Axioloids are three-dimensional (3D) structures derived from pluripotent stem cells (PSCs) that model key aspects of human somitogenesis and early axis development in vitro. These mesoderm-based aggregates recapitulate essential morphogenetic features of the segmentation process, including axial elongation, sequential formation of epithelial somites with proper rostrocaudal patterning, and oscillatory activity of the segmentation clock. They further reproduce the spatiotemporal organization characteristic of early axial development, such as opposing FGF/WNT and retinoic acid signaling gradients and anteroposterior HOX gene expression patterns similar to those observed in vivo. Together, these attributes highlight axioloids as a robust and versatile platform for studying human axial development and congenital disorders of the spine and axial skeleton. In this chapter, we provide a concise, step-by-step protocol for generating axioloids, along with key developmental readouts and troubleshooting strategies to address common problems and issues encountered. This protocol provides a reproducible framework for producing and characterizing axioloids as an in vitro model of human axial development and disease.

Cardiovascular system develops from the lateral plate mesoderm. Its three primary cell lineages (hematopoietic, endothelial, and muscular) are specified by the sequential actions of conserved transcriptional factors.ETV2, a master regulator of mammalian hemangioblast development, however, is absent in the chicken genome and acts downstream ofNPAS4Lin zebrafish. Here, we investigated the epistatic relationship between NPAS4L and ETV2 in avian hemangioblast development. We showed thatETV2is deleted in all 363 avian genomes analyzed. Mouse ETV2 induced LMO2, but not NPAS4L or SCL, expression in chicken mesoderm. Squamate (lizards, geckos, and snakes) genomes contain bothNPAS4LandETV2. In Madagascar ground gecko, both genes were expressed in developing hemangioblasts. Gecko ETV2 induced only LMO2 in chicken mesoderm. We propose that bothNPAS4LandETV2were present in ancestral amniote, with ETV2 acting downstream of NPAS4L in endothelial lineage specification. ETV2 may have acted as a pioneer factor by promoting chromatin accessibility of endothelial-specific genes and, in parallel withNPAS4Lloss in ancestral mammals, has gained similar function in regulating blood-specific genes.

Abstract Here we describe a step-by-step protocol for the derivation of axioloids, a pluripotent stem cell (PSC)-based 3D in vitro model of human segmentation and somitogenesis. Mesoderm-based axioloids capture reproducibly core features of the segmentation process in vitro including axial elongation, sequential formation of properly patterned epithelial somites and coordinated oscillatory activity of the segmentation clock. Axioloids recapitulate furthermore key morphogenetic and molecular features of post-gastrulation human embryos, including temporally coordinated emergence and spatial organization of somitogenesis-associated mesodermal cell populations, presence of opposing FGF/WNT and RA signaling gradients and spatiotemporally controlled expression of HOX genes. In addition to increasing our still limited understanding of human axial development, axioloids can be used to model and study congenital diseases of the human spine, when utilizing patient-like iPSCs with defined pathogenic mutations in genes such as HES7 or MESP2.

High mobility group nucleosome‐binding protein 3 (HMGN3), a member of the HMGN family, modulates the structure of chromatin and regulates transcription through transcription factors. HMGN3 has been implicated in the development of various cancers; however, the underlying mechanisms remain unclear. We herein demonstrated that the high expression of HMGN3 correlated with the metastasis of liver fluke infection‐induced cholangiocarcinoma (CCA) in patients in northeastern Thailand. The knockdown of HMGN3 in CCA cells significantly impaired the oncogenic properties of colony formation, migration, and invasion. HMGN3 inhibited the expression of and blocked the intracellular polarities of epithelial regulator genes, such as the CDH1/E‐cadherin and TJAP1 genes in CCA cells. A chromatin immunoprecipitation sequencing analysis revealed that HMGN3 required the transcription factor SNAI2 to bind to and repress the expression of epithelial regulator genes, at least in part, due to histone deacetylases (HDACs), the pharmacological inhibition of which reactivated these epithelial regulators in CCA, leading to impairing the cell migration capacity. Therefore, the overexpression of HMGN3 represses the transcription of and blocks the polarities of epithelial regulators in CCA cells in a manner that is dependent on the SNAI2 gene and HDACs.

The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms, but remains largely elusive in humans due to ethical and technical limitations. Despite recent advances with pluripotent stem cell (PSC)-based approaches1–5, a system that robustly recapitulates human somitogenesis in both space and time remains missing. Here, we introduce a PSC-derived mesoderm-based 3D model of human segmentation and somitogenesis, which we termed Axioloids, that captures accurately the oscillatory dynamics of the segmentation clock as well as the morphological and molecular characteristics of segmentation and sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF/WNT signaling gradients and Retinoic Acid (RA) signaling components. We identify an unexpected critical role of RA signaling in the stabilization of forming segments, indicating distinct, but also synergistic effects of RA and extracellular matrix (ECM) on the formation and epithelialization of somites. Importantly, comparative analysis demonstrates striking similarities of Axioloids to the human embryo, further validated by the presence of the HOX code in Axioloids. Lastly, we demonstrate the utility of our Axioloid system to study the pathogenesis of human congenital spine diseases, by using patient-like iPSC cells with mutations in HES7 and MESP2, which revealed disease-associated phenotypes including loss of epithelial somite formation and abnormal rostrocaudal patterning. These results suggest that Axioloids represent a promising novel platform to study axial development and disease in humans.

The successful derivation and culture of pluripotent stem cells (PSCs) is tightly connected with the study of embryonic development, and was made largely possible by advances in in vitro fertilization and blastocyst culture during the latter half of the last century [1,2]. Since then, embryonic and induced pluripotent stem cells have been extensively used to derive a plethora of functional cell types in vitro, heavily relying on and utilizing insights into cellular differentiation won from developmental biological studies in model organisms. Excitingly, PSCs are now being increasingly used to reconstitute and analyze complex aspects of mouse and human embryonic development. These bottom-up approaches are starting to provide novel insights into core developmental processes and biological questions and may ultimately help decipher the biological principles that underlie the emergence of form and function during development. This mini review summarizes the latest advances and recent breakthroughs in this rapidly growing field of research on PSC-based in vitro models of early embryonic development.

From fertilization to onset of gastrulation, a mammalian embryo goes through several rounds of cellular morphogenesis resembling phenomena of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), collectively referred to as EMTs. How these EMT events play a role in shaping the three-dimensional (3-D) architecture of the developing embryo is not well-understood. In this review, we present a model in which cellular morphogenesis, represented primarily by dynamic changes in its epithelialization status, is the driving force of embryonic 3-D organization. This is achieved through the integration of three key components of mammalian early development, the pluripotency regulation, morphogenetic signaling, and biomechanical force anisotropy. Although cells in an early embryo do not exhibit full mesenchymal characteristics, our model underscores the importance of investigating molecular regulation of epithelial cell polarity and partial EMT/MET in understanding mammalian early development.

As our understanding of Epithelial Mesenchymal Transition (EMT) increases, the original binary concept of E versus M no longer fits with experimental evidence. Re-definition of the EMT paradigm as spectral transitions between a full epithelium and a full mesenchyme suggests the existence of a virtual infinity of intermediate cellular states. The new challenge is to develop technical tools needed to contextualize each of these states and identify biologically significant cellular mechanisms that could be targeted in combatting EMT-related diseases.

The pluripotent epiblast gives rise to all tissues and organs in the adult body. Its differentiation starts at gastrulation, when the epiblast generates mesoderm and endoderm germ layers through epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in vitro can adopt either mesenchymal or epithelial morphology. The relationship between epiblast cellular morphology and its pluripotency is not well understood. Here, using chicken epiblast and mammalian pluripotency stem cell (PSC) models, we show that PSCs undergo a mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and its subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible initiation of pluripotency exit, whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and that epiblast pluripotency is restricted to an intermediate cellular state residing between the fully mesenchymal and fully epithelial states.

Epiblast is composed of pluripotent cells which will give rise to all cell lineages in a human body. It forms a single-cell layered epithelium conserved among all amniotic vertebrates (birds, reptiles and mammals) and undergoes complex morphogenesis both before and during gastrulation. Our knowledge of the amniote epiblast is based on data acquired through cellular and molecular analyses of early chick and mouse embryos in vivo and mammalian pluripotent stem cells (PSCs) in vitro. Very few studies have been published on biomechanical characteristics of the amniote epiblast, largely due to lack of experimental tools for measuring and perturbing biomechanical properties. Also missing is a conceptual framework that can integrate both biomechanical and molecular parameters of the epiblast. This review is aimed at providing a background based on which epiblast morphogenesis, including its transition between the epithelial and mesenchymal states, can be understood from a biomechanical perspective. This simple developmental biology system is suitable for testing a multitude of theoretical models in biomechanics, leading to a better understanding of biomechanical logics and constraints governing multicellular organization.

Apolipoprotein E (ApoE) belongs to a class of cellular proteins involved in lipid metabolism. ApoE is a polymorphic protein produced primarily in macrophages and astrocytes. Different isoforms of ApoE have been associated with susceptibility to various diseases including Alzheimer's and cardiovascular diseases. ApoE expression has also been found to affect susceptibility to several viral diseases, including Hepatitis C and E, but its effect on the life cycle of HIV-1 remains obscure. In this study, we initially found that HIV-1 infection selectively up-regulated ApoE in human monocyte-derived macrophages (MDMs). Interestingly, ApoE knockdown in MDMs enhanced the production and infectivity of HIV-1, and was associated with increased localization of viral envelope (Env) proteins to the cell surface. Consistent with this, ApoE over-expression in 293T cells suppressed Env expression and viral infectivity, which was also observed with HIV-2 Env, but not with VSV-G Env. Mechanistic studies revealed that the C-terminal region of ApoE was required for its inhibitory effect on HIV-1 Env expression. Moreover, we found that ApoE and Env co-localized in the cells, and ApoE associated with gp160, the precursor form of Env, and that the suppression of Env expression by ApoE was cancelled by the treatment with lysosomal inhibitors. Overall, our study revealed that ApoE is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages that exerts its anti-HIV-1 activity through association with gp160 Env via the C-terminal region, which results in subsequent degradation of gp160 Env in the lysosomes.

Epithelial-mesenchymal transition (EMT) is a morphogenetic process of cells that adopt an epithelial organization in their developmental ontogeny or homeostatic maintenance. Abnormalities in EMT regulation result in many malignant tumours in the human body. Tumours associated with the haematopoietic system, however, are traditionally not considered to involve EMT and haematopoietic stem cells (HSCs) are generally not associated with epithelial characteristics. In this review, we discuss the ontogeny and homeostasis of adult HSCs in the context of EMT intermediate states. We provide evidence that cell polarity regulation is critical for both HSC formation from embryonic dorsal aorta and HSC self-renewal and differentiation in adult bone marrow. HSC polarity is controlled by the same set of surface and transcriptional regulators as those described in canonical EMT processes. With an emphasis on partial EMT, we propose that the concept of EMT can be similarly applied in the study of HSC generation, maintenance and pathogenesis.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Fucoidan promotes early step of cardiac differentiation from human embryonic stem cells and long term maintenance of beating areas. Sofiane Hamidi, Didier Letourneur, Rachida Aid, Antonio Di Stefano, William Vainchenker, Francoise Norol, Catherine Le Visage

Somatic stem cells require specific niches and three-dimensional scaffolds provide ways to mimic this microenvironment. Here, we studied a scaffold based on Fucoidan, a sulfated polysaccharide known to influence morphogen gradients during embryonic development, to support human embryonic stem cells (hESCs) differentiation toward the cardiac lineage. A macroporous (pore 200 μm) Fucoidan scaffold was selected to support hESCs attachment and proliferation. Using a protocol based on the cardiogenic morphogen bone morphogenic protein 2 (BMP2) and transforming growth factor (TGFβ) followed by tumor necrosis factor (TNFα), an effector of cardiopoietic priming, we examined the cardiac differentiation in the scaffold compared to culture dishes and embryoid bodies (EBs). At day 8, Fucoidan scaffolds supported a significantly higher expression of the 3 genes encoding for transcription factors marking the early step of embryonic cardiac differentiation NKX2.5 (p<0.05), MEF2C (p<0.01), and GATA4 (p<0.01), confirmed by flow cytometry analysis for MEF2C and NKX2.5. The ability of Fucoidan scaffolds to locally concentrate and slowly release TGFβ and TNFα was confirmed by Luminex technology. We also found that Fucoidan scaffolds supported the late stage of embryonic cardiac differentiation marked by a significantly higher atrial natriuretic factor (ANF) expression (p<0.001), although only rare beating areas were observed. We postulated that absence of mechanical stress in the soft hydrogel impaired sarcomere formation, as confirmed by molecular analysis of the cardiac muscle myosin MYH6 and immunohistological staining of sarcomeric α-actinin. Nevertheless, Fucoidan scaffolds contributed to the development of thin filaments connecting beating areas through promotion of smooth muscle cells, thus enabling maintenance of beating areas for up to 6 months. In conclusion, Fucoidan scaffolds appear as a very promising biomaterial to control cardiac differentiation from hESCs that could be further combined with mechanical stress to promote sarcomere formation at terminal stages of differentiation.

JAK2(V617F) is the predominant mutation in myeloproliferative neoplasms (MPN). Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS) cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2(V617F), respectively. In the patient with homozygous JAK2(V617F), additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO) between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO)-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA(+)CD41(+) cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy.

Induced pluripotent stem cells were discovered at the end of 2006. They are derived from somatic cells and are functionally close to embryonic stem cells, i.e. characterized by self-renewal and pluripotency properties. They are offering new opportunities in fundamental research and in applied research such as disease modeling, pharmacology research and toxicology. In clinic, they could be used in cell and gene therapies.

Abstract Abstract 1758 During the last several years, iPS cells have been described as a new powerful tool for disease modeling and drug screening. It became possible to reprogram adult patients cells into specific pluripotent cell lines harboring inherited and/or acquired genetic abnormalities. We have focused our work on modeling Jak2V617F positive MPN patients in iPS cells. By retroviral infection (OCT4, SOX2, KLF4, c-Myc), several iPS cell lines were generated from CD34+ cells of a healthy donor (control), a heterozygous JAK2 V617F patient and a homozygous JAK2 V617F patient. All cell lines expressed pluripotent surface markers (TRA1–81, SSEA4), pluripotent genes (NANOG, OCT4, SOX2) and were capable to induce teratomas in immunodeficient mice. Moreover, they were silenced for the expression of the 4 exogenous transgenes and showed no new clonal karyotypic abnormalities. All the iPS cell lines derived from the JAK2V617F heterozygous and homozygous patients were also heterozygous and homozygous for JAK2V617F. No JAK2 wild type iPS could be obtained from the patients CD34+ cells. Interestingly all the homozygous JAK2V617Fhad no 20q deletion by cytogenetic and CGH analysis demonstrating that this deletion was a secondary event in this MPN. JAK2V617F mutation did not elicit a significant increase in hematopoiesis in comparison to the control iPS. However, marked differences in cytokine sensitivity were found. Homozygous iPS-derived erythroid progenitors had spontaneous growth, hypersensitivity to EPO and gave rise to slightly larger erythroid colonies in methylcellulose assays as compared to the control, whereas erythroid progenitors from JAK2V617F heterozygous iPS has similar Epo-response than those from the control. Interestingly, Megakaryocytes (MK) progenitors from JAK2V617F heterozygous iPS had hypersensitivity to TPO whereas those from homozygous iPS showed a complete TPO independence. Those cytokine-response profiles recapitulate the disease's primary features and validate the iPS as a good tool for JAK2V617F MPN modeling. MK differentiation showed a maturation defect with an excess of immature MKs only in the JAK2 V617Fhomozygous lines, who was derived with a post-PV myelofibrosis, suggesting the existence of intrinsic abnormalities of the MK lineage. Finally, we tested several JAK2, PI3K and ERK inhibitors on the JAK2V617F-mediated erythroid growth and showed that only JAK2 and PI3K inhibitors were able to block the erythroid spontaneous growth. iPS technology recapitulates the cytokine hypersensitivity of JAK2V617F MPNs with marked differences between heterozygous and homozygous mutations, enabling us to screen various pharmaceutical inhibitors. In an addition it can also be used to study the clonal hierarchy in a MPN and in the future to study the synergistic effects of different mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3416 The relationship between monocytes and mesenchymal stromal cells remains a controversial issue. During embryonic development, the two cell types emerge early and share a large pattern of tissue expression. Using human embryonic stem cells (hES), we recently reported that embryonic monocytes/macrophages were endowed mainly with anti-inflammatory and remodeling functions. Here, we show that a subset of embryonic monocytic cells can give rise to stromal cells. Mesoderm and hematopoietic specification of hES were achieved from embryoid bodies in Iscove's Modified Dulbecco's medium (IMDM) supplemented with 15% fetal bovine serum (FBS) in presence of Bone Morphogenetic Protein 4 (BMP-4, 10 ng/ml) and Vascular Endothelial Growth Factor (VEGF, 5 ng/ml), followed by fetal liver tyrosine kinase 3 ligand (FLT3-ligand, 10 ng/ml), stem cell factor (SCF, 50 ng/ml), interleukin-3 (IL-3, 100 U/ml) and thrombopoietin (TPO, 10 ng/ml). Between day 14–21 of culture, CD45+14+ cells were sorted, cultured for 4 days in presence of Monocyte-Colony Stimulating Factor (M-CSF, 50 ng/ml), Granulocyte-Macrophage CSF (GM-CSF, 20 ng/ml) and IL-3, and subsequently seeded on fibronectin. After culture in Endothelial Cell Growth Medium supplemented with Endothelial GF (EGF), VEGF (25 ng/ml) and bFibroblast GF (bFGF) (1 ng/ml) for 14 days, clones of adherent cells with typical fibroblast-like morphology emerged at a frequency of 2/104 plated embryonic monocytic cells. In order to eliminate contaminating stromal cells before seeding on fibronectin, CD34low43+ hematopoietic cells were sorted at day 10 and CD45+14+ cells were sorted 7 days later. These cells were cultured with the previously described growth factors, but in serum free medium that does not support stromal cell proliferation. In these conditions, we observed that stromal cell developed from CD45+CD14+ embryonic monocytes. These EM-SCs shared several phenotypic and functional characteristics with adult mesenchymal stem cells (MSCs). They could be expanded in vitro in complete alpha–modified Eagle's medium (MEMa) supplemented with 10% FBS, by successive cycles of dissociation. At a density of 500 and 1000 per cm2, EM-SCs formed small colonies of CFU-F. EM-SCs did not express the hematopoietic surface markers CD14 and CD45, nor the endothelial markers CD31 and KDR, and strongly expressed CD105, CD73, CD13 and CD90. In contrast with adult MSCs, they expressed CD133 and low levels of CD34. EM-SCs could not elicit a proliferative response in the presence of allogeneic lymphocytes, and exhibited a suppressive effect on T-cell proliferation in mixed lymphocyte reaction. Under appropriate conditions, EM-SCs displayed osteogenic, chondrogenic and adipogenic differentiation. They could also adopt a smooth muscle cell but not an endothelial or a cardiac phenotype. Compared to adult MSCs, EM-SCs did not expressed telomerase reverse transcriptase, but demonstrated longer telomeres and enhanced expression of genes encoding growth factors, adhesion proteins, tissue degrading enzymes, and anti-inflammatory chemokines. EM-SCs also secreted high amounts of proteins involved in tissue remodeling and angiogenesis. Thus, a rare subset of embryonic monocytic cells can give rise to a population of stromal cells with high immunosuppressive and remodeling functions. A large body of evidence shows that macrophages and stromal cells are involved in tumor development. It remains to be explored whether stromal cells with remodelling potential could derive from tumor-infiltrating macrophages as it can derive from embryonic macrophages. hES cells offer a valuable experimental model for in vitro studies of these differentiation pathways. Disclosures: No relevant conflicts of interest to declare.