of both colonies and cells extracted from the phase-contrast images of human embryonic stem cell line H9, control human induced pluripotent stem cell (hiPSC) line AD3, and hiPSC line HPCASRi002-A (CaSR) in various passages during their growth for 120 h. The morphological phenotype of each colony was classified using a visual analysis and associated with its potential for pluripotency and clonality maintenance, thus defining the colony phenotype as the control parameter. Using the analysis of variance for the morphological parameters of each line, we showed that selected parameters carried information about different cell lines and different phenotypes within each line. We demonstrated that a model of classification of colonies and cells by phenotype, built on the selected parameters as predictors, recognized the phenotype with an accuracy of 70–75%. In addition, we performed a qRT-PCR analysis of eleven pluripotency markers genes. By analyzing the variance of their expression in samples from different lines and with different phenotypes, we identified group-specific sets of genes that could be used as the most informative ones for the separation of the best clones. Our results indicated the fundamental possibility of constructing a morphological portrait of a colony informative for the automatic identification of the phenotype and for linking this portrait to the expression of pluripotency markers. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem cells (mESCs) go through self-renewal in the existence of the cytokine leukemia inhibitory factor (LIF). LIF is added to the mouse stem cells culture medium, and its removal results in fast differentiation. Dimethyl sulfoxide (DMSO) is one of the most used solvents in drug test. We exposed 4-day mESC cultures to different concentrations of DMSO (0.1%, 0.5%, 1.0%, and 2.0%) to identify the safest dose exhibiting efficacy as a solvent. mESCs grown under general pluripotency conditions in the absence of LIF were treated with DMSO. In addition, as a control for differentiation, mESCs were grown in the absence of LIF. DMSO upregulated the mRNA expression level of pluripotency markers. Moreover, DMSO reduced the mRNA expression levels of ectodermal marker (β-tubulin3), mesodermal marker (Hand1), and endodermal markers (Foxa2 and Sox17) in mESCs. These results indicate that DMSO treatment enhances the pluripotency and disrupts the differentiation of mESCs. We also show that members of the Tet oncogene family are critical to inhibiting the differentiation and methylation of mESCs. DMSO is appropriate to sustain the pluripotency of mESCs in the absence of LIF, and that mESCs can be sustained in an undifferentiated state using DMSO. Therefore, DMSO may, in part, function as a substitute for LIF. [ABSTRACT FROM AUTHOR]

Germ cell tumours of ovary (GCT) are rapidly growing neoplasms derived from pleuripotent primordial germ cells. They comprise 15–20% of all ovarian neoplasms of which 95% are mature cystic teratomas and 5% are malignant ovarian germ cell tumours (MOGCT). MOGCT continues to be the cause of diverse, diagnostically challenging issues for the pathologist, and they are academically interesting because of the biological diversity exhibited by these tumours in the two gonads (testis and ovaries). Their correct diagnosis often has major important therapeutic and prognostic implications. This review intends to provide a refresher and update on the diagnostic aspects MOGCT including clinical, morphological, immunocytochemical and where relevant the molecular features. Somatic malignancies with a germ cell components and the use of new pluripotency markers that have improved diagnostic accuracy have also been included. [ABSTRACT FROM AUTHOR]

It is thought that the spleen contains stem cells that differentiate into somatic cells other than immune cells. We investigated the presence of these hypothetical splenic cells with stem cell characteristics and identified adherent cells forming densely‐packed colonies (Splenic Adherent Colony‐forming Cell; SACC) in the spleen. Splenic Adherent Colony‐forming Cell was positive for alkaline phosphatase staining and stage‐specific embryonic antigen (SSEA)‐1 antigen. However, the self‐renewal properties of SACCs were limited because they stopped cell proliferation once colonies visible to the naked eye were formed. Gene expression analyses by semi‐quantitative RT‐PCR revealed the significant expression of c‐Myc and Klf4, whereas faint or no expression was evident for Nanog, Oct3/4, and Sox2. Global expression analyses by DNA microarray and subsequent gene ontology analyses revealed that the expression levels of genes related to the immune system were significantly lower in SACCs than in control splenic cells. In contrast, genes unrelated to the immune system, such as those involved in cell adhesion and axon guidance, were relatively highly expressed in SACCs compared with control splenic cells. Taken together, we identified a novel cell type residing in the spleen that is different from the hypothetical splenic stem cell, but which bears some, but not all, characteristics that represent an undifferentiated state. [ABSTRACT FROM AUTHOR]

Changes in morphology and molecular genetic profile of human retinal pigment epithelial (RPE) cells in vitro were studied when exposed to the basic fibroblast growth factor (bFGF). The cell morphology was estimated by cell area and perimeter, spreading and polarization coefficients. It was demonstrated that the number of elongated (fibroblast-like) cells and the number of cells, which size was less than in the control increased in 48 h after adding the factor to the culture. At the same time, the cell proliferative activity decreased (according to MTT test). Immunocytochemical analysis demonstrated a decrease in the staining for connexin Cx43 and an increase in the intensity of staining for protein the Otx2 neuroepithelium protein. Simultaneously, the number of nestin-positive cells and βIII-tubulin positive cells increased. Using quantitative real-time PCR method, an increase in mRNA expression of the KLF4, OCT4, NANOG, OTX2, and NES and decrease in mRNA expression of the MITF and KRT18 were detected in RPE cells treated with bFGF; this indicates enhancement of cell dedifferentiation. These data are confirmed by a decrease in the COL1A1 mRNA expression indicating a decrease in synthetic cell activity. The results indicate that single (short-time) effect by bFGF is sufficient to activate the mechanism, which decreases the cell differentiation level toward neuroepithelium. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cells (iPSCs) have the potential to repair/regenerate smooth muscle cells (SMCs) in different organs. However, there are many challenges in their translation to clinical therapies. In this study, we describe our observations of in vitro SMC differentiation in three iPSC lines derived from human fibroblasts using retroviral, episomal, and mRNA/miRNA reprogramming methods. We sought to elucidate correlations between differentiation characteristics and efficiencies that can facilitate large-scale production of differentiated cells for clinical applications, and to report differences in pluripotency marker expression in differentiated cells from different iPSC lines. A standardized SMC differentiation protocol was used to induce the CD31+/CD34+ vascular progenitor cell phenotype. These were sorted by magnetic-activated (MACS) and fluorescence-activated cell sorting (FACS), and then treated with PDGF-BB and smooth muscle growth medium for further differentiation into smooth muscle progenitor cells (pSMCs). The expression of SMC and pluripotency markers in early- and late-passage (P1 and P4) pSMCs was analyzed. A total of 36 differentiation runs was performed on the three patient iPSC lines. All pSMC populations expressed SMC markers and Ki67 consistent with the progenitor phenotype. Initial iPSC density correlated positively with the sorted cell FACS efficiency, and this correlation could be fit to a quadratic equation. We also observed that a specific "honeycomb" pattern of the starting cultured iPSCs cultured correlated with higher efficiency in all three iPSC lines. Pluripotency marker expression decreased significantly to nearly undetectable levels in all three lines. There was no significant change in SMC and pluripotent marker expression between passage 1 and 4. In summary, our observations suggest that the method of iPSC reprogramming does not affect iPSC differentiation into pSMCs. Protocol efficiency can be modeled mathematically and coupled with the initial "honeycomb" cell pattern to optimize production of large cell numbers for clinical therapies. [ABSTRACT FROM AUTHOR]

During development, cellular differentiation programs need tight regulation for proper display of the activity of multiple factors in time and space. Chromatin adaptors of the BET family (Brd2, Brd3, Brd4 and Brdt in vertebrates) are transcription co-regulators tightly associated with the progression of the cell cycle. A key question regarding their function is whether they work as part of the general transcription machinery or, on the contrary, they are precisely recruited to the chromatin through specific transcription factors. Here, we report the selective recruitment of Brd2 to the chromatin by the transcription factor Lyar. We show that Lyar downregulation results in Brd2 dissociation from a number of promoters studied. On the contrary, dissociation of BET proteins from the chromatin has no effect on Lyar occupancy. Under differentiation conditions, the absence of Lyar leads to impaired downregulation of the pluripotency gene Nanog , with concomitant reduction in the upregulation of differentiation markers. Interestingly, following the induction of differentiation, Brd2 depletion exhibits the same effects as expressing a truncated Lyar molecule lacking the Brd2 interacting domain. Both approaches result in stronger Nanog repression, indicating that Lyar-mediated recruitment of Brd2 moderates Nanog downregulation when differentiation is triggered. Moreover, expression of truncated Lyar leads to impaired differentiation and increased apoptosis. Thus, Lyar-mediated recruitment of Brd2 would participate in preserving a proper timing for Nanog silencing ensuring the appropriate establishment of the differentiation program. [ABSTRACT FROM AUTHOR]

Background We investigated the effect of long-term lithium treatment on very-small embryonic-like stem cells (VSELs) and the mRNA expression of pluripotency and glial markers, in peripheral blood, in patients with bipolar disorder (BD). Methods Fifteen BD patients (aged 53 ± 7 years) not treated with lithium, with duration of illness > 10 years, 15 BD patients (aged 55 ± 6 years) treated with lithium for 8–40 years (mean 16 years) and 15 control subjects (aged 50 ± 5 years) were included. The number of VSELs was measured by flow cytometric analysis. Assessment of the mRNA levels of pluripotency markers (Oct-4, Sox 2 and Nanog) and glial markers (glial fibrillary acidic protein - GFAP, Olig1 and Olig2) was performed, using the Real-time quantitative reverse transcription PCR. Results In BD patients not taking lithium, the number of VSELs was significantly higher than in control subjects and correlated with the duration of illness. The expression of pluripotency markers was significantly higher than in the controls and correlated with the number of VSELs. The mRNA levels of the Olig1 and Olig 2 were higher than in the controls and those of the GFAP were higher than in patients receiving lithium. In lithium-treated BD patients the number of VSELs was similar to controls and correlated negatively with the duration of lithium treatment and serum lithium concentration. The mRNA levels of Oct-4, Sox-2, GFAP and Olig1 were not different from controls. The mRNA expression of Nanog was significantly higher and correlated with the number of VSELs. The mRNA expression of Olig 2 was higher than in the BD patients not taking lithium. Conclusion Long-term treatment with lithium may suppress the activation of regenerative processes by reducing the number of VSELs circulating in PB. These cells, in BD patients not treated with lithium, may provide a new potential biological marker of the illness and its clinical progress. The higher expression of peripheral mRNA markers in BD patients may involve ongoing inflammatory process, compensatory mechanisms and regenerative responses. Long-term lithium treatment may attenuate these mechanisms, especially in relation to the transcription factors Oct-4, Sox-2, GFAP and Olig1. [ABSTRACT FROM AUTHOR]

Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem cells (CD133+/CD24lo), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology, as did the nonexpressing cells. Depletion experiments showed that after the complete removal of the subpopulations of NANOG- and REX1-expressing NPCs, the expression of these genes appeared in other NPCs within a few days. The percentage of NANOG- and REX1-expressing cells returned to that observed before depletion. Our results are best explained by a model in which there is stochastic transient expression of pluripotency-associated genes in proliferating NPCs. [ABSTRACT FROM AUTHOR]

The search for effective and safe methods to generate induced pluripotent stem cells is especially urgent. In the paper murine embryonic fibro blasts were reprogrammed towards actively proliferating colonies with typical induced pluripotent stem cells morphology by means of Sleeping beauty transposon-based vector system. The obtained clones were checked for the expression of various pluripotency markers: alkaline phosphatase, Oct4 and Sox2 genes, SSEA-1 expression in various clones was evaluated. Also the reactivation of endogenous pluripotency factors Nanog and Rex1 was indicated. The data obtained is analyzed and compared to the established pluripotent stem cell line. It is shown that somatic cells are reprogrammed towards pluripotency by means of Sleeping beauty transposon system. Therefore, the system is a new perspective biotechnological tool to generate pluripotent cells.

Objective Wharton's jelly (WJ), an appropriate source of mesenchymal stem cells (MSCs), has been shown to have a wide array of therapeutic applications. However, the WJ-derived MSCs are very heterogeneous and have limited expression of pluripotency markers. Hence, improvement of their culture condition would promote the efficiency of WJ-MSCs. This study aims to employ a simple method of cultivation to obtain WJ-MSCs which express more pluripotency markers. Methods CD105+ cells were separated by magnetic-associated (activated) cell sorting from umbilical cord mucous tissue. CD105+ cells were added to Methocult medium diluted in α-minimum essential medium (α-MEM) and seeded in poly(2-hydroxyethyl methacrylate) (poly-HEMA)-coated plates for suspension culture preparation. Differentiation capacity of isolated cells was evaluated in the presence of differentiation-inducing media. The expression of pluripotency markers such as Oct3/4, Nanog, and Sox2 was also analyzed by RT-PCR and western blot techniques. Moreover, immunocytochemistry was performed to detect alpha-smooth muscle actin (antigene) (α-SMA) protein. Results WJ-MSCs grew homogeneously and formed colonies when cultured under suspension culture conditions (Non-adhesive WJ-MSCs). They maintained their growth ability in both adherent and suspension cultures for several passages. Non-adhesive WJ-MSCs expressed Oct3/4, Nanog, and Sox2 both at transcriptional and translational levels in comparison to those cultured in conventional adherent cultures. They also expressed α-SMA protein. Discussion In this study, we isolated WJ-MSCs using a slightly modified culture condition. Our simple non-genetic method resulted in a homogeneous population of WJ-MSCs, which highly expressed pluripotency markers. Conclusion In the future, more multipotent WJ-MSCs can be harnessed as a non-embryonic source of MSCs in MSC-based cell therapy. [ABSTRACT FROM AUTHOR]

Introduction Human term placenta has attracted increasing attention as an alternative source of stem cells for regenerative medicine since it is accessible without ethical objections. The amniotic membrane (AM) contains at least two stem cell types from different embryological origins: ectodermal amniotic epithelial stem cells, and mesodermal mesenchymal stromal cells. Among the second group we studied the characteristics of amniotic mesenchymal cells (AMC) versus the ones enriched for the commonly used surface marker c-Kit (amniotic progenitor/stem cells-ASC), a stem cell factor receptor with crucial functions in a variety of biological systems and presents in early progenitors of different origin, as been already demonstrated in the enriched chorionic stem cells. Methods After isolation, cells from the amniotic membranes (amniotic cells-AC) were selected for c-Kit (ASC) and compared these cells with c-Kit unselected (AMC), evaluating the expression of other stem cell markers (Oct-4, Tra-1-81, SSEA-4), CD271 and Slug. Results Immunofluorescence analysis showed that ASC cells exhibited greater stem cell marker expression and included more CD271 and Slug positive cells. This was consistent with the interpretation that c-Kit enriched AC show greater stemness capacity compared to c-Kit unselected AMC. Discussion AMC and ASC can both differentiate into various cell types including adipogenic, osteogenic, chondrogenic, neurogenic and hepatic lineages, but the enrichment in c-Kit improved stemness and differentiation potential of ASC. [ABSTRACT FROM AUTHOR]

Purpose: The levels and timing of expression of genes like BCLXL, HDAC1 and pluripotency marker genes namely, OCT4, SOX2, NANOG and KLF4 are known to influence preimplantation embryo development. Despite this information, precise understanding of their influence during preimplantation embryo development is lacking. The present study attempts to compare the expression of these genes in the in vivo and in vitro developed preimplantation embryos. Methods: The in vivo and in vitro developed rabbit embryos collected at distinct developmental stages namely, pronuclear, 2 cell, 4 cell, 8 cell, 16 cell, Morula and blastocyst were compared at the transcriptional and translational levels using Real Time PCR and immunocytochemical studies respectively. Results: The study establishes the altered levels of candidate genes at the transcriptional level and translational level with reference to the zygotic genome activation (ZGA) phase of embryo development in the in vivo and in vitro developed embryos. The expression of OCT4, KLF4, NANOG and SOX2 genes were higher in the in vitro developed embryos whereas and HDAC1 was lower. BCLXL expression had its peak at ZGA in in vivo developed embryos. Protein expression of all the candidate genes was observed in the embryos. BCLXL, KLF4 and NANOG exhibited diffused localisation whereas HDAC1, OCT4, and SOX2 exhibited nuclear localisation. Conclusions: This study leads to conclude that BCLXL peak expression at the ZGA phase may be a requirement for embryo development. Further expression of all the candidate genes was influenced by ZGA phase of development at the transcript level, but not at the protein level. [ABSTRACT FROM AUTHOR]

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2-5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs. [ABSTRACT FROM AUTHOR]

The search for effective and safe methods to generate induced pluripotent stem cells is especially urgent. In the paper murine embryonic fibro blasts were reprogrammed towards actively proliferating colonies with typical induced pluripotent stem cells morphology by means of Sleeping beauty transposon-based vector system. The obtained clones were checked for the expression of various pluripotency markers: alkaline phosphatase, Oct4 and Sox2 genes, SSEA-1 expression in various clones was evaluated. Also the reactivation of endogenous pluripotency factors Nanog and Rex1 was indicated. The data obtained is analyzed and compared to the established pluripotent stem cell line. It is shown that somatic cells are reprogrammed towards pluripotency by means of Sleeping beauty transposon system. Therefore, the system is a new perspective biotechnological tool to generate pluripotent cells. [ABSTRACT FROM AUTHOR]

Testicular germ cell tumours (TGCTs), the most frequent solid tumour of the young men, originate from the primitive germ cells. They share some pluripotency stem-cell markers which may help to distinguish between seminoma, the most frequent TGCTs and non-seminoma tumours, such as embryonal carcinoma, teratocarcinoma or choriocarcinoma. Due probably to the propensity of seminoma to apoptosis, only two cell lines originated from pure testicular seminoma, TCam-2 and JKT-1 have been up to now, established, maintained and proposed as representative models of human testicular seminoma. However, both seem, following recent reports, to be able to drift. Thus, the molecular signature of embryonic stem-cell markers of the JKT-1 cells cultured in our laboratory, were studied by RT-PCR, Western blot and immunofluorescence (IF). JKT-1 cells analysed after 30 passages, expressed placenta alkaline phosphatase but not alphafoetoprotein (αFP) nor beta-human chorionic gonadotropin. JKT-1 cells also expressed markers of pluripotency such as NANOG and OCT3/4 and more specific seminoma markers, such as AP2γ and HIWI. However, protein expression of OCT3/4 and AP2y was weak and these JKT-1 cells expressed SOX2, a marker of embryonal carcinoma and did not express c-KIT usually expressed in most seminoma. Possible derivation through in vitro culture conditions was supported by looking at later passages (61) which showed a decrease of NANOG and HIWI protein expression. JKT-1 cells express a signature of markers which is still near from the one express by seminoma cells, allowing carcinogenetic studies. However, because of their great ability to drift as shown for TCam-2, it is recommended to verify and to precise this molecular signature before reporting functional results. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) hold a great potential for therapeutic regenerative medicine. The aim of this study was to generate induced pluripotent stem cells from goat embryonic cardiac tissue derived fibroblasts. The isolated cardiac fibroblasts from the cardiac tissue of goat embryos were positive for alfa smooth muscle actin, vimentin and discoidin domain receptor2. From these cells, we generated transgene free iPSCs using piggyBac transposons / transposase using five transcription factors (Oct4, Sox2, Klf, Myc and Lin 28). The generated iPSCs were SSEA1, SSEA4 and Oct4 positive. They were cultured on neofeeders using 20% Serum replacement - IMDM with bFGF. They could form cystic and compact embryoid bodies that showed differentiated ectodermal and mesodermal like cells when cultured using 20% FBS-IMDM without bFGF. The iPSCs, generated in the frame of this approach were produced without the use of integrating virus and the reprogramming transgenes were removed at the end of the process. Though there were limitations in the approach used, a substantial sign of reprogramming was obtained., Competing Interests: None, (Copyright © Journal of Stem Cells and Regenerative Medicine.)

Various classifications have been proposed for human amniotic subpopulations, including classification of spindle-shaped (SS) and round-shaped (RS) cells, as well as the more referred triple-category of epithelioid (E-type) cells, amniotic fluid-specific (AF-type) cells and fibroblastoid (F-type) cells. The present study aims to investigate these amniotic subpopulations regarding the expression of some stem cell markers, including OCT4, NANOG, SOX2, C-KIT (CD117), C-MYC, KLF4, and THY1 (CD90). Flow cytometry was performed to characterize the isolated clonal subpopulations for a hematopoietic and a mesenchymal marker using PE-CD31 and FITC-CD90, respectively. A semi-quantitative RT-PCR analysis was carried out on the isolates in the second half of their lifespan when the cells were at the stationary phase of the growth curve. Characterization of isolated cells demonstrated that all clones including both epithelioid and fibroblastoid cells, had mesenchymal, not hematopoietic, lineage. RT-PCR analysis also revealed a higher expression of the target genes in epithelioid cells. Furthermore, the expression pattern of the genes and their correlations were remarkably different between primary- and long term-cultured amniocytes. Taken together, our results showed that the primary-cultured cells express the stemness genes equally, whereas the long term-cultured amniocytes exhibited a highly variable manner in the expression pattern of the genes. Diverse derivation site of amniocytes and individual genetic background can potentially explain the observed variation in the expression level of the target genes. These can also explain why amniocytes differ in many respects observed in our study, including survival rate, plastic adhesion, and growth characteristics., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2020 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Quality of in vitro-produced embryos is influenced by changes in gene expression in response to adverse conditions. Gene markers for predicting 'good embryos' do not exist at present. We propose that the expression of pluripotency markers OCT4-SOX2-NANOG in D9 (day 9) bovine demi-embryos correlated with development at D13 (day 13). Day 8 in vitro-produced blastocysts were split in two cloned halves, one half (D9) was subjected to analysis of pluripotency markers and the other was kept in culture until D13 of development. Embryo development was scored and correlated with its own status at D9 and assigned to one of two categories: G1, arrested/dead; or G2, development up to D13. SOX2 and NANOG expression levels were significantly higher in embryos from G1 and there was also negative correlation between SOX2 and embryo survival to D13 (G3; r = -0.37; P = 0.03). We observed a significant reduction in the expression of the three studied genes from D9 to D13. Furthermore, there was a correlation between the expression of pluripotency markers at D9 and embryo diameter and the expression of trophoblastic markers at D13 (TP1-EOMES-FGF4-CDX2-TKDP1). Finally, the quotient between the relative expression of SOX2 and OCT4 in the D9 blastocysts from G1 and G2 showed that embryos that were considered as competent (G2) had a quotient close to one, while the other group had a quotient of 2.3 due to a higher expression of SOX2. These results might indicate that overexpression of SOX2 at the blastocyst stage had a negative effect on the control of embryonic developmental potential.

Background: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs., Results: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth., Conclusions: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs., Methods: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients ( n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

The capacity to regenerate entire body parts, tissues, and organs had generally been thought to be lost in evolution with very few exceptions (e.g. the liver) surviving in mammals. The discovery of the MRL mouse and the elucidation of the underlying molecular pathway centering around hypoxia inducible factor, HIF-1α, has allowed a drug and materials approach to regeneration in mice and hopefully humans. The HIF-1α pathway is ancient and permitted the transition from unicellular to multicellular organisms. Furthermore, HIF-1α and its regulation by PHDs, important oxygen sensors in the cell, provides a perfect drug target. We review the historical background of regeneration biology, the discovery of the MRL mouse, and its underlying biology, and novel approaches to drugs, targets, and delivery systems (see Fig. 1)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aim: Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs)., Methods: Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers - alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry., Results: In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein., Conclusion: Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage.

Preeclampsia (PE) and intrauterine growth retardation (IUGR) are rare but severe pregnancy complications that are associated with placental insufficiency often resulting in premature birth. The clinical pathologies are related to gross placental pathologies and trophoblastic deficiencies that might derive from inflammatory processes and oxidative stress injury. The mesenchymal core of placental villi has been identified as a possible niche for trophoblast progenitor cells that are called upon to replenish the injured syncytiotrophoblast layer. These progenitor cells are known to express trophoblast stem cell (CDX2) and pluripotency (SOX2, NANOG and OCT4A) markers, however only little data is available characterizing the expression of these transcription factors beyond the blastocyst stage. We aimed to describe the expression of these factors in healthy 1st and 3rd trimester placentae as well as PE, IUGR and combined PE+IUGR placentae. We analyzed 8 respective samples derived from 1st trimester (elective abortions), and 3rd trimester (healthy controls, PE, IUGR and combined PE+IUGR). We accomplished immunoperoxidase staining to detect the stem cell markers: CDX2 (trophectoderm), SOX2, NANOG and OCT4A (embryonal). Immunoreative scoring was used for objective analyses of staining patterns. All markers display clearly elevated signals in 1st trimester villous samples as compared to healthy 3rd trimester counterparts. Especially CDX2 and NANOG were specific to the cytotrophoblast layer and the mesenchymal core. Specific and differential expression patterns were visible in the villous/extravillous compartment of each placenta-associated pregnancy complication (PE: pan elevated expression; IUGR elevated SOX2 in basal plate; combined PE+IUGR pan loss of expression). Reduction of stem cell transcription factor expression in term placentae indicates temporal regulation, and probably a specific function which is yet to be elucidated. The differential expression patterns within placentae complicated with placenta-associated pregnancy complications indicate that PE, IUGR and combined PE+IUGR are separate entities. It is unclear whether the alterations are the cause or the effect of the clinical pathology.

Objective: Wharton's jelly (WJ), an appropriate source of mesenchymal stem cells (MSCs), has been shown to have a wide array of therapeutic applications. However, the WJ-derived MSCs are very heterogeneous and have limited expression of pluripotency markers. Hence, improvement of their culture condition would promote the efficiency of WJ-MSCs. This study aims to employ a simple method of cultivation to obtain WJ-MSCs which express more pluripotency markers., Methods: CD105(+) cells were separated by magnetic-associated (activated) cell sorting from umbilical cord mucous tissue. CD105(+) cells were added to Methocult medium diluted in α-minimum essential medium (α-MEM) and seeded in poly(2-hydroxyethyl methacrylate) (poly-HEMA)-coated plates for suspension culture preparation. Differentiation capacity of isolated cells was evaluated in the presence of differentiation-inducing media. The expression of pluripotency markers such as Oct3/4, Nanog, and Sox2 was also analyzed by RT-PCR and western blot techniques. Moreover, immunocytochemistry was performed to detect alpha-smooth muscle actin (antigene) (α-SMA) protein., Results: WJ-MSCs grew homogeneously and formed colonies when cultured under suspension culture conditions (Non-adhesive WJ-MSCs). They maintained their growth ability in both adherent and suspension cultures for several passages. Non-adhesive WJ-MSCs expressed Oct3/4, Nanog, and Sox2 both at transcriptional and translational levels in comparison to those cultured in conventional adherent cultures. They also expressed α-SMA protein., Discussion: In this study, we isolated WJ-MSCs using a slightly modified culture condition. Our simple non-genetic method resulted in a homogeneous population of WJ-MSCs, which highly expressed pluripotency markers., Conclusion: In the future, more multipotent WJ-MSCs can be harnessed as a non-embryonic source of MSCs in MSC-based cell therapy.

Embryonic stem (ES) cells are a valuable system to study early development of mammals. A lot of researchers are dedicated to comprehensive study of the ES cells. Today ES cells have been isolated from mice, human and some other mammalian species. A set of the American mink ES cell lines had been isolated from mink morulas and blastocysts in our group. The formation of embryoid bodies and teratomas in athymic mice suggested that these cells are pluripotent. It is known that several transcription factors are essential for the ES cell identity, including two homeodomain proteins, Nanog and Oct-4, and a HMG factor, Sox-2. These factors influence on other transcription factors and control expression of each other establishing regulatory network that maintains ES cell pluripotency and self-renewal. In this study, we analyzed the expression of the Oct-4 and Sox-2 genes in the American mink ES cells and determined their chromosomal localization. First we determined the primary structure of the American mink Oct-4 and Sox-2 gene fragments. Using the Chinese hamster-American mink somatic cell hybrids panel, consisting of 14 clones, we localized Oct-4 and Sox-2 genes to mink chromosomes one and six, respectively, with 100% concordance between the marker and the specific chromosome. To analyze the expression of Oct-4 in the mink ES cell lines (MES-12, MES-12neo and MES-13) we used RT-PCR analysis and immunostaining. Both methods revealed that the ES cells expressed Oct-4. As for Sox-2 with use of RT-PCR we have found the low level of expression in the ES cells. The expression of pluripotency markers confirms the pluripotent capacities of examined ES cells. [ABSTRACT FROM AUTHOR]

Background and Objectives: Recent findings suggest that therapeutic potential of mesenchymal stem cells (MSCs) could be increased through aggregation into three-dimensional (3D) bodies, and different culture methods have been employed to obtain 3D spheroids of MSCs. In the current study we report accidentally encountered spontaneous formation of adipose-derived stem cell (ASC) bodies in standard ASC culture of a single donor., Methods and Results: Human ASCs from passages 1 to 3, cultured in a medium containing 5% autologous serum (AS), spontaneously clustered and formed floating 3D bodies. After a transfer of floating ASC bodies onto new adherent plastic dish, they attached to the surface and gradual migration of spindle-shaped ASCs out of the bodies was detected. A substitution of AS with allogeneic sera did not hinder this ability, but commercial medium containing fetal bovine serum delayed the process. Substantial part of ASCs surrounding transferred ASC bodies showed alkaline phosphatase (AP) activity, while ASC aggregates were AP negative. Similar 3D bodies formed when ASCs were grown on an uncoated glass surface. These ASC aggregates as well as clusters of ASCs, where formation of the 3D bodies is initiated, expressed pluripotency marker NANOG, but the expression of OCT4A was not detected., Conclusions: Obtained results suggest that spontaneously formed ASC aggregates may represent a more primitive cell subpopulation within the individual ASC culture. The ability to form 3D aggregates, the expression of NANOG, and the lack of the AP activity may be used to enrich ASC cultures with potentially more primitive cells serving as an excellent basis for therapeutic applications.