Archetypal Architecture Construction, Patterning, and Scaling Invariance in a 3D Embryoid Body Differentiation Model.

Self-organized patterning and architecture construction studying is a priority goal for fundamental developmental and stem cell biology. To study the spatiotemporal patterning of pluripotent stem cells of different origins, we developed a three-dimensional embryoid body (EB) differentiation model quantifying volumetric parameters and investigated how the EB architecture formation, patterning, and scaling depend on the proliferation, cavitation, and differentiation dynamics, external environmental factors, and cell numbers. We identified three similar spatiotemporal patterns in the EB architectures, regardless of cell origin, which constitute the EB archetype and mimick the pre-gastrulation embryonic patterns. We found that the EB patterning depends strongly on cellular positional information, culture media factor/morphogen content, and free diffusion from the external environment and between EB cell layers. However, the EB archetype formation is independent of the EB size and initial cell numbers forming EBs; therefore, it is capable of scaling invariance and patterning regulation. Our findings indicate that the underlying principles of reaction-diffusion and positional information concepts can serve as the basis for EB architecture construction, patterning, and scaling. Thus, the 3D EB differentiation model represents a highly reproducible and reliable platform for experimental and theoretical research on developmental and stem cell biology issues.

High Content Image Analysis of Spatiotemporal Proliferation and Differentiation Patterns in 3D Embryoid Body Differentiation Model.

The 3D embryoid body (EB) differentiation model is a promising tool for fundamental cell biology and drug discovery studies assessing the compound effects on mammalian and human development. This 3D cell model allows for analyzing spatiotemporal changes during morphogenesis and differentiation. A combination of confocal microscopy with high content image analysis (HCIA) can significantly improve the study of spatiotemporal patterns of early embryonic lineages and compound efficacy and toxicity testing by enhancing the identification and quantification of various cell types. HCIA can be used to assess the EB architecture through quantitative and qualitative characteristics, such as viability and apoptosis, identification, localization, ratio and timing for various types of early embryonic cells, dimensions of compartments of proliferating and differentiating cells, changes in the size and shape of EBs, and translocation of individual cells and cell layers. This chapter describes a comprehensive framework for HCIA for 3D EB differentiation model that allows investigators to analyze EB growth, differentiation, and morphogenetic dynamics.

5-Hydroxytryptophan (5-HTP)-induced intracellular syndrome in mouse non-neural embryonic cells is associated with inhibited proliferation and cell death.

Biogenic monoamines are involved in the regulation of various processes in both neural and non-neural cells during development. The present study aimed to identify the regulatory effects of serotonin (5-HT) and its precursors (l-tryptophan and 5-hydroxytryptophan, 5-HTP) on proliferation and cell death in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs and 3T3 cells). The concentration-dependent cell growth and viability of the ESCs, MEFs and 3T3 cells were analyzed after treatment with l-tryptophan, 5-HTP and 5-HT in the concentration range 10-6 - 10-2 M. Treating the cells with 5-HTP, but not l-tryptophan and 5-HT, induced reversible toxic effects. 5-HTP treatment (10-3 - 10-2 M) significantly inhibited cell proliferation through blocking of the S-phase of the cell cycle and increasing apoptotic and necrotic cell death. Moreover, 5-HTP treatment stimulated a reorganization of the actin and tubulin networks and upregulated the gene expression of enzymes involved in 5-HT synthesis and metabolism: aromatic amino acid decarboxylase (Aadc/Ddc), monoamine oxidase A (Maoa), and transglutaminase 2 (Tgm2). HPLC analysis found no changes in the intracellular and extracellular levels of 5-HT after 5-HTP treatment, but a significant increase of intracellular 5-HTP levels. However, inhibition of AADC with NSD-1015 or transglutaminase with cystamine prevented 5-HTP-induced cell growth impairment and attenuated the toxic effects of 5-HTP treatment. Our results suggest that 5-HTP can induce toxic effects through cell cycle arrest and cell death in embryonic stem and somatic cells by enhancing the levels of 5-HT-mediated protein modifications. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

Comparative assessment of toxic responses in 3D embryoid body differentiation model and mouse early embryos treated with 5-hydroxytryptophan.

Pluripotent stem cells recapitulate in vitro the early developmental stages and are considered promising cell models for predictive developmental toxicity studies. To investigate the consistency between adverse drug effects on early development and the early stages of embryonic stem cell differentiation in three-dimensional (3D) in vitro culture, the toxic responses to 5-hydroxytryptophan (5-HTP; 0.5-2 mM) were evaluated in early mouse embryos and the embryoid body (EB) differentiation model. 3D architectures, developmental and differentiation dynamics and the cell death rates were analyzed in early mouse embryos (E2.5-E5.5) and EBs at 1 and 6 days of differentiation using a combination of confocal immunofluorescence microscopy with high content imaging analysis and quantitative gene expression analysis. Comparative analysis of toxic responses in early embryos and EBs revealed a similar dose- and stage-dependent decrease in the 5-HTP toxic effects during development and differentiation. The integral toxic responses in the early embryos and EBs were significantly dependent on their 3D architecture and cellular composition. Treatment with 5-HTP (1 mM and above) induced developmental arrest, growth inhibition, and increased cell death in the early embryos without the trophoblasts (E2.5) and those with impaired trophoblasts and in early EBs, whereas later embryos and EBs were more resistant due to the protection of the extraembryonic tissues. This study demonstrates that the EB differentiation model is a relevant 3D-model of early mammalian development and can be useful for the predictive evaluation of toxic and teratogenic effects in embryos at the preimplantation and early post-implantation developmental stages.

Combined anti-HER2 and hormonal treatment in a patient with HR+HER2+ clear-cell uterine carcinoma: A case report.

Clear-cell carcinoma (CCC) of the uterus is an aggressive disease. Current international guidelines on the treatment of uterine carcinomas predominantly cover cancer with endometrioid histology, and clinicians tend to use the same approach for patients with non-endometrioid histology due to the absence of separate guidelines for these rare tumor types. At present, molecular analysis enables the assessment of novel and non-standard treatment options based on the individual characteristics of a tumor. The present report presents a clinical case of successful treatment of a patient with clear cell uterine carcinoma with HER2 and ER expression. Non-toxic targeted treatment was used based on immunohistochemistry (IHC) data. The patient received anti-HER2 and hormonal treatment and demonstrated an excellent response. The follow-up period was 47 months and the patient remained stable during treatment without significant toxicity. Therefore, this approach demonstrated the potential for selecting highly-specific therapy for rare tumors, which lack distinct recommendations for their treatment.

TGFß Family Signaling Pathways in Pluripotent and Teratocarcinoma Stem Cells' Fate Decisions: Balancing Between Self-Renewal, Differentiation, and Cancer.

The transforming growth factor-ß (TGFß) family factors induce pleiotropic effects and are involved in the regulation of most normal and pathological cellular processes. The activity of different branches of the TGFß family signaling pathways and their interplay with other signaling pathways govern the fine regulation of the self-renewal, differentiation onset and specialization of pluripotent stem cells in various cell derivatives. TGFß family signaling pathways play a pivotal role in balancing basic cellular processes in pluripotent stem cells and their derivatives, although disturbances in their genome integrity induce the rearrangements of signaling pathways and lead to functional impairments and malignant transformation into cancer stem cells. Therefore, the identification of critical nodes and targets in the regulatory cascades of TGFß family factors and other signaling pathways, and analysis of the rearrangements of the signal regulatory network during stem cell state transitions and interconversions, are key issues for understanding the fundamental mechanisms of both stem cell biology and cancer initiation and progression, as well as for clinical applications. This review summarizes recent advances in our understanding of TGFß family functions in naїve and primed pluripotent stem cells and discusses how these pathways are involved in perturbations in the signaling network of malignant teratocarcinoma stem cells with impaired differentiation potential.

Expression dynamics of Mage family genes during self-renewal and differentiation of mouse pluripotent stem and teratocarcinoma cells.

The biological roles of cancer-testis antigens of the Melanoma antigen (Mage) family in mammalian development, stem cell differentiation and carcinogenesis are largely unknown. In order to understand the involvement of the Mage family genes in maintenance of normal and cancer stem cells, the expression patterns of Mage-a, Mage-b, Mage-d, Mage-e, Mage-h and Mage-l gene subfamilies were analyzed during the self-renewal and differentiation of mouse pluripotent stem and teratocarcinoma cells. Clustering analysis based on the gene expression profiles of undifferentiated and differentiating cell populations revealed strong correlations between Mage expression patterns and differentiation and malignant states. Gene co-expression analysis disclosed the potential contributions of Mage family members in self-renewal and differentiation of pluripotent stem and teratocarcinoma cells. Two gene clusters including Mage-a4 and Mage-a8, Mageb1, Mage-d1, Mage-d2, Mage-e1, Mage-l2 were identified as functional antagonists with opposing roles in the regulation of proliferation and differentiation of mouse pluripotent stem and teratocarcinoma cells. The identified aberrant expression patterns of Mage-a2, Mage-a6, Mage-b4, Mageb-16 and Mage-h1 in teratocarcinoma cells can be considered as specific teratocarcinoma biomarkers promoted the malignant phenotype. Our study first provides a model for the involvement of Mage family members in regulatory networks during the self-renewal and early differentiation of normal and cancerous stem cells for further research of the predicted functional modules and the development of new cancer treatment strategies.

Cancer-testis antigens: Unique cancer stem cell biomarkers and targets for cancer therapy.

Cancer-testis antigens (CTAs) are considered as unique and promising cancer biomarkers and targets for cancer therapy. CTAs are multifunctional protein group with specific expression patterns in normal embryonic and adult cells and various types of cancer cells. CTAs are involved in regulating of the basic cellular processes during development, stem cell differentiation and carcinogenesis though the biological roles and cell functions of CTA families remain largely unclear. Analysis of CTA expression patterns in embryonic germ and somatic cells, pluripotent and multipotent stem cells, cancer stem cells and their cell descendants indicates that rearrangements of characteristic CTA profiles (aberrant expression) could be associated with cancer transformation and failure of the developmental program of cell lineage specification and germ line restriction. Therefore, aberrant CTA profiles can be used as panels of biomarkers for diagnoses and the selection of cancer treatment strategies. Moreover, immunogenic CTAs are prospective targets for cancer immunotherapy. Clinical trials testing broad range of cancer therapeutic vaccines against antigens of MAGEA and NY-ESO-1 families for treating various cancers have shown mixed clinical efficiency, safety and tolerability, suggesting the requirement of in-depth research of CTA expression in normal and cancer stem cells and extensive clinical trials for improving cancer immunotherapy technologies. This review focuses on recent advancement in study of CTAs in normal and cancer cells, particularly in normal and cancer stem cells, and provides a new insight into CTA expression patterns during normal and cancer stem cell lineage development. Additionally, new approaches in development of effective CTA-based therapies exclusively targeting cancer stem cells will be discussed.

Dose-reduced first cycle of chemotherapy for prevention of life-threatening acute complications in nonseminomatous germ cell tumor patients with ultra high tumor markers and/or poor performance status.

PURPOSE: Patients with metastatic nonseminomatous germ cell tumors (mNSGCT) and a high tumor burden or a poor performance status at initial diagnosis are at risk from potentially life-threatening early complications during or after the first chemotherapy cycle. The outcomes with dose-reduced first cycle of chemotherapy in this population of patients are not well established. METHODS: We performed a retrospective analysis of patients with mNSGCT and International Germ Cell Cancer Collaborative Group (IGCCCG) poor risk features. All patients received cisplatin and etoposide-based combinations as first-line treatment. Ultra high tumor marker levels were defined as α-fetoprotein ≥ 100,000 ng/ml or human chorionic gonadotropin ≥ 200,000 mIU/ml. Before 2005, the first treatment cycle was administered at a full dose in our center. After 2005, we used an abbreviated course of cisplatin and etoposide (EP) for the first cycle, followed by subsequent full-dose administration. RESULTS: From 1987 to 2012, 265 patients with poor risk features according to IGCCCG received first-line chemotherapy. Among them, 63 out of 265 (24%) patients had ultra high tumor marker levels and/or ECOG performance status of 3-4. Dose reduction of the first chemotherapy cycle was associated with a significant decrease of life-threatening complications from 76 to 44% (p = 0.01), but not with the overall survival (HR 0.99, 95% CI 0.44-2.26). CONCLUSIONS: Dose reduction of the first EP cycle by 40-60% in the subgroup of poor risk patients with ultra high tumor marker levels and/or ECOG performance status 3-4 is associated with significantly lowered acute complication rates but not with overall survival.

Tumorigenic and Differentiation Potentials of Embryonic Stem Cells Depend on TGFß Family Signaling: Lessons from Teratocarcinoma Cells Stimulated to Differentiate with Retinoic Acid.

A significant challenge for the development of safe pluripotent stem cell-based therapies is the incomplete in vitro differentiation of the pluripotent stem cells and the presence of residual undifferentiated cells initiating teratoma development after transplantation in recipients. To understand the mechanisms of incomplete differentiation, a comparative study of retinoic acid-induced differentiation of mouse embryonic stem (ES) and teratocarcinoma (EC) cells was conducted. The present study identified differences in proliferative activity, differentiation, and tumorigenic potentials between ES and EC cells. Higher expression of Nanog and Mvh, as well as Activin A and BMP4, was found in undifferentiated ES cells than in EC cells. However, the expression levels of Activin A and BMP4 increased more sharply in the EC cells during retinoic acid-induced differentiation. Stimulation of the Activin/Nodal and BMP signaling cascades and inhibition of the MEK/ERK and PI3K/Act signaling pathways resulted in a significant decrease in the number of Oct4-expressing ES cells and a loss of tumorigenicity, similar to retinoic acid-stimulated EC cells. Thus, this study demonstrates that a differentiation strategy that modulates prodifferentiation and antiproliferative signaling in ES cells may be effective for eliminating tumorigenic cells and may represent a valuable tool for the development of safe stem cell therapeutics.

Expression patterns of cancer-testis antigens in human embryonic stem cells and their cell derivatives indicate lineage tracks.

Pluripotent stem cells can differentiate into various lineages but undergo genetic and epigenetic changes during long-term cultivation and, therefore, require regular monitoring. The expression patterns of cancer-testis antigens (CTAs) MAGE-A2, -A3, -A4, -A6, -A8, -B2, and GAGE were examined in undifferentiated human embryonic stem (hES) cells, their differentiated derivatives, teratocarcinoma (hEC) cells, and cancer cell lines of neuroectodermal and mesodermal origin. Undifferentiated hES cells and embryoid body cells expressed MAGE-A3, -A6, -A4, -A8, and GAGEs while later differentiated derivatives expressed only MAGE-A8 or MAGE-A4. Likewise, mouse pluripotent stem cells also express CTAs of Magea but not Mageb family. Despite similarity of the hES and hEC cell expression patterns, MAGE-A2 and MAGE-B2 were detected only in hEC cells but not in hES cells. Moreover, our analysis has shown that CTAs are aberrantly expressed in cancer cell lines and display low tissue specificity. The identification of CTA expression patterns in pluripotent stem cells and their derivatives may be useful for isolation of abnormally CTA-expressing cells to improve the safety of stem-cell based therapy.

Normal and pathological development of pluripotent stem cells.

Pluripotent cells of the early embryo originate all types of somatic cells and germ cells of adult organism. Pluripotent stem cell lines were derived from mammalian embryos and adult tissues using different techniques and from different sources--inner cell mass of the blastocyst, primordial germ cells, parthenogenetic oocytes, and mature spermatogonia--as well as by transgenic modification of various adult somatic cells. Despite different origin, all pluripotent stem cell lines demonstrate considerable similarity of the major biological properties: unlimited self-renewal and differentiation into various somatic and germ cells in vitro and in vivo, similar gene expression profiles, and similar cell cycle structure. Their malignant counterpart embryonal teratocarcinoma stem cell lines have restricted developmental potentials caused by genetic disturbances that result in deregulation of proliferation and differentiation balance. Numerous studies on the stability of different pluripotent stem cell lines demonstrated that, irrespective of their origin, long-term in vitro cultivation leads to the accumulation of chromosomal and gene mutations as well as epigenetic changes that can cause oncogenic transformation of cells. Our research of signaling pathways and pattern of specific gene expression in pluripotent stem cells and teratocarcinoma cells is focused on discovery of fundamental mechanisms that regulate normal development of pluripotent cells into different lineages and are disrupted in cancer initiating cells. Analysis gene expression profiles, differentiation potentials and cell cycle of normal and mutant pluripotent stem cells provide new data to search molecular targets to eliminate malignant cells in tumors.