BACKGROUND

Three-dimensional (3D) cultures recapitulate the microenvironment of tissue-resident stem cells and enable them to modulate their properties. We determined whether salivary gland-resident stem cells (SGSCs) are primed by a 3D spheroid culture prior to treating irradiation-induced salivary hypofunction using in-vitro coculture and in-vivo transplant models.

METHODS

3D spheroid-derived SGSCs (SGSCs3D) were obtained from 3D culture in microwells consisting of a nanofiber bottom and cell-repellent hydrogel walls, and were examined for salivary stem or epithelial gene/protein expression, differentiation potential, and paracrine secretory function compared with monolayer-cultured SGSCs (SGSCs2D) in vitro and in vivo.

RESULTS

SGSCs3D expressed increased salivary stem cell markers (LGR5 and THY1) and pluripotency markers (POU5F1 and NANOG) compared with SGSCs2D. Also, SGSCs3D exhibited enhanced potential to differentiate into salivary epithelial cells upon differentiation induction and increased paracrine secretion as compared to SGSCs2D. Wnt signaling was activated by 3D spheroid formation in the microwells and suppression of the Wnt/β-catenin pathway led to reduced stemness of SGSCs3D. Enhanced radioprotective properties of SGSCs3D against radiation-induced salivary hypofunction was confirmed by an organotypic 3D coculture and in-vivo transplantation experiments.

CONCLUSIONS

The 3D spheroid culture of SGSCs in nanofibrous microwells promotes stem cell properties via activation of Wnt signaling. This may contribute to SGSC priming prior to regenerative therapy to restore salivary hypofunction after radiotherapy.

Testicular germ cell tumors (TGCTs) represent a well curable malignity due to their exceptional response to cisplatin (CDDP). Despite remarkable treatment results, approximately 5% of TGCT patients develop CDDP resistance and die. Exceptional curability makes TGCTs a highly valuable model system for studying the molecular mechanisms of CDDP sensitivity. Our study was aimed at revealing difference in gene expression between the CDDP-resistant and -sensitive TGCT cell lines, and hence at identifying candidate genes that could serve as potential biomarkers of CDDP response. Using gene expression array, we identified 281 genes that are differentially expressed in CDDP-resistant compared to -sensitive TGCT cell lines. The expression of 25 genes with the highest fold change was validated by RT-qPCR. Of them, DNMT3L, GAL, IGFBP2, IGFBP7, L1TD1, NANOG, NTF3, POU5F1, SOX2, WNT6, ZFP42, ID2, PCP4, SLC40A1 and TRIB3, displayed comparable expression change in gene expression array and RT-qPCR, when all CDDP-resistant TGCT cell lines were pairwise combined with all -sensitive ones. Products of the identified genes are pluripotency factors, or are involved in processes, such as cell metabolism, proliferation or migration. We propose that, after clinical validation, these genes could serve as prognostic biomarkers for early detection of CDDP response in TGCT patients.

Keywords: cisplatin; gene expression array; pluripotency factors; prognostic biomarkers; testicular germ cell tumors.

Transcriptional enhancers are critical for development and phenotype evolution and are often mutated in disease contexts; however, even in well-studied cell types, the sequence code conferring enhancer activity remains unknown. To examine the enhancer regulatory code for pluripotent stem cells, we identified genomic regions with conserved binding of multiple transcription factors in mouse and human embryonic stem cells (ESCs). Examination of these regions revealed that they contain on average 12.6 conserved transcription factor binding site (TFBS) sequences. Enriched TFBSs are a diverse repertoire of 70 different sequences representing the binding sequences of both known and novel ESC regulators. Using a diverse set of TFBSs from this repertoire was sufficient to construct short synthetic enhancers with activity comparable to native enhancers. Site-directed mutagenesis of conserved TFBSs in endogenous enhancers or TFBS deletion from synthetic sequences revealed a requirement for 10 or more different TFBSs. Furthermore, specific TFBSs, including the POU5F1:SOX2 comotif, are dispensable, despite cobinding the POU5F1 (also known as OCT4), SOX2, and NANOG master regulators of pluripotency. These findings reveal that a TFBS sequence diversity threshold overrides the need for optimized regulatory grammar and individual TFBSs that recruit specific master regulators.

Study question: What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model?

Summary answer: POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation.

What is known already: Clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage.

Study design, size, duration: The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain-B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos.

Participants/materials, setting, methods: A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing.

Main results and the role of chance: Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts.

Limitations, reasons for caution: One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes.

Wider implications of the findings: Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis.

Study funding/competing interest(s): The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests.

Trial registration number: N/A.

Keywords: CRISPR-Cas9; CRISPR-associated genes; POU class 5 homeobox 1; clustered regularly interspaced; genome editing; human embryo; mouse embryo; post-implantation development; pre-implantation development; short palindromic repeats.

Human spermatogonial stem cells (hSSCs) have potential in fertility preservation of prepubertal boys or in treatment of male adults suffering from meiotic arrest. Prior to therapeutic application, in vitro propagation of rare hSSCs is mandatory. As the published data points to epigenetic alterations in long-term cell culture of spermatogonia (SPG), an initial characterisation of their DNA methylation state is important. Testicular biopsies from five adult normogonadotropic patients were converted into aggregate-free cell suspensions. FGFR3-positive (FGFR3+) SPG, resembling a very early stem cell state, were labelled with magnetic beads and isolated in addition to unlabelled SPG (FGFR3-). DNA methylation was assessed by limiting dilution bisulfite pyrosequencing for paternally imprinted (H19 and MEG3), maternally imprinted (KCNQ1OT1, PEG3, and SNRPN), pluripotency (POU5F1/OCT4 and NANOG), and spermatogonial/hSSC marker (FGFR3, GFRA1, PLZF, and L1TD1) genes on either single cells or pools of 10 cells. Both spermatogonial subpopulations exhibited a methylation pattern largely equivalent to sperm, with hypomethylation of hSSC marker and maternally imprinted genes and hypermethylation of pluripotency and paternally imprinted genes. Interestingly, we detected fine differences between the two spermatogonial subpopulations, which were reflected by an inverse methylation pattern of imprinted genes, i.e. decreasing methylation in hypomethylated genes and increasing methylation in hypermethylated genes, from FGFR3+ through FGFR3- SPG to sperm. Limitations of this study are due to it not being performed on a genome-wide level and being based on previously published regulatory gene regions. However, the concordance of DNA methylation between SPG and sperm implies that hSSC regulation and germ cell differentiation do not occur at the DNA methylation level.

Recovery from ischemic tissue injury can be promoted by cell proliferation and neovascularization. Transient expression of four pluripotency factors (Pou5f1, Sox2, Myc, and Klf4) has been used to convert cell types but never been tested as a means to promote functional recovery from ischemic injury. Here we aimed to determine whether transient in situ pluripotency factor expression can improve neurobehavioral function. Cerebral ischemia was induced by transient bilateral common carotid artery occlusion, after which the four pluripotency factors were expressed through either doxycycline administration into the lateral ventricle in transgenic mice in which the four factors are expressed in a doxycycline-inducible manner. Histologic evaluation showed that this transient expression induced the proliferative generation of astrocytes and/or neural progenitors, but not neurons or glial scar, and increased neovascularization with upregulation of angiogenic factors. Furthermore, in vivo pluripotency factor expression caused neuroprotective effects such as increased numbers of mature neurons and levels of synaptic markers in the striatum. Dysplasia or tumor development was not observed. Importantly, neurobehavioral evaluations such as rotarod and ladder walking tests showed that the expression of the four factors dramatically promoted functional restoration from ischemic injury. These results provide a basis for novel therapeutic modality development for cerebral ischemia.

SummaryOxidative stress is a major cause of defective embryo development during in vitro culture. Retinoids are recognized as non-enzymatic antioxidants and may have an important role in the regulation of cell differentiation and vertebrate development. However, there are not enough reports discussing the antioxidant and developmental capacity of retinoids, including retinol (RT), on the in vitro development of embryos recovered from livestock animals, particularly in rabbit species. Therefore, morula embryos obtained from nulliparous Red Baladi rabbit does were cultured for 48 h in TCM199 medium in the absence of RT (control group) or in the presence of RT at concentrations of 10, 100 and 1000 nM. The developmental capacity to the hatched blastocyst stage, the antioxidant biomarker assay and the expression of several selected genes were analyzed in each RT group. The data show that RT significantly (P<0.001) promoted the embryo hatchability rate at the concentration of 1000 nM to 69.44% versus 29.71% for the control. The activity of malondialdehyde (MDA) level was significantly (P<0.05) lower in the RT groups than in the control group, while the total antioxidant capacity (TAC), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were significantly (P<0.05) higher following treatment with RT. Furthermore, RT treatment considerably upregulated the relative expression of gap junction protein alpha 1 (GJA1), POU class 5 homeobox 1 (POU5F1) and superoxide dismutase 1 (SOD1) genes compared with the control group. The current study highlights the potential effects of RT as antioxidant in the culture medium on the in vitro development of rabbit embryos.

Multiple myeloma (MM) is a malignancy of plasma cells usually infiltrating the bone marrow, associated with the production of a monoclonal immunoglobulin (M protein) which can be detected in the blood and/or urine. Multiple lines of evidence suggest that genetic factors are involved in MM pathogenesis, and several studies have identified single nucleotide polymorphisms (SNPs) associated with the susceptibility to the disease. SNPs within miRNA-binding sites in target genes (miRSNPs) may alter the strength of miRNA-mRNA interactions, thus deregulating protein expression. MiRSNPs are known to be associated with risk of various types of cancer, but they have never been investigated in MM. We performed an in silico genome-wide search for miRSNPs predicted to alter binding of miRNAs to their target sequences. We selected 12 miRSNPs and tested their association with MM risk. Our study population consisted of 1,832 controls and 2,894 MM cases recruited from seven European countries and Israel in the context of the IMMEnSE (International Multiple Myeloma rESEarch) consortium. In this population two SNPs showed an association with p < 0.05: rs286595 (located in gene MRLP22) and rs14191881 (located in gene TCF19). Results from IMMEnSE were meta-analyzed with data from a previously published genome-wide association study (GWAS). The SNPs rs13409 (located in the 3'UTR of the POU5F1 gene), rs1419881 (TCF19), rs1049633, rs1049623 (both in DDR1) showed significant associations with MM risk. In conclusion, we sought to identify genetic polymorphisms associated with MM risk starting from genome-wide prediction of miRSNPs. For some mirSNPs, we have shown promising associations with MM risk.

BACKGROUND

Recently, the capacity of mesenchymal stem/stromal cells (MSCs) to migrate into damaged tissues has been reported. For MSCs to be a promising tool for tissue engineering and cell and gene therapy, it is essential to know their migration ability according to their tissue of origin. However, little is known about the molecular mechanisms regulating porcine MSC chemotaxis. The aim of this study was to examine the migratory properties in an inflammatory environment of porcine MSC lines from different tissue origins: subcutaneous adipose tissue (SCA-MSCs), abdominal adipose tissue (AA-MSCs), dermal skin tissue (DS-MSCs) and peripheral blood (PB-MSCs).

METHODS

SCA-MSCs, AA-MSCs, DS-MSCs and PB-MSCs were isolated and analyzed in terms of morphological features, alkaline phosphatase activity, expression of cell surface and intracellular markers of pluripotency, proliferation, in vitro chondrogenic, osteogenic and adipogenic differentiation capacities, as well as their ability to migrate in response to inflammatory cytokines.

RESULTS

SCA-MSCs, AA-MSCs, DS-MSCs and PB-MSCs were isolated and showed plastic adhesion with a fibroblast-like morphology. All MSC lines were positive for CD44, CD105, CD90 and vimentin, characteristic markers of MSCs. The cytokeratin marker was also detected in DS-MSCs. No expression of MHCII or CD34 was detected in any of the four types of MSC. In terms of pluripotency features, all MSC lines expressed POU5F1 and showed alkaline phosphatase activity. SCA-MSCs had a higher growth rate compared to the rest of the cell lines, while the AA-MSC cell line had a longer population doubling time. All MSC lines cultured under adipogenic, chondrogenic and osteogenic conditions showed differentiation capacity to the previously mentioned mesodermal lineages. All MSC lines showed migration ability in an agarose drop assay. DS-MSCs migrated greater distances than the rest of the cell lines both in nonstimulated conditions and in the presence of the inflammatory cytokines TNF-α and IL-1β. SCA-MSCs and DS-MSCs increased their migration capacity in the presence of IL-1β as compared to PBS control.

CONCLUSIONS

This study describes the isolation and characterization of porcine cell lines from different tissue origin, with clear MSC properties. We show for the first time a comparative study of the migration capacity induced by inflammatory mediators of porcine MSCs of different tissue origin.

Nonobstructive azoospermia (NOA) is a severe form of male infertility, with limited effective treatments. Urine-derived stem cells (USCs) possess multipotent differentiation capacity and paracrine effects, and participate in tissue repair and regeneration. The aim of this study is to investigate whether the transplantation of USCs or USC exosomes (USC-exos) could promote endogenous spermatogenesis restoration in a busulfan-induced NOA mice model. USCs were cultured and characterized by flow cytometry. High-density USCs were cultured in a hollow fiber bioreactor for exosomes collection. USC-exos were isolated from USCs conditional media and identified by transmission electron microscopy, western blotting, and Flow NanoAnalyzer analysis. USC-exos exhibited sphere- or cup-shaped morphology with a mean diameter of 66.5 ± 16.0 nm, and expressed CD63 and CD9. USCs and USC-exos were transplanted into the interstitial space in the testes of NOA mice per the following groups: normal group; groups treated with no injection, phosphate-buffered saline (PBS), USCs or USC-exos on days 3 and 36 after busulfan administration, respectively. Thirty days after USCs and USC-exos transplantation, spermatogenesis was restored by both USCs and USC-exos in NOA mice 36 days after busulfan treatment as confirmed by immunofluorescence staining and hematoxylin and eosin staining. Moreover, spermatogenic genes (Pou5f1, Prm1, SYCP3, and DAZL) and the spermatogenic protein UCHL1 were significantly increased in both the USCs 36 and USC-exos36 groups compared with the PBS group, as demonstrated using quantitative real-time polymerase chain reaction and western blot analysis. However, the transplantation of USCs or USC-exos at day 3 after busulfan treatment did not improve spermatogenesis in NOA mice. Our study demonstrated that USCs could facilitate endogenous spermatogenesis restoration of busulfan-induced NOA mice through paracrine exosomes but could not protect the mouse testicles at the early stage of destruction caused by busulfan. This study provides a novel insight into the treatment of NOA.

Buffalo is an economically important livestock species in Asia. Little is known about male germ line technology owing to lack of sufficient understanding regarding expression of germ- and somatic-cell specific-proteins in the testis. In this study, we identified UCHL-1 (PGP 9.5) and lectin- Dolichos biflorus agglutinin (DBA) as specific markers for spermatogonia in buffalo testis. Expression of germ-cell and pluripotency-specific proteins such as DDX4 (VASA) and POU5F1 (OCT3/4) were also present in spermatogonia. Interestingly, the expression of somatic cell-specific proteins such as VIMENTIN and GATA4 were also detected in germ cells. Using two-step enzymatic digestion followed by differential plating and Percoll density-gradient centrifugation, an approximately 55% spermatogonia-enriched cell population could be obtained from the prepubertal buffalo testis. Isolated spermatogonia could survive and proliferate in vitro in DMEM/F12 medium containing 10% fetal bovine serum in the absence of any specific growth factors for a week. Cultured spermatogonia showed DBA affinity and expressed DDX4 and POU5F1. These results may help to establish a long-term culture system for buffalo spermatogonia.

Thirteen independent induced bovine trophectroderm (iBT) cell lines were established by reprogramming bovine fetal liver-derived fibroblasts after viral-vector transduction with either six or eight factors, including POU5F1 (OCT4), KLF4, SOX2, MYC, NANOG, LIN28, SV40 large T antigen, and hTERT. Light- and electron-microscopy analysis showed that the iBT cells had epithelial cell morphology typical of bovine trophectoderm cells. Reverse-transcription-PCR assays indicated that all of the cell lines expressed interferon-tau (IFNT) at passages 1 or 2. At later passages (≥ passage 8), however, immunoblot and antiviral activity assays revealed that more than half of the iBT cell lines had stopped expressing IFNT. Messenger RNAs specific to trophectoderm differentiation and function were found in the iBT cell lines, and 2-dimensional-gel analysis for cellular proteins showed an expression pattern similar to that of trophectoderm cell lines derived from bovine blastocysts. Integration of some of the human reprogramming factors, including POU5F1, KLF4, SOX2, MYC, NANOG, and LIN28, were detected by PCR, but their transcription was mostly absent in the iBT cell lines. Gene expression assessment of endogenous bovine reprogramming factor orthologs revealed endogenous bLIN28 and bMYC transcripts in all; bSOX2 and bNANOG in none; and bKLF4 and bPOU5F1 in less than half of the iBT cell lines. These results demonstrate that bovine trophectoderm can be induced via reprogramming factor expression from bovine liver-derived fibroblasts, although other fibroblast populations-e.g., derived from fetal thigh tissue-may produce similar results, albeit at lower frequencies.

It is essential to enhance the in vitro maturation (IVM) condition for immature oocytes after cryopreservation, particularly if limited numbers of oocytes collected from specific donors. The objective of this study was to determine if quality of vitrified porcine immature oocytes was enhanced by coculturing with fresh oocytes during IVM. To distinguish fresh versus vitrified oocytes, we used two types of coculture systems: (a) transwell two-chamber coculture; (b) labeling and tracing fresh oocytes with CellTracker™ Green CMFDA during conventional culture. Coculture systems significantly accelerated meiotic progression of vitrified oocytes and significantly increased blastocyst formation rates following parthenogenetic activation and somatic cell nuclear transfer. Reactive oxygen species generation in vitrified oocytes was ameliorated by the coculture conditions, with no significant difference between fresh and vitrified oocytes for intracellular glutathione level. Both coculture systems significantly increased rate of normal mitochondrial distribution in vitrified oocytes, but did not affect fluorescence intensity of mitochondria. The percentage of oocytes with normal endoplasmic reticulum (ER) distribution and ER fluorescence intensity were significantly higher in vitrified oocytes cocultured with fresh oocytes. After 20 hr of IVM, mRNA expression of COX2, HAS2, PTX3, and TNFAIP6 remained significantly higher in cumulus cells derived from vitrified oocytes and coculture systems significantly decreased the expression of these genes. Additionally, coculture methods prevented the reduction of mRNA expression for BMP15, ZAR1, POU5F1, and DNMT3A in vitrified oocytes. In conclusion, oocyte quality and subsequent embryo development of vitrified porcine immature oocytes were significantly improved by fresh oocyte coculture during IVM.

Direct generation of skeletal muscle cells from human pluripotent stem cells (hPSCs) would be beneficial for drug testing, drug discovery, and disease modelling in vitro. Here we show a rapid and robust method to induce myogenic differentiation of hPSCs by introducing mRNA encoding MYOD1 together with siRNA-mediated knockdown of POU5F1 (also known as OCT4 or OCT3/4). This integration-free approach generates functional skeletal myotubes with sarcomere-like structure and a fusion capacity in several days. The POU5F1 silencing facilitates MYOD1 recruitment to the target promoters, which results in the significant activation of myogenic genes in hPSCs. Furthermore, deep sequencing transcriptome analyses demonstrated that POU5F1-knockdown upregulates the genes associated with IGF- and FGF-signaling and extracellular matrix that may also support myogenic differentiation. This rapid and direct differentiation method may have potential applications in regenerative medicine and disease therapeutics for muscle disorders such as muscular dystrophy.

As one of the most powerful natural antioxidants, astaxanthin (Ax) has begun to be applied to the field of reproductive biology. Here we used porcine oocyte as a model to explore how Ax improves the oocyte potential during in vitro maturation (IVM), and we also investigated the cytoprotective effects of Ax on the vitrified oocytes. Ax supplementation (final concentration of 2.5 μM) was subjected for immature oocytes during vitrification and subsequent IVM; fresh oocytes were also matured in vitro in the presence or absence of 2.5 μM Ax. Our results showed that Ax significantly increased the survival rate of vitrified oocytes, and promoted the blastocyst yield of both fresh and vitrified oocytes after parthenogenetic activation and somatic cell nuclear transfer. The oocytes treated with Ax displayed significantly lower reactive oxygen species generation and higher glutathione level. Vitrification of oocytes had no impact on caspase-3, cathepsin B and autophagic activities; Ax significantly decreased the cathepsin B activity in both fresh and vitrified oocytes. Moreover, the relative fluorescence intensity of lysosomes was significantly increased in vitrified oocytes, which was recovered by Ax treatment. The mitochondrial activity did not differ between fresh and vitrified oocytes, and was significantly enhanced in Ax-treated oocytes. Furthermore, Ax significantly restored the decreased expression of BMP15, ZAR1, POU5F1, GPX4 and LAMP2 genes in vitrified oocytes. Both fresh and vitrified oocytes treated with Ax showed significantly higher mRNA levels of GDF9, POU5F1, SOD2, NRF2 and ATG5. Taken together, this study provides new perspectives in understanding the mechanisms by which Ax improves the developmental competence of both fresh and vitrified porcine oocytes.

Keywords: Astaxanthin; Embryo development; In vitro maturation; Oocyte quality; Porcine oocyte; Vitrification.

Drug resistance remains a serious issue of clinical importance and is a consequence of cancer stemness. In this study, we showed that the level of Aldolase A (ALDOA) expression is significantly associated with the IC50 value of chemotherapy drugs in lung cancer. Our data revealed that ALDOA overexpression resulted in a significant increase of lung tumor spheres. The use of ingenuity pathway analysis (IPA) resulted in the identification of POU5F1 (Oct4) as the leading transcription factor of ALDOA. We observed high expression of ALDOA, Oct4 and stemness markers in collected spheroid cells. DUSP4 and TRAF4 were confirmed as major downstream targets of the ALDOA-Oct4 axis. Knockdown of these molecules significantly decreased the stemness ability of cells. In addition, we investigated whether miR-145 targets the 3'-UTR of Oct4 and is regulated by ALDOA due to the involvement of ALDOA in glycolysis and metabolic reprogramming. Furthermore, we constructed several mutant forms of ALDOA that disrupted its enzymatic activity and showed that they still induced significant in vitro sphere formation and in vivo tumorigenicity. These results demonstrated that ALDOA-mediated spheroid formation is independent of its enzymatic activity. In the clinical component, we also showed that the combination of ALDOA and TRAF4 or DUSP4 is positively correlated with poor overall survival in a xenograft model and cancer patients through immunohistochemical analyses. The results of our study revealed novel functional roles of ALDOA in inducing cancer stemness via the inhibition of miR-145 expression and the activation of Oct4 transcription. These findings offer new therapeutic strategies for modulation of lung cancer stemness to enhance chemotherapeutic responses in lung cancer patients.

Somatic cell nuclear transfer in mammalian cloning suffers from a faulty epigenetic reprogramming, which is believed to cause developmental failures in cloned embryos. Regulating the epigenetic-modifying enzymes can rescue the chromatin of cloned embryos from aberrant epigenetic status, thereby potentially promoting cloning efficiency. In this study, we investigated the effect of two histone methyltransferase inhibitors, namely, DZNep and UNC0642, on the in vitro developmental competence of cloned pig embryos. We found that (1) treatment with 10 nM DZNep or 5 nM UNC0642 for 24 h after activation had the best promoting effect on the development of cloned embryos (blastocyst rate 10.32% vs 18.08% for DZNep, and 10.44% vs 18.14% for UNC0642); (2) 10 nM DZNep and 5 nM UNC0642 significantly decreased the levels of H3K27me3 and H3K9me2, respectively, at the 2-cell, 4-cell and blastocyst stages; (3) the apoptosis level was lower in the treatment groups than in untreated control; and (4) the transcriptional expression of epigenetic genes (EZH2, GLP, G9a, Setdb1, Setdb2, Suv39h1 and Suv39h2) was decreased and pluripotency genes (Nanog, Pou5f1, Sox2 and Bmp4) was increased in treatment groups compared with control. These results indicated that treatment with DZNep and UNC0642 improves the epigenetic reprogramming of cloned embryos, which could render beneficial effect on the embryo quality and aberrant gene expression, and finally improve the developmental competence of cloned pig embryos.

Human amniotic fluid stem cells (hAFSCs) have features intermediate between embryonic and adult SCs, can differentiate into lineages of all three germ layers, and do not develop into tumors in vivo. Moreover, hAFSCs can be easily obtained in routine procedures and there is no ethical or legal limitations regarding their use for clinical and experimental applications. The aim of this study was to assess the effect of slow freezing/thawing and two different concentrations of DMSO (10% DMSO + 90% fetal bovine serum [FBS] and 5% DMSO + 95% FBS) on the survival of hAFSCs. hAFSCs were obtained from 5 pregnant women during amniocentesis at 16-22 weeks of gestation. The expression of pluripotency markers (Octamer-binding transcription factor 4 [Oct4] and NANOG) by reverse transcription polymerase chain reaction and cell surface markers (cluster of differentiation [CD31], CD44, CD45, and CD90) by flow cytometry was analyzed before and after the slow-freezing. Cell viability was assessed by trypan blue exclusion or MTT assay. Quantitative mRNA expression of Oct4, NANOG, cyclin D1 and p21 was determined by real-time PCR before and after the slow-freezing. Pluripotency of hAFSCs was confirmed by NANOG and POU5F1 (Oct4) gene expression before and after slow-freezing. All hAFSC cultures were positive for CD44 and CD90. A higher viability of hAFSCs was observed after freezing with 90% FBS + 10% DMSO. There was increased expression of NANOG and decreased expression of POU5F1 gene after freezing, compared to control cells (before freezing). DMSO and the process of freezing did not significantly change the expression of p21 and cyclin D1 genes in hAFSCs. Overall, our results indicate the applicability of slow-freezing and DMSO in cryopreservation of SCs.

Despite significant recent advances in characterizing the molecular pathogenesis of undifferentiated round cell neoplasms, rare cases remain unclassified. Here, we report two distinctive undifferentiated round cell tumors occurring in young adults. One tumor presented intrabdominally and the other arose within the abdominal wall. One patient died of disease following local and distance recurrence, despite aggressive chemotherapy and radiotherapy. Morphologically, both tumors were similarly composed of primitive round to epithelioid cells arranged in nests, sheets, and trabecular patterns. The cytoplasm was scant and amphophilic, while the nuclei were round and uniform with brisk mitotic activity. Focal necrosis was present. Immunohistochemically, both tumors were variably positive for S100 and EMA, and one case focally expressed cytokeratin and TLE1. Targeted RNA sequencing revealed in both an identical SS18-POU5F1 fusion gene. Fluorescence in situ hybridization was performed which confirmed SS18 and POU5F1 gene rearrangements. Expression data, relative to over 200 other mesenchymal neoplasms that had undergone targeted RNA sequencing on the same platform, suggested the SS18-POU5F1 tumors cluster with EWSR1/FUS-POU5F1-positive myoepithelial tumors. In view of our limited sample size, additional studies are needed to characterize the breadth of clinical and pathologic findings in these neoplasms. In addition, further investigation is necessary to determine whether this entity represents a clinically aggressive and phenotypically undifferentiated variant of myoepithelial tumors, or perhaps an altogether novel category of undifferentiated round cell sarcoma.

Keywords: POU5F1; SS18; fusion; myoepithelial tumor; sarcoma; synovial sarcoma.

Background: Current studies have enlightened the rosy prospects of human pluripotent stem cell (hPSC)-derived mesenchymal stem/stromal cells (MSCs) in regenerative medicine. However, systematic investigation of their signatures and applications with alternative biomaterials in osteoarthritis (OA) remains indistinct.

Methods: Herein, we initially took advantage of a small molecule library-mediated programming strategy for hPSC-MSC induction. Then, with the aid of multifaceted analyses such as flow cytometry (FCM), chromosome karyocyte and cell vitality, wound healing and microtubule formation assay and coculturing with T lymphocytes, we systematically evaluated the characterizations of signatures in vitro and the in vivo efficacy of hPSC-MSCs and HA hydrogel composite on rabbit osteoarthritis model.

Results: We found the combination of LLY-507 and AZD5153 was sufficient for high-efficiency CD73⁺CD90⁺CD105⁺CD31^-CD34^-CD45^-HLA-DR^- MSC induction from both hESCs and hiPSCs with stemness (POU5F1/SOX2/NANOG). The programmed hPSC-MSCs revealed conservative transcriptome variations and went through a heterogeneous intermediate-stage with mesenchymal-associated gene expression (NT5E, ENG, VIM and FN1) as well as displayed typical cytomorphology, immunophenotypes and normal karyotyping, multilineage differentiation potential, favorable cell vitality, proangiogenic and immunoregulatory properties in vitro. Meanwhile, the cell population exhibited preferable restorative and ameliorative function on OA rabbits with HA hydrogel in vivo.

Conclusions: Collectively, we established a rapid and convenient procedure for hPSC-MSC generation without redundant manipulations. The fundamental and clinical studies upon osteoarthritis (OA) treatment would benefit tremendously from the combination of the inexhaustible hPSC-MSCs and advantageous biomaterials.

Keywords: Genetic variation; HA hydrogel; Immunoregulation; Osteoarthritis; Programming; hPSC-MSCs.

Cognitive decline is among the most feared aspects of ageing. We have generated induced pluripotent stem cells (iPSCs) from 24 people from the Lothian Birth Cohort 1936, whose cognitive ability was tested in childhood and in older age. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using non-integrating oriP/EBNA1 backbone plasmids expressing six iPSC reprogramming factors (OCT3/4 (POU5F1), SOX2, KLF4, L-Myc, shp53, Lin28, SV40LT). All lines demonstrated STR matched karyotype and pluripotency was validated by multiple methods. These iPSC lines are a valuable resource to study molecular mechanisms underlying individual differences in cognitive ageing and resilience to age-related neurodegenerative diseases.

Oocyte vitrification preserves the female genetic resources of elite dromedary camels. In the current study, we aimed to explore the effects of vitrification of camel oocytes on mitochondrial activity, redox stress, and expression of genes related to mitochondrial function, apoptosis, pluripotency, and cytoskeleton. Moreover, we investigated developmental competence of vitrified oocytes after parthenogenetic activation. Oocytes vitrified with the Cryotop method were compared with the fresh oocytes. Our results showed that vitrification led to increased ROS production in oocytes as evidenced by an increase in the DCFDHA fluorescence intensity, and lower mitochondrial activity. At the molecular level, vitrification reduced mRNA expression of many genes, including those related to mitochondrial function (TFAM, MT-CO1, MFN1, ATP1A1, NRF1), pluripotency (SOX2 and POU5F1), and apoptosis (p53 and BAX). In contrast, expression of KLF4 and cytoskeleton-related genes (ACTB and KRT8) was not affected. However, we found no difference in the rates of oocyte survival, cleavage, and blastocyst development, and blastocyst hatching between fresh and vitrified oocytes after warming. Our results indicate that although vitrification of camel metaphase II (MII) oocytes adversely affected mitochondrial functions, the effect was transient without compromising the developmental potential of the oocytes after parthenogenetic activation.

Keywords: Camel; Mitochondria; Oocytes; Reactive oxygen species; Real-time polymerase chain reaction; Vitrification.

In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulation. Here we report the successful production of rabbit embryonic stem cells (ESCs) from oocytes produced by in vitro culture of immature follicles and subsequent in vitro maturation treatment. In total, we obtained 53 blastocysts from oocytes that received intracytoplasmic sperm injection followed by in vitro culture. Although only weak expression of POU5f1 was observed in the inner cell masses of in-vitro-cultured follicle-derived embryos, repeated careful cloning enabled establishment of 3 stable ESC lines. These ESC lines displayed the morphological characteristics of primed pluripotent stem cells. The ESC lines also expressed the pluripotent markers Nanog, POU5f1, and Sox2. Further, these ESCs could be differentiated into each of the 3 different germ layers both in vitro and in vivo. These results demonstrate that immature follicles from rabbits can be used to generate ESCs. Moreover, the use of rabbit oocytes as a cell source provides an experimental system that closely matches human reproductive and stem cell physiology.