Pou5f1/Oct4 is a key transcription factor for the induction of pluripotency and totipotency in preimplantation mouse embryos. In mice, loss or gain of function experiments have demonstrated an important role for Oct4 in preimplantation and developmental ability. In this study, using mouse preimplantation embryos as a model for the evaluation of Oct4 function, we constructed Oct4 overexpression embryos with various mutations at the N-terminal transactivation domain. Developmental competency and molecular biological phenotypes depended on the type of mutation. The replacement of serine 106 with alanine resulted in more severe phenotypes similar to that of wild type Oct4, indicating that this alteration using alanine is negligible for Oct4 function. In contrast, we found that Oct4-specific antibodies could not recognize Oct4 protein when this residue was replaced by aspartic acid (Oct4-S106D). Oct4-S106D overexpressing embryos did not show developmental arrest and aberrant chromatin structure. Thus, these results demonstrated that the Ser-106 residue within the N-terminal transactivation domain is crucial for Oct4 function and suggested that this mutation might affect Oct4 protein conformation.

The type and pattern of epigenetic modification in donor cells can significantly affect the developmental competency of somatic cell nuclear transfer (SCNT) embryos. Here, we investigated the developmental capacity, gene expression, and epigenetic modifications of SCNT embryos derived from porcine bone marrow-derived mesenchymal stem cells (BMSCs) and fetal fibroblasts (FFs) donor cells compared to embryos obtained from in vitro fertilization (IVF). Compared to FFs, the donor BMSCs had more active epigenetic markers (Histone H3 modifications: H3K9Ac, H3K4me3, and H3K4me2) and fewer repressive epigenetic markers (H3K9me3, H3K9me2, and DNA methyltransferase 1). Embryos derived from BMSC nuclear-transfer (BMSC-NT embryos) and IVF embryos had significantly higher cleavage and blastocyst rates (BMSC-NT: 71.3 ± 3.4%, 29.1 ± 2.3%; IVF: 69.2 ± 2.2%, 30.2 ± 3.3%; respectively) than FF-NT embryos (58.1 ± 3.4%, 15.1 ± 1.5%, respectively). Bisulfite sequencing revealed that DNA methylation at the promoter regions of NANOG and POU5F1 was lower in BMSC-NT embryos (30.0%, 9.8%, respectively) than those in FF-NT embryos (34.2%, 28.0%, respectively). We also found that BMSC-NT embryos had more H3K9Ac and less H3K9me3 and 5-methylcytosine than FF-NT embryos. In conclusion, our finding comparing BMSCs versus FFs as donors for nuclear transfer revealed that differences in the initial epigenetic state of donor cells have a remarkable effect on overall nuclear reprogramming of SCNT embryos, wherein donor cells possessing a more open chromatin state are more conducive to nuclear reprogramming.

The objective of this study was to investigate the survival and development of porcine cloned embryos vitrified by Cryotop carrier at the zygote, 2- and 4-cell stages. The quality of resultant blastocysts was evaluated according to their total cell number, apoptotic cell rate, reactive oxygen species (ROS) production, glutathione (GSH) content and mRNA expression levels of genes related to embryonic development. The survival rates of zygotes, 2- and 4-cell embryos after vitrification did not differ from those of their fresh counterparts. Vitrification still resulted in significantly decreased blastocyst formation rates of these early-stage embryos. Moreover, the total cells, apoptotic rate, ROS and GSH levels in resultant blastocysts were unaffected by vitrification. The mRNA expression levels of PCNA, CPT1, POU5F1 and DNMT3B in the blastocysts derived from vitrified early-stage embryos were significantly higher than those in the fresh blastocysts, but there was no change in expression of CDX2 and DNMT3A genes. In conclusion, our data demonstrate that the early-stage porcine cloned embryos including zygotes, 2- and 4-cells can be successfully vitrified, with respectable blastocyst yield and quality.

Keywords: Cloned embryo; Development; Early-stage; Pig; Vitrification.

The postwarming recovery culture, as one of the steps in cryopreservation process, is directly correlated with the survival and quality of embryos. Generally, recovery medium includes undefined serum or serum components that may cause the instability of results and other problems. The objective of this study was to evaluate the effect of knockout serum replacement (KSR) as a substitute for serum during recovery culture on the development and quality of vitrified parthenogenetic porcine blastocysts. Fetal bovine serum (FBS) was used as a positive control. The expanded blastocysts on day 5 were vitrified by the Cryotop method, and recovered with 10% (v/v) KSR or 10% (v/v) FBS for 48 hours after warming. Survival and hatching rates of vitrified blastocysts were significantly increased by KSR or FBS supplementation. The vitrified blastocysts recovered in KSR or FBS exhibited significantly decreased percentages of membrane damage and apoptosis, and increased total cells. Addition of KSR or FBS during recovery culture significantly reduced reactive oxygen species levels, and improved mitochondrial activity and adenosine triphosphates content in the vitrified blastocysts. Vitrification did not affect the gene expression of PCNA, CDX2, and CPT1, but significantly increased mRNA levels of POU5F1 and uPA. KSR added to the recovery medium significantly upregulated mRNA levels of PCNA and CPT1, and downregulated POU5F1 mRNA levels. The expression levels of PCNA, CDX2, CPT1, and uPA in vitrified blastocysts were significantly upregulated by addition of FBS to recovery medium. Moreover, the BAX: BCL2L1 ratio was similar between fresh and vitrified blastocysts, and KSR or FBS supplementation had no effect on the value. In conclusion, our data showed that KSR supplementation during recovery culture can improve the development and quality of vitrified parthenogenetic porcine blastocysts. These findings provide a useful reference that KSR could be used to replace FBS as a defined serum supplement for recovery culture of vitrified blastocysts.

Background: Breast tumors enriched with breast cancer stem cells (BCSCs), play a crucial role in metastasis and tumor relapse. Hence, targeting BCSCs may lead to efficacious breast cancer therapy. BCSCs have a unique expression of stemness markers, including Nanog, POU5F1, SOX2, and CD44, which play a vital role in cancer stem cell properties. However, the regulation of microRNAs (miRNAs)-mediated cancer stem cell marker expressions is largely unclear.

Methods: MIENTURNET was used to predict miRNA-target interactions. miR-TV, UALCAN and GEPIA databases were used to analyze the expression of miR-145-5p and SOX2. Survival analysis was obtained by cBioportal, KM plotter and Breast Cancer Gene-Expression Miner. RNAComposer was used to perform miRNA-mRNA duplex prediction. In vitro mRNA and miRNA analysis was performed by qRT-PCR.

Results: It was observed that miR-145-5p was the common miRNA targeting stemness markers. miR-145-5p expression was found to be lower in breast cancer patients compared to healthy subjects. Based on survival analysis, low expression of miR-145-5p and high expression of SOX2 led to a poor overall survival rate in breast cancer patients. Pathway enrichment analysis indicated that SOX2 was highly enriched with transcription factors. Moreover, SOX2 expression level was also upregulated in axillary metastatic lymph nodules. Further, in vitro ectopic expression of miR-145-5p by its mimic downregulated the SOX2 expression compared to the control mimic. Overall, SOX2 was a direct target for miR-145-5p as per the binding and minimal-free energy.

Conclusions: In this study, miR-145-5p targeting SOX2 was identified as a potential predictive biomarker for breast cancer stemness.

Keywords: Biomarker; Breast cancer stemness; SOX2; miR-145–5p; miRNAs.

Liver kinase B1 (LKB1), a serine/threonine protein, is a key regulator in stem cell function and energy metabolism. Herein, we describe the role of LKB1 in modulating the differentiation of synovium-derived stem cells (SDSCs) toward chondrogenic, adipogenic, and osteogenic lineages. Human fetal SDSCs were transduced with CRISPR associated protein 9 (Cas9)-single-guide RNA vectors to knockout or lentiviral vectors to overexpress the LKB1 gene. Analyses including ICE (Inference of CRISPR Edits) data from Sanger sequencing and quantitative polymerase chain reaction (qPCR) as well as Western blot demonstrated successful knockout (KO) or overexpression (OE) of LKB1 in human fetal SDSCs without any detectable side effects in morphology, proliferation rate, and cell cycle. LKB1 KO increased CD146 expression; interestingly, LKB1 OE increased SSEA4 level. The qPCR data showed that LKB1 KO upregulated the levels of SOX2 and NANOG while LKB1 OE lowered the expression of POU5F1 and KLF4. Furthermore, LKB1 KO enhanced, and LKB1 OE inhibited, chondrogenic and adipogenic differentiation potential. However, perhaps due to the inherent inability to achieve osteogenesis, LKB1 did not obviously affect osteogenic differentiation. These data demonstrate that LKB1 plays a significant role in determining human SDSCs' adipogenic and chondrogenic differentiation, which might provide an approach for fine-tuning the direction of stem cell differentiation in tissue engineering and regeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

The present study examined the comparative expression and secretory profile of vital signaling molecules in buffalo fetal fibroblasts (BFF) and Wharton's jelly (BWJ) feeder layers at different passages. Both feeder layers were expanded up to 8th passage. Signaling molecules viz. bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGFB1) and pluripotency-associated transcriptional factors (POU5F1, SOX2, NANOG, KLF4, MYC and FOXD3) were immunolocalized in the both feeder types. A clear variation in the expression pattern of key signaling molecules with passaging was registered in both feeders compared to primary culture (0 passage). The conditioned media (CM) was collected from different passages (2, 4, 6, 8) of both the feeder layers and was quantified using enzyme-linked immunosorbent assay (ELISA). Concomitant to expression profile, protein quantification also revealed differences in the concentration of signaling molecules at different time points. Conjointly, expression and secretory profile revealed that 2nd passage of BFF and 6th passage of BWJ exhibit optimal levels of key signaling molecules thus may be selected as best passages for embryonic stem cells (ESCs) propagation. Further, the effect of mitomycin-C (MMC) treatment on the expression profile of signaling molecules in the selected passages of BFF and BWJ revealed that MMC modulates the expression profile of these molecules. In conclusion, the results indicate that feeder layers vary in expression and secretory pattern of vital signaling molecules with passaging. Based on these findings, the appropriate feeder passages may be selected for the quality propagation of buffalo ESCs.

Germ cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell-intrinsic β-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in β-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of β-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of Znrf3, a negative regulator of WNT/β-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that β-catenin is a central gatekeeper in ovarian differentiation and gametogenesis.

Keywords: POU5F1/OCT4; WNT/β-catenin; differentiation; germ cells; ovary.

POU5F1 (POU class 5 homeobox 1) is a transcription factor that is critically involved in the self-renewal of undifferentiated embryonic stem cells. In this present study, we have developed our study to analyze the expression of the POU5F1 in the neonatal and adult mice testis section and isolated spermatogonial stem cells (SSCs). We also examine POU5F1 protein localization by three various kinds of antibodies. In this experimental research, to enhance our understanding of the POU5F1 expression levels, protein localization, and function in testicular germ cell, we used immunohistochemistry, immunocytochemistry, and Fluidigm real-time polymerase chain reaction (RT-PCR) analysis in the mouse testis section and neonatal and adult SSCs, and also we used protein-protein network analysis and gene enrichment analysis for genes involved in testicular development. Counting POU5F1-positive cells represented significantly higher expression (p < 0.05) of POU5F1 in the adult testis in comparison to the neonate. Finally, Fluidigm RT-PCR showed a significant expression (p < 0.05) level of germ cells gene POU5F1 in neonate SSCs (1-2 week) than 16-24 week SSCs. The illustrated results identify POU5F1 as a necessary transcription factor of testicular germ cells and can be supportive for the investigation of the development and differentiation of SSCs.

Keywords: POU5F1 (OCT4); analysis; and adult germ cells; neonate; seminiferous tubules; spermatogonial stem cells; transcription factor.

Prenatal ethanol exposure can result in loss of neural stem cells (NSCs) and decreased brain growth. Here, we assessed whether a noncoding RNA (ncRNA) related to the NSC self-renewal factor Oct4/Pou5f1, and transcribed from a processed pseudogene locus on mouse chromosome 9 (mOct4pg9), contributed to the loss of NSCs due to ethanol. Mouse fetal cortical-derived NSCs, cultured ex vivo to mimic the early neurogenic environment of the fetal telencephalon, expressed mOct4pg9 ncRNA at significantly higher levels than the parent Oct4/Pou5f1 mRNA. Ethanol exposure increased expression of mOct4pg9 ncRNA, but decreased expression of OCT4/POU5F1. Gain- and loss-of-function analyses indicated that mOct4pg9 overexpression generally mimicked effects of ethanol exposure, resulting in increased proliferation and expression of transcripts associated with neural maturation. Moreover, mOct4pg9 associated with Ago2 and with miRNAs, including the anti-proliferative miR-328-3p, whose levels were reduced following mOct4pg9 overexpression. Finally, mOct4pg9 inhibited Oct4/Pou5f1-3'UTR-dependent protein translation. Consistent with these observations, data from single-cell transcriptome analysis showed that mOct4pg9-expressing progenitors lack Oct4/Pou5f1, but instead overexpress transcripts for increased mitosis, suggesting initiation of transit amplification. Collectively, these data suggest that the inhibitory effects of ethanol on brain development are explained, in part, by a novel ncRNA which promotes loss of NSC identity and maturation.

Keywords: FASD; Fetal neural progenitor cells; Neural development; Oct4/Pou5f1; Pseudogene; miRNAs; ncRNA.

The molecular interaction network of Oct-4 (POU5F1) and NANOG connected to regulation and growth of mesenchymal stem cells (MSCs) were supplemented with information of miRNA to find an important micro-RNAs and supplemented molecular interaction network. Following co-culturing of Bone marrow mesenchymal stem cells (BMMSCs) with SKOV3 ovarian cancer cell lines and undifferentiated BMMSCs, MTT was analyzed for cell cytotoxicity. The analyses of the expression of miRNA were performed either after oesteogenesis (hsa-miR-34 and hsa-miR-335) or chondrogenic (hsa-miR-145 and hsa-miR-455) differentiation. This molecular interaction network was imaged in using software. The results from these findings gave an understanding of the main molecular mechanisms regulating MSCs therapeutic activity and their undifferentiated state maintenance. We recommend that the downregulation of miR-335 is crucial role for tissue homeostasis.

Chromosomal abnormalities, as a frequent phenomenon in cultured embryonic stem cells (ESCs), is a major obstacle in the ESC application in regenerative medicine. Recent studies showed that aneuploid embryonic stem cells of humans and mice are more vulnerable to anticancer drugs, compared with normal cells. The aim of the current study was to evaluate effects of three anticancer drugs, paclitaxel, lapatinib and bortezomib, on mouse embryonic stem cells (mESCs) as a suitable and available model. To assess in vitro cell toxicity, two mESC lines were treated with the aforementioned drugs. Using G-band karyotyping and micronucleus assay, the effect of anticancer drugs in terms of reduction of chromosomal instability in the mESCs was evaluated in control and treatment groups. Also, apoptosis rate of both groups was estimated by FITC-Annexin V/Propidium Iodide (PI) double staining. In addition, the effect of these three drugs in maintaining the pluripotency was assessed through alkaline phosphatase assay and quantification of the expression of three key pluripotency genes, Nanog, Pou5f1 and Sox-2 was performed using Real Time PCR. The rate of numerical abnormalities after treatment with paclitaxel and lapatinib was lower than the control group. The expression level of pluripotency genes exhibited no significant difference between control and treatment groups. Administration of paclitaxel and lapatinib to the mESCs culture at an appropriate dose and in a timely manner could decrease chromosome stability without affecting pluripotency.

Keywords: Bortezomib; Chromosomal instability; Lapatinib; Mouse embryonic stem cells; Paclitaxel.

Aims: Folliculogenesis contains gonadotropin-independent and -dependent stage. Disruption in any of this process would induce failure in retrieving capable oocytes during clinical treatment. However, there is still limited understanding of the molecular components specifically regulating this process.

Material and methods: Ovaries of P3, P20 and exogenous gonadotropin-treated P22 mice were sampled and underwent RNA-seq to investigate the transcriptome variance during mouse folliculogenesis.

Key findings: In our dataset, 1883 and 626 DEGs were captured for each stage respectively, which were further clustered into eight expression patterns. Pathway enrichment analysis identified distinct biological processes enriched in two stages, with the most prominent being the pathways related to metabolism, gene expression, cell cycle, immune system and DNA methylation. Transcriptional regulator inference yielded eight master transcription factors (i.e. Runx1, Stat3, Sox3, Pou5f1, Gata4, Foxl2, Cebpb, and Esr1) driving folliculogenesis.

Significance: Our study revealed the temporal transcriptional reprogramming and gene expression dynamics during folliculogenesis mediated by extra hormone treatment, which could provide novel insights to controlled ovarian stimulation in future infertility treatment.

Keywords: Folliculogenesis; Infertility; Ovary; RNA-seq; Transcription factor.

The LIF-JAK2-STAT3 pathway is the central signal transducer that maintains undifferentiated mouse ESCs (mESCs), which is achieved by the recruitment of activated STAT3 to the master pluripotency genes and activation of the gene transcriptions. It remains unclear, however, how the epigenetic status required for the master gene transcriptions is built into LIF-treated mESC cultures. In this study, Jak2, but not Stat3, in the LIF canonical pathway, establishes an open epigenetic status in the pluripotency gene promoter regions. Upon LIF activation, cytosolic JAK2 was translocalized into the nucleus of mESCs, and reduced DNA methylation (5mC levels) along with increasing DNA hydroxymethylation (5hmC) in the pluripotent gene (Nanog/Pou5f1) promoter regions. In addition, the repressive histone codes H3K9m3/H3K27m3 were reduced by JAK2. Activated JAK2 directly interacted with the core epigenetic enzymes TET1 and JMJD2, modulating its activity and promotes the DNA and histone demethylation, respectively. The JAK2 effects were attained by tyrosine phosphorylation on the epigenetic enzymes. The effects of JAK2 phosphorylation on the enzymes were diverse, but all were merged to the epigenetic signatures associated with open DNA/chromatin structures. Taken together, these results reveal a previously unrecognized epigenetic regulatory role of JAK2 as an important mediator of mESC maintenance. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: This study reveals underappreciated JAK2-mediated epigenetic control in maintaining mESC pluripotency. JAK2 activation by LIF induce JAK2 translocation to nucleus where it directly interacts with epigenetic regulator protein which ultimately affect the DNA and histone methylation of pluripotent genes. Briefly, JAK2 primed DNMT2 for degradation, while inducing activation of TET1 and JMJD2 that ultimately open the epigenetic status in the pluripotent genes promoter regions.

Keywords: Embryonic Stem Cells (ESCs); Epigenetics; Janus kinase (JAK); LIF.

Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of POU5F1 and SOX2. We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.

Keywords: CRISPR activator; CRISPR/dCas9; gene activation; reprogramming factor.

Solid tumors are complex collections of cells surrounded by benign tissues that influence and are influenced by the tumor. These surrounding cells include vasculature, immune cells, neurons, and other cell types, and are collectively known as the tumor microenvironment. Tumors manipulate their microenvironment for the benefit of the tumor. Autonomic neurons innervate and drive malignant growth in a variety of solid tumors. However, the mechanisms underlying neuron-tumor relationships are not well understood. Recently, Amit et al. described that trophic relationships between oral cavity squamous cell carcinomas (OCSCCs) and nearby autonomic neurons arise through direct signaling between tumors and local neurons. An inducible tumor model in which 4NQO was introduced into the drinking water of Trp53 knockout mice was used to model OCSCC-microenvironment interactions. Using this model, this group discovered that loss of p53 expression in OCSCC tumors resulted in increased nerve density within these tumors. This neuritogenesis was controlled by tumor-derived microRNA-laden extracellular vesicles (EVs). Specifically, EV-delivered miR-34a inhibited neuritogenesis, whereas EV-delivered miR-21 and miR-324 increased neuritogenesis. The neurons innervating p53-deficient OCSCC tumors were predominantly adrenergic and arose through the transdifferentiation of trigeminal sensory nerve fibers to adrenergic nerve fibers. This transdifferentiation corresponded with increased expression of neuron-reprogramming transcription factors, including POU5F1, KLF4, and ASCL1, which were overexpressed in the p53-deficient samples, and are proposed targets of miR-34a-mediated regulation. Human OCSCC samples enriched in adrenergic neuron markers are associated strongly with poor outcomes, thus demonstrating the relevance of these findings to cancer patients.

Keywords: MicroRNA; adrenergic neurons; microenvironment; neuron-tumor crosstalk; neurotrophic growth; solid tumors.

Preimplantation development is a complicated process, which involves many genes. We have investigated the expression patterns of 17 developmentally important genes and isoforms in early mouse embryos as well as in single cells of the mouse embryo. The comparison is an excellent example for showing the importance of studying heterogeneity among cell populations on the RNA level, which is being increasingly addressed in basic research and medical sciences, particularly with a link to diagnostics (e.g. the analysis of circulating tumor cells and their progenitors). The ubiquitously expressed histone variant H3f3a and the transcription factor Pou5f1 generated mRNA-derived products in all analyzed preimplantation embryos (up to the morula stage) and in all analyzed blastomeres from 16-cell embryos, indicating a rather uniform reactivation of pluripotency gene expression during mouse preimplantation development. In contrast, genes that have been implicated in epigenetic genome reprogramming, such as DNA methyltransferases, methylcytosine-binding proteins, or base excision repair genes revealed considerable variation between individual cells from the same embryo and even higher variability between cells from different embryos. We conclude that at a given point of time, the transcriptome encoding the reprogramming machinery and, by extrapolation, genome reprogramming differs between blastomeres. It is tempting to speculate that cells expressing the reprogramming machinery have a higher developmental potential.

The present study was designed to describe the development of germ cell neoplasia in situ in Chinchilla rabbit by administration of estradiol. The study was performed in rabbits distributed into two groups: control and 17 β-estradiol. The determination of histological alterations and POU5F1 and c-kit proteins employed as biomarkers for the diagnosis of this neoplasia was carried out. Testicular descent and complete spermatogenesis were observed in the control group. The protein biomarkers were negative. However, in the rabbits treated with estradiol, the testes remained undescended with the gonocytes undifferentiated to spermatogonia. There were histological lesions owing to germ cell neoplasia in situ and positive to POU5F1 and c-kit proteins. These findings indicate that the chinchilla rabbit is an ideal model to study this neoplasia in which the histological characteristics and biomarkers of the disease could be clearly observed. Using this model we suggested that the persisting gonocytes could be responsible for the development of germ cell neoplasia in situ.

Marsupials have long attracted scientific interest because of their unique biological features and their position in mammalian evolution. Mesenchymal stem cells (MSCs) are of considerable research interest in translational medicine due to their immunomodulatory, anti-inflammatory and regenerative properties. MSCs have been harvested from various tissues in numerous eutherian species; however, there are no descriptions of MSCs derived from a marsupial. In this study, we have generated Tasmanian devil (Sarcophilus harrisii) MSCs from devil induced pluripotent stem cells (iPSCs), thus providing an unlimited source of devil MSCs and circumventing the need to harvest tissues from live animals. Devil iPSCs were differentiated into MSCs (iMSCs) through both embryoid body formation assays (EB-iMSCs) and via inhibition of the transforming growth factor beta (TGF-β)/Activin signalling pathway (SB-iMSCs). Both EB-iMSCs and SB-iMSCs are highly proliferative and express the MSC-specific surface proteins CD73, CD90 and CD105, in addition to the pluripotency transcription factors OCT4/POU5F1, SOX2 and NANOG. Expression of the marsupial pluripotency factor POU5F3, a paralogue of OCT4/POU5F1, is significantly reduced in association with the transition from pluripotency to multipotency. Devil iMSCs readily differentiate along the adipogenic, osteogenic and chondrogenic pathways in vitro, confirming their trilineage differentiation potential. Importantly, in vitro teratoma assays confirmed their multipotency, rather than pluripotency, since the iMSCs only formed derivatives of the mesodermal germ layer. Devil iMSCs show a tropism towards medium conditioned by devil facial tumour cells and express a range of immunomodulatory and anti-inflammatory factors. Therefore, devil iMSCs will be a valuable tool for further studies on marsupial biology and may facilitate the development of an MSC-based treatment strategy against Devil Facial Tumour Disease.

A recent study showned that complementary medicine is gradually gaining wide acceptance. In the present study, the herbal mixture extract (H3) composed of 3 oriental herbal plants was investigated for anticancer activity in vitro and in vivo. H3 inhibited PANC1 cell growth by promoting G0/G1 arrest (11% increase) and apoptotic cell death (9% increase). H3 also suppressed stem cell-like side population cells (4% decrease) and migration activity (24% decrease). In contrast, gemcitabine decreased side population cells and migration activity by 3 and 11%, respectively. These effects of H3 and gemcitabine were further studied by examining the expression of apoptosis-associated genes (CXCR4, JAK2 and XIAP) and stem cell-associated genes (ABCG2, POU5F1 and SOX2). We also found that H3 suppressed tumor growth by 46% in a PANC1‑xenograft model, while gemcitabine caused a 36% decrease. The antitumor effects of H3 were confirmed by western blot analysis for COX-2 and cytochrome c expression. Furthermore, necrotic cell death and erythrocyte-containing cavities were detected in tumor tissue by hematoxylin and eosin (H&E) staining. Notably, the combinatorial therapy (H3 and gemcitabine) increased tumor growth compared to that in the control. In conclusion, the present study shows that H3 has promise as a therapeutic agent against pancreatic cancer and its cancer stem cells.

The development of three-dimensional cell culture techniques has allowed cancer researchers to study the stemness properties of cancer cells in in vitro culture. However, a method to grow PAX3-FOXO1 fusion-positive rhabdomyosarcoma (FP-RMS) - an aggressive soft tissue sarcoma of childhood - has to date not been reported, hampering efforts to identify the dysregulated signaling pathways that underlie FP-RMS stemness. Here, we first examine the expression of canonical stem cell markers in human RMS tumors and cell lines. We then describe a method to grow FP-RMS cell lines as rhabdospheres and demonstrate that these spheres are enriched in expression of canonical stemness factors as well as Notch signaling components. Specifically, FP-RMS rhabdospheres have increased expression of SOX2, POU5F1 (OCT4), and NANOG, and several receptors and transcriptional regulators in the Notch signaling pathway. FP-RMS rhabdospheres also exhibit functional stemness characteristics including multipotency, increased tumorigenicity in vivo, and chemoresistance. This method provides a novel practical tool to support research into FP-RMS stemness and chemoresistance signaling mechanisms.

Keywords: Fusion-positive rhabdomyosarcoma; NANOG; NOTCH; POU5F1/OCT4; SOX2; Spheres; Stemness.

The pluripotency factor OCT4 is essential for the maintenance of naive pluripotent stem cells in vitro and in vivo. However, the specific role of OCT4 in this process remains unknown. Here, we developed a rapid protein-level OCT4 depletion system that demonstrates that the immediate downstream response to loss of OCT4 is reduced expression of key pluripotency factors. Our data show a requirement for OCT4 for the efficient transcription of several key pluripotency factors and suggest that expression of trophectoderm markers is a subsequent event. In addition, we find that NANOG is able to bind to the genome in the absence of OCT4, and this binding is in fact enhanced. Globally, however, the active enhancer-associated histone mark H3K27ac is depleted. Our work establishes that, while OCT4 is required for the maintenance of the naive transcription factor network, at a normal embryonic stem cell levels it antagonizes this network through inhibition of NANOG binding.

Keywords: Nanog; Oct4; Pou5f1; auxin-inducible degron; embryonic stem cells; mouse nPSCs.

The objective of this study was to investigate the effect of milrinone supplementation as a phosphodiesterase 3A inhibitor during in vitro maturation (IVM) to coordinate the cytoplasmic and nuclear maturation of porcine oocytes and subsequent development of porcine cloned embryos. Brilliant cresyl blue (BCB)-stained (BCB +) oocytes, classified as well-developed, and BCB- oocytes were used in parthenogenesis (PA) and cloning, and their preimplantation development was compared. In PA embryos, BCB + oocytes had significantly higher rates of development than BCB- oocytes in terms of maturation (87.5 vs. 71.3%), cleavage (88.6 vs. 76.3%), and blastocyst development (34.3 vs. 25.3%) and also had higher cell numbers (46.9 vs. 38.9%), respectively (p < 0.05). In cloned embryos, the BCB + group also had a significantly higher blastocyst formation rate than the BCB- group (30.6 vs. 20.1%; p < 0.05). Supplementation with 75 μM milrinone during IVM of BCB- oocytes showed improvement in maturation and blastocyst development rates, which may be due to the coordinated maturation of the cytoplasm with the nucleus as an effect of milrinone. Moreover, the analysis of nuclear reprogramming via the examination of the expression levels of the reprogramming-related genes POU5F1, DPPA2, and NDP52IL in milrinone-supplemented BCB- oocytes showed higher expression levels than that in non-treated BCB- oocytes. These findings demonstrate that milrinone is useful in improving developmental competence in less competent oocytes during IVM and for proper nuclear reprogramming in the production of porcine cloned embryos by coordinating cytoplasmic and nucleus maturation.

Keywords: cloning; development; meiosis; milrinone; oocyte.

Extracellular vesicles (EVs) carry bioactive cargoes involved in the early preimplantation development. This study investigated the effects of EVs obtained from an oviductal epithelial cell (OEC) conditioned medium on the developmental competence of in parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) porcine embryos. The OEC-EV-treated group showed significant increases in blastocyst formation and hatching rates compared to the control group (40.8% ± 2.2% and 20.1% ± 2.1% vs. 24.9% ± 2.0% and 5.3% ± 1.1%; p < 0.05), respectively. The 7 day OEC-EVs treatment group significantly increased blastocyst formation rate than the 3 day and 0 day-groups (45.0 ± 0.8 vs. 33.0 ± 0.7 and 26.7 ± 0.5; p < 0.05), respectively. SCNT revealed that the OEC-EV increased blastocyst formation rate compared to that of oviductal fluid EVs (OF-EVs) (35.4% ± 1.4% vs. 29.3% ± 1.3%; p < 0.05). Reactive oxygen species levels, apoptosis, and blastocyst lipid content were significantly decreased in the OEC-EVs group compared with the control group. OEC-EV group showed a significantly decreased BAX and increased BCL2, SOD1, POU5F1, SOX2, NANOG, GATA6, PNPLA2, LIPE, and MGLL gene expression than the control group (p < 0.05). In conclusion, OEC-EVs supplementation in embryo culture media improved the quality of porcine embryos, potentially helping porcine-cloned embryonic development possibly through transfer of messenger RNA and proteins to the early embryos.

Keywords: embryo; extracellular vesicles; oviduct epithelial cells; parthenogenesis; porcine; somatic cell nuclear transfer.