Embryoid Bodies and Related Proliferations in Ovarian Germ Cell Tumors.

We investigated the frequency and associated pathology of embryoid bodies in ovarian tumors by evaluating neoplasms in which they are known to occur: 100 immature teratomas, 125 malignant mixed germ cell tumors, and 6 polyembryomas. Three immature teratomas contained a single relatively well-formed embryoid body, whereas these and 11 others showed foci we categorized as embryoid body remnants consisting of microscopic aggregates of embryonal or yolk sac-type epithelium associated with spaces consistent with yolk sac or amniotic cavity but lacking a classic embryoid body structure. Teratomas with these foci were all high grade. A well-formed embryoid body was found in only 1 malignant mixed tumor, but embryoid body remnants were present in 25%, invariably associated with foci of immature teratoma (100%) and often with yolk sac tumor (97%), embryonal carcinoma (35%), or both (32%). These foci usually took the form of round to oval aggregates, often well-circumscribed, for which the term "polyembryoma background" has been proposed. The polyembryomas were typically grossly hemorrhagic and occurred in patients from 9 to 43 years of age. The embryoid bodies in them generally grew in lobules within an edematous to occasionally myxoid stroma. Four tumors contained liver-like cells, 4 numerous glands likely recapitulating the allantois, 3 syncytiotrophoblast cells, 2 prominent cysts, and 2 striking vascular proliferations. This study indicates that (1) typical embryoid bodies are rare in immature teratomas but about 14% of them have embryoid body remnants. (2) Embryoid body remnants are seen in 25% of malignant mixed germ cell tumors with a teratomatous component and often proliferate to form yolk sac tumor and embryonal carcinoma. (3) Well-formed embryoid bodies growing in a confluent manner (polyembryoma) are rare, and minor foci of teratoma, yolk sac tumor, or embryonal carcinoma are almost always present, indicating that these are fundamentally malignant mixed germ cell tumors but the polyembryoma component is dominant and distinctive which, in our opinion, justifies its own nomenclature. (4) Embryoid bodies are not a feature of other germ cell tumors.

Ovarian Malignant Mixed Germ Cell Tumor With Prominent Embryoid Bodies (Polyembryoma Background): A Case Report and Literature Review.

Ovarian malignant mixed germ cell tumors are rare tumors occurring in young women. The presence of prominent embryoid bodies in these tumors is extremely uncommon. Herein, we report such a case, with a histomorphologic description and immunohistochemical and fluorescence in situ hybridization analyses.

MicroRNAs in Differentiation of Embryoid Bodies and the Teratoma Subtype of Testicular Cancer.

BACKGROUND: Testicular germ cell tumours (TGCTs) are the most frequent tumour type among young, adult men. TGCTs can be efficiently treated, but metastases of the teratoma subtype, for which there are no circulating biomarkers, represent a challenge. MATERIALS AND METHODS: Global microRNA expression in teratoma tissue and embryoid bodies was assessed using next-generation sequencing. Levels of microRNAs identified as potential biomarkers were obtained from serum of patients with teratoma and matched healthy men. RESULTS: We identified miR-222-5p, miR-200a-5p, miR-196b-3p and miR-454-5p as biomarker candidates from the tumour tissue and embryoid body screening but the expression of these microRNAs was very low in serum and not statistically different between patients and controls. miR-375-3p was highly expressed, being highest in patients with teratoma (p=0.012) but the levels of expression in serum from these patients and healthy controls overlapped. miR-371a-3p was not expressed in serum from patients with pure teratoma, only in patients with mixed tumours. CONCLUSION: The microRNA profiles of the teratoma subtype of TGCT and embryoid bodies were obtained and assessed for candidate circulating biomarkers, but none with high sensitivity and specificity for teratoma were identified in our study. We conclude that neither the proposed teratoma marker miR-375-3p nor miR-371a-3p are suitable as circulating teratoma markers.

Characterization of in vitro 3D cultures.

Over the past decade, 3D culture models of human and animal cells have found their way into tissue differentiation, drug development, personalized medicine and tumour behaviour studies. Embryoid bodies (EBs) are in vitro 3D cultures established from murine pluripotential stem cells, whereas tumoroids are patient-derived in vitro 3D cultures. This thesis aims to describe a new implication of an embryoid body model and to characterize the patient-specific microenvironment of the parental tumour in relation to tumoroid growth rate. In this thesis, we described a high-throughput monitoring method, where EBs are used as a dynamic angiogenesis model. In this model, digital image analysis (DIA) is implemented on immunohistochemistry (IHC) stained sections of the cultures over time. Furthermore, we have investigated the correlation between the genetic profile and inflammatory microenvironment of parental tumours on the in vitro growth rate of tumoroids. The EBs were cultured in spinner flasks. The samples were collected at days 4, 6, 9, 14, 18 and 21, dehydrated and embedded in paraffin. The histological sections were IHC stained for the endothelial marker CD31 and digitally scanned. The virtual whole-image slides were digitally analysed by Visiopharm® software. Histological evaluation showed vascular-like structures over time. The quantitative DIA was plausible to monitor significant increase in the total area of the EBs and an increase in endothelial differentiation. The tumoroids were established from 32 colorectal adenocarcinomas. The in vitro growth rate of the tumoroids was followed by automated microscopy over an 11-day period. The parental tumours were analysed by next-generation sequencing for KRAS, TP53, PIK3CA, SMAD4, MAP2K1, BRAF, FGFR3 and FBXW7 status. The tumoroids established from KRAS-mutated parental tumours showed a significantly higher growth rate compared to their wild-type counterparts. The density of CD3+ T lymphocytes and CD68+ macrophages was calculated in the centre of the tumours and at the invasive margin of the tumours. The high density of CD3+ cells and the low density of CD68+ cells showed a significant correlation with a higher growth rate of the tumoroids. In conclusion, a novel approach for histological monitoring of endothelial differentiation is presented in the stem cell-derived EBs. Furthermore, the KRAS status and density of CD3+ T cells and macrophages in the parental tumour influence the growth rate of the tumoroids. Our results indicate that these parameters should be included when tumoroids are to be implemented in personalized medicine.

Histone demethylase Kdm2a regulates germ cell genes and endogenous retroviruses in embryonic stem cells.

Aim: To investigate the function of Kdm2a in embryonic stem cells (ESCs). Materials & methods: Expression profile analysis after Kdm2a knockout. Analysis of Kdm2a, H3K4me3 and H3K27me3 ChIP-seq data in ESCs. qPCR analysis and ChIP-qPCR analysis of epigenetic changes after Kdm2a loss. Results:Kdm2a was dispensable for pluripotency maintenance in ESCs. Kdm2a genomic binding profile was positively correlated with that of H3K4me3, Zfx and Tet1. Kdm2a directly regulated germ cell genes in primordial germ cell-like cells. Kdm2a loss led to the reduced expression of endogenous retrovirus IAPEy and resulted in the gain of H3K36me2 and loss of H3K4me3 on IAPEy. Conclusion: Kdm2a regulates germ cell genes and endogenous retroviruses in ESCs possibly through demethylating H3K36me2 and influencing H3K4me3 deposition.

The biocompatibility of polyaniline and polypyrrole: A comparative study of their cytotoxicity, embryotoxicity and impurity profile.

Conducting polymers (CP), namely polyaniline (PANI) and polypyrrole (PPy), are promising materials applicable for the use as biointerfaces as they intrinsically combine electronic and ionic conductivity. Although a number of works have employed PANI or PPy in the preparation of copolymers, composites, and blends with other polymers, there is no systematic study dealing with the comparison of their fundamental biological properties. The present study, therefore, compares the biocompatibility of PANI and PPy in terms of cytotoxicity (using NIH/3T3 fibroblasts and embryonic stem cells) and embryotoxicity (their impact on erythropoiesis and cardiomyogenesis within embryonic bodies). The novelty of the study lies not only in the fact that embryotoxicity is presented for the first time for both studied polymers, but also in the elimination of inter-laboratory variations within the testing, such variation making the comparison of previously published works difficult. The results clearly show that there is a bigger difference between the biocompatibility of the respective polymers in their salt and base forms than between PANI and PPy as such. PANI and PPy can, therefore, be similarly applied in biomedicine when solely their biological properties are considered. Impurity content detected by mass spectroscopy is presented. These results can change the generally accepted opinion of the scientific community on better biocompatibility of PPy in comparison with PANI.

Critical roles of hMAGEA2 in induced pluripotent stem cell pluripotency, proliferation, and differentiation.

Induced pluripotent stem (iPS) cells are important for clinical application and stem cell research. Although human melanoma-associated antigen A2 (hMAGEA2) expression is known to affect differentiation in embryonic stem cells, its specific role in iPS cells remains unclear. To evaluate the function of hMAGEA2 and its characteristics in iPS cells, we produced hMAGEA2-overexpressing iPS cells from hMAGEA2-overexpressing transgenic mice. Although the iPS cells with overexpressed hMAGEA2 did not differ in morphology, their pluripotency, and self-renewal related genes (Nanog, Oct3/4, Sox2, and Stat3), expression level was significantly upregulated. Moreover, hMAGEA2 contributed to the promotion of cell cycle progression, thereby accelerating cell proliferation. Through embryoid body formation in vitro and teratoma formation in vivo, we demonstrated that hMAGEA2 critically decreases the differentiation ability of iPS cells. These data indicate that hMAGEA2 intensifies the self-renewal, pluripotency, and degree of proliferation of iPS cells, while significantly repressing their differentiation efficiency. Therefore, our findings prove that hMAGEA2 plays key roles in iPS cells.

A feeder- and xeno-free human induced pluripotent stem cell line obtained from primary human dermal fibroblasts with epigenetic repression of reprogramming factors expression: GPCCi001-A.

The primary human dermal fibroblasts (PHDFs) from breast cancer patient were obtained to generate the human induced pluripotent stem cell line GPCCi001-A via lentiviral transfection. Thus, a modified EF1a-hSTEMCCA-loxP with tetO operator which regulates transgene expression was used. This method takes advantage of epigenetic regulation of transcription and allows for stable silencing of the reprogramming factors in obtained hiPS cells. To increase the potential utility of hiPSCs for clinical applications, they were adapted to feeder- and xeno-free conditions. The pluripotency of GPCCi001-A cell line and ability to differentiate into three germ layers was confirmed.

Derivation of the human induced pluripotent stem cell line MUi017-A from a patient with homozygous Hemoglobin Constant Spring.

Hemoglobin Constant Spring (HbCS, HBA2: c.427T>C) is a common nondeletional α-thalassemia resulting from a nucleotide substitution at the termination codon of the HBA2 gene. Homozygosity for HbCS is characterized with mild anemia, jaundice, and splenomegaly. In this study, the human induced pluripotent stem cell line MUi017-A was successfully generated from peripheral blood CD34+ hematopoietic progenitors of a 52year old female with homozygous HbCS. The MUi017-A cell line exhibited embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of differentiating into the three germ layers. The cell line may be used for the disease modeling.

Generation of induced pluripotent stem cells from peripheral blood CD34+ hematopoietic progenitors of a 31year old healthy woman.

The MUi019-A human induced pluripotent stem cell line was generated from peripheral blood CD34+ hematopoietic progenitors of a healthy woman using a non-integrative reprogramming method. Episomal vectors carrying reprogramming factors OCT4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 and EBNA-1 were delivered using electroporation. The iPSC line can be used as a control in studying disease mechanisms. Furthermore, gene editing approaches can be used to introduce specific mutations into the MUi019-A to model disease while the cell type affected by the disease is inaccessible.

Generation of human iPSCs from an essential thrombocythemia patient carrying a V501L mutation in the MPL gene.

Activating point mutations in the MPL gene encoding the thrombopoietin receptor are found in 3%-10% of essential thrombocythemia (ET) and myelofibrosis patients. Here, we report the derivation of induced pluripotent stem cells (iPSCs) from an ET patient with a heterozygous MPL V501L mutation. Peripheral blood CD34+ progenitor cells were reprogrammed by transient plasmid expression of OCT4, SOX2, KLF4, c-MYC plus BCL2L1 (BCL-xL) genes. The derived line M494 carries a MPL V501L mutation, displays typical iPSC morphology and characteristics, are pluripotent and karyotypically normal. Upon differentiation, the iPSCs are able to differentiate into cells derived from three germ layers.

Generation of non-integrated induced pluripotent stem cells from a 59-year-old female with multiple endocrine neoplasia type 1 syndrome.

Urine resource cells were collected from a 59-year-old female patient with multiple endocrine neoplasia type 1 syndrome (MEN1) for generating iPS cells with episomal plasmids carrying Oct4, Sox2, Klf4 and miR-302-367. The patient sustained a heterozygous G>T transition mutation on the exon 9 of Men1 gene that was confirmed by sequencing analysis on the obtained iPSC lines. Karyotyping indicated the chromosomes with normal appearances and numbers. Their pluripotency was demonstrated by gene expression, as well as their abilities for differentiating into three germ layers. This cell line provides an ideal model for studying MEN1.

Creating a patient carried Men1 gene point mutation on wild type iPSCs locus mediated by CRISPR/Cas9 and ssODN.

A patient specific point mutation (c.1288G>T) of Men1 gene was introduced into wide type iPSC line with CRISPR/Cas9 and single-stranded donor oligonucleotides carrying the mutation. The mutated iPSC line has a heterozygous c.1288G>T mutation on exon-9 of Men1 that was confirmed by sequencing analysis. The karyotype of this line was normal and the pluripotency was demonstrated by its ability to differentiate into three germ layers. These artificially created Men1 mutation in wild type iPSC line will help to dissect out the molecular basis of two patients carried the same mutation from one family who were differentially represented hypoglycemia.

Generation of non-integrated induced pluripotent stem cells from a 23-year-old male with multiple endocrine neoplasia type 1 syndrome.

Urine resource cells were collected from a 23-year-old male with multiple endocrine neoplasia type 1 syndrome (MEN1) for generating iPS cells with episomal plasmids. Two stable iPSC lines with free of episomal plasmid were established. The patient has a heterozygous G>T mutation on the exon 9 of Men1 gene that was confirmed by sequencing analysis on all resulted cell lines. Karyotyping indicated the chromosomes with normal appearances and numbers. Their pluripotency was demonstrated by gene expression and their abilities for differentiating into three germ layers. These iPSC lines provide valuable in vitro resources for pathological study on MEN1 syndrome.

SMYD5 regulates H4K20me3-marked heterochromatin to safeguard ES cell self-renewal and prevent spurious differentiation.

BACKGROUND: Epigenetic regulation of chromatin states is thought to control the self-renewal and differentiation of embryonic stem (ES) cells. However, the roles of repressive histone modifications such as trimethylated histone 4 lysine 20 (H4K20me3) in pluripotency and development are largely unknown. RESULTS: Here, we show that the histone lysine methyltransferase SMYD5 mediates H4K20me3 at heterochromatin regions. Depletion of SMYD5 leads to compromised self-renewal, including dysregulated expression of OCT4 targets, and perturbed differentiation. SMYD5-bound regions are enriched with repetitive DNA elements. Knockdown of SMYD5 results in a global decrease of H4K20me3 levels, a redistribution of heterochromatin constituents including H3K9me3/2, G9a, and HP1α, and de-repression of endogenous retroelements. A loss of SMYD5-dependent silencing of heterochromatin nearby genic regions leads to upregulated expression of lineage-specific genes, thus contributing to the decreased self-renewal and perturbed differentiation of SMYD5-depleted ES cells. CONCLUSIONS: Altogether, these findings implicate a role for SMYD5 in regulating ES cell self-renewal and H4K20me3-marked heterochromatin.

Integrin and dystroglycan compensate each other to mediate laminin-dependent basement membrane assembly and epiblast polarization.

During early embryogenesis, endodermal γ1-laminin expression is required for basement membrane (BM) assembly, promoting conversion of non-polar pluripotent cells into polarized epiblast. The influence of laminin-111 (Lm111) and its integrin and dystroglycan (DG) receptors on epiblast in embryoid bodies (EBs), a model for differentiation of the embryonic plate, was further investigated. Lm111 added to the medium of EBs initiated conversion of inner nonpolar cell to the polarized epiblast epithelium with an exterior-to-central basal-to-apical orientation. Microinjection of Lm111 into EB interiors resulted in an interior BM with complete inversion of cell polarity. Lm111 assembled a BM on integrin-ß1 null EBs with induction of polarization at reduced efficiency. ß-Integrin compensation was not detected in these nulls with integrin adaptor proteins failing to assemble. A dimer of laminin LG domains 4-5 (LZE3) engineered to strongly bind to α-dystroglycan almost completely inhibited laminin accumulation on integrin ß1-null EBs, reducing BM and ablating cell polarization. When Lm111 was incubated with integrin-ß1/dystroglycan double-knockout EBs, laminin failed to accumulate on the EBs, the EBs did not differentiate, and the EBs underwent apoptosis. Collectively the findings support the hypotheses that the locus of laminin cell surface assembly can determine the axis of epithelial polarity. This requires integrin- and/or dystroglycan-dependent binding to laminin LG domains with the highest efficiency achieved when both receptors are present. Finally, EBs that cannot assemble a matrix undergo apoptosis.

A human VE-cadherin-tdTomato and CD43-green fluorescent protein dual reporter cell line for study endothelial to hematopoietic transition.

Human embryonic stem cell line WA01 was genetically modified using zinc-finger nucleases and the PiggyBac/transponson system to introduce a fluorescence reporter for VE-cadherin (VEC; tdTomato) and CD43 (eGFP). Phenotypic and functional assays for pluripotency revealed the modified hES cell reporter lines remained normal. When the cells were differentiated into hematoendothelial lineages, either by directed differentiation or direct reprogramming, flow cytometric and fluorescence microscopy showed that VEC+ endothelial cells express tdTomato and CD43+ hematopoietic progenitors express eGFP.

Regulation of WNT Signaling by VSX2 During Optic Vesicle Patterning in Human Induced Pluripotent Stem Cells.

Few gene targets of Visual System Homeobox 2 (VSX2) have been identified despite its broad and critical role in the maintenance of neural retina (NR) fate during early retinogenesis. We performed VSX2 ChIP-seq and ChIP-PCR assays on early stage optic vesicle-like structures (OVs) derived from human iPS cells (hiPSCs), which highlighted WNT pathway genes as direct regulatory targets of VSX2. Examination of early NR patterning in hiPSC-OVs from a patient with a functional null mutation in VSX2 revealed mis-expression and upregulation of WNT pathway components and retinal pigmented epithelium (RPE) markers in comparison to control hiPSC-OVs. Furthermore, pharmacological inhibition of WNT signaling rescued the early mutant phenotype, whereas augmentation of WNT signaling in control hiPSC-OVs phenocopied the mutant. These findings reveal an important role for VSX2 as a regulator of WNT signaling and suggest that VSX2 may act to maintain NR identity at the expense of RPE in part by direct repression of WNT pathway constituents. Stem Cells 2016;34:2625-2634.

Generation of Equine-Induced Pluripotent Stem Cells and Analysis of Their Therapeutic Potential for Muscle Injuries.

Horse health has become a major concern with the expansion of horse-related industries and sports; the importance of healthy muscles for horse performance and daily activities is undisputed. Here we generated equine-induced pluripotent stem cells (E-iPSCs) by reprogramming equine adipose-derived stem cells (E-ADSCs) into iPSCs using a polycistronic lentiviral vector encoding four transcription factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and then examined their pluripotent characteristics. Subsequently, established E-iPSCs were transplanted into muscle-injured Rag/ mdx mice. The histopathology results showed that E-iPSC-transplanted mice exhibited enhanced muscle regeneration compared to controls. In addition, E-iPSC-derived myofibers were observed in the injured muscles. In conclusion, we show that E-iPSCs could be successfully generated from equine ADSCs and transplanted into injured muscles and that E-iPSCs have the capacity to induce regeneration of injured muscles.