scholarly journals Gastruloids generated without exogenous Wnt activation develop anterior neural tissues

2020 ◽  
Author(s):  
Mehmet U. Girgin ◽  
Matthias P. Lutolf
Keyword(s):  
Stem Cells ◽  
Wnt Pathway ◽  
Embryonic Stem ◽  
Break Symmetry ◽  
Chemical Stimulation ◽  
Mouse Escs ◽  
Neural Tissues ◽  
The Brain ◽  
Post Implantation

AbstractWhen stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used bioengineered hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions.

scholarly journals Mouse Embryonic Stem Cells Expressing GDNF Show Enhanced Dopaminergic Differentiation and Promote Behavioral Recovery After Grafting in Parkinsonian Rats

2021 ◽  
Vol 9 ◽  
Author(s):  
Rolando Lara-Rodarte ◽  
Daniel Cortés ◽  
Karla Soriano ◽  
Francia Carmona ◽  
Luisa Rocha ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Substantia Nigra Pars Compacta ◽  
Mouse Escs ◽  
Human Escs ◽  
6 Hydroxydopamine ◽  
The Brain ◽  
Da Release

Parkinson’s disease (PD) is characterized by the progressive loss of midbrain dopaminergic neurons (DaNs) of the substantia nigra pars compacta and the decrease of dopamine in the brain. Grafting DaN differentiated from embryonic stem cells (ESCs) has been proposed as an alternative therapy for current pharmacological treatments. Intrastriatal grafting of such DaNs differentiated from mouse or human ESCs improves motor performance, restores DA release, and suppresses dopamine receptor super-sensitivity. However, a low percentage of grafted neurons survive in the brain. Glial cell line-derived neurotrophic factor (GDNF) is a strong survival factor for DaNs. GDNF has proved to be neurotrophic for DaNs in vitro and in vivo, and induces axonal sprouting and maturation. Here, we engineered mouse ESCs to constitutively produce human GDNF, to analyze DaN differentiation and the possible neuroprotection by transgenic GDNF after toxic challenges in vitro, or after grafting differentiated DaNs into the striatum of Parkinsonian rats. GDNF overexpression throughout in vitro differentiation of mouse ESCs increases the proportion of midbrain DaNs. These transgenic cells were less sensitive than control cells to 6-hydroxydopamine in vitro. After grafting control or GDNF transgenic DaNs in hemi-Parkinsonian rats, we observed significant recoveries in both pharmacological and non-pharmacological behavioral tests, as well as increased striatal DA release, indicating that DaNs are functional in the brain. The graft volume, the number of surviving neurons, the number of DaNs present in the striatum, and the proportion of DaNs in the grafts were significantly higher in rats transplanted with GDNF-expressing cells, when compared to control cells. Interestingly, no morphological alterations in the brain of rats were found after grafting of GDNF-expressing cells. This approach is novel, because previous works have use co-grafting of DaNs with other cell types that express GDNF, or viral transduction in the host tissue before or after grafting of DaNs. In conclusion, GDNF production by mouse ESCs contributes to enhanced midbrain differentiation and permits a higher number of surviving DaNs after a 6-hydroxydopamine challenge in vitro, as well as post-grafting in the lesioned striatum. These GDNF-expressing ESCs can be useful to improve neuronal survival after transplantation.

scholarly journals Formative pluripotent stem cells show features of epiblast cells poised for gastrulation

Cell Research ◽  
2021 ◽  
Author(s):  
Xiaoxiao Wang ◽  
Yunlong Xiang ◽  
Yang Yu ◽  
Ran Wang ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Large Set ◽  
Mouse Escs ◽  
Epiblast Stem Cells ◽  
Early Gastrula

AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

scholarly journals Induction of functional hypothalamus and pituitary tissues from pluripotent stem cells for regenerative medicine

2020 ◽  
Author(s):  
Mayuko Kano ◽  
Hidetaka Suga ◽  
Hiroshi Arima
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Pluripotent Stem Cells ◽  
Hormone Replacement ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Hormone Deficiency ◽  
Adrenal Crisis ◽  
Mouse Escs

Abstract The hypothalamus and pituitary have been identified to play essential roles in maintaining homeostasis. Various diseases can disrupt the functions of these systems, which can often result in serious lifelong symptoms. The current treatment for hypopituitarism involves hormone replacement therapy. However, exogenous drug administration cannot mimic the physiological changes that are a result of hormone requirements. Therefore, patients are at a high risk of severe hormone deficiency, including adrenal crisis. Pluripotent stem cells (PSCs) self-proliferate and differentiate into all types of cells. The generation of endocrine tissues from PSCs has been considered as another new treatment for hypopituitarism. Our colleagues established a three-dimensional culture method for embryonic stem cells (ESCs). In this culture, the ESC-derived aggregates exhibit self-organization and spontaneous formation of highly ordered patterning. Recent results have shown that strict removal of exogenous patterning factors during early differentiation efficiently induces rostral hypothalamic progenitors from mouse ESCs. These hypothalamic progenitors generate vasopressinergic neurons, which release neuropeptides upon exogenous stimulation. Subsequently, we reported adenohypophysis tissue self-formation in three-dimensional cultures of mouse ESCs. The ESCs were found to differentiate into both non-neural oral ectoderm and hypothalamic neuroectoderm in adjacent layers. Interactions between the two tissues appear to be critically important for in vitro induction of a Rathke's pouch-like developing embryo. Various endocrine cells were differentiated from non-neural ectoderm. The induced corticotrophs efficiently secreted adrenocorticotropic hormone when engrafted in vivo, which rescued hypopituitary hosts. For future regenerative medicine, generation of hypothalamic and pituitary tissues from human PSCs is necessary. We and other groups succeeded in establishing a differentiation method with the use of human PSCs. Researchers could use these methods for models of human diseases to elucidate disease pathology or screen potential therapeutics.

230 GENERATION OF PUTATIVE RABBIT EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 231
Author(s):  
S. Wang ◽  
X. Tang ◽  
Y. Niu ◽  
H. Chen ◽  
T. Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
High Speed ◽  
Es Cells ◽  
Research Work ◽  
Embryonic Stem ◽  
Morphological Characteristics ◽  
Mouse Escs

The rabbit, as a laboratory animal model, has several advantages in the study of human physiological disorders. In this study, stable putative pluripotent rabbit embryonic stem cells (rESCs) were derived from in vivo-fertilized and in vitro-cultured blastocysts. The rabbit ICMs were obtained by 0.05% trypsin–0.008% EDTA treatment and mechanical separation; the ES-like cell colonies seen several days later. ICM-derived outgrowths which were treated with 5 mg/mL-1 dispase, followed by 0.05% trypsin–0.008% EDTA, were mechanically disaggregated into small clumps and reseeded on MEFs. The putative ES cell lines maintained expression of pluripotent cells markers and normal XY karyotype for long periods of culture (>1 month). The putative rESCs expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and tumor-related antigens (TRA-1-60 and TRA-1-81). The morphological characteristics of the putative ESCs are closer to those of human ESCs; their high speed of proliferation, however, is closer to that of mouse ESCs. Putative rabbit ESCs were induced to differentiate into many cell types including trophoblast cells, similar to primate ESCs, in vitro, and formed teratomas with derivatives of the 3 major germ layers in vivo when injected into SCID mice. Using RT-PCR measurement, but with some differences in ligands and inhibitors, and comparing with human and mouse ESCs, the putative rabbit ESCs expressed similar genes related to pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and similar genes of FGF, WNT, and TGF signaling pathways related to the proliferation and self-renewal. Our further research work showed that TGF beta and FGF pathways cooperate to maintain pluripotency of rabbit ESCs similar to those of human ES cells.

scholarly journals An in vitro stem cell model of human epiblast and yolk sac interaction

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kirsty ML Mackinlay ◽  
Bailey AT Weatherbee ◽  
Viviane Souza Rosa ◽  
Charlotte E Handford ◽  
George Hudson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Human Embryo ◽  
Cell Model ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryonic Cells ◽  
Post Implantation

Human embryogenesis entails complex signalling interactions between embryonic and extra-embryonic cells. However, how extra-embryonic cells direct morphogenesis within the human embryo remains largely unknown due to a lack of relevant stem cell models. Here, we have established conditions to differentiate human pluripotent stem cells (hPSCs) into yolk sac-like cells (YSLCs) that resemble the post-implantation human hypoblast molecularly and functionally. YSLCs induce the expression of pluripotency and anterior ectoderm markers in human embryonic stem cells (hESCs) at the expense of mesoderm and endoderm markers. This activity is mediated by the release of BMP and WNT signalling pathway inhibitors, and, therefore, resembles the functioning of the anterior visceral endoderm signalling centre of the mouse embryo, which establishes the anterior-posterior axis. Our results implicate the yolk sac in epiblast cell fate specification in the human embryo and propose YSLCs as a tool for studying post-implantation human embryo development in vitro.

Generating gastruloids with somite-like structures from mouse embryonic stem cells

2020 ◽  
Author(s):  
Vincent van Batenburg ◽  
Susanne Carina van den Brink ◽  
Marloes Blotenburg ◽  
Anna Alemany ◽  
Naomi Moris ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cultured Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Hands On ◽  
Somite Formation ◽  
Key Aspects ◽  
Post Implantation

Abstract Gastruloids are aggregates of mouse embryonic stem cells that can be used to study key aspects of mammalian post-implantation development in vitro1–4. Gastruloids generated with previously published protocols do not generate somite-like structures4–6. Here, we describe a modified version of the gastruloids culture protocol5,6 that results in gastruloids that do generate somite-like structures in vitro (van den Brink et al., Nature, 2020)7. Under these conditions, about 50% of the gastruloids generated form structures with features that are characteristic of somites7.This protocol takes 6 days, with relatively little hands-on time. The protocol starts with the aggregation of the cultured cells. Then, the Wnt-agonist Chiron is added 2 days (48h) later. The medium of the aggregates is replaced 3 days (72h) after aggregation. To induce somite-formation, gastruloids are embedded in Matrigel 4 days (96h) after aggregation. After 5 days (120h) of culture, gastruloids resemble E8.5 mouse embryos. At this timepoint they can be fixed (fixative is added on day 5 and washed away on day 6 after overnight incubation in PFA) to prepare them for staining or microscopy experiments.

scholarly journals Retinoic acid combined with spermatogonial stem cell conditions facilitate the generation of mouse germ-like cells

2017 ◽  
Vol 37 (2) ◽  
Author(s):  
Guoyi Dong ◽  
Zhouchun Shang ◽  
Longqi Liu ◽  
Chuanyu Liu ◽  
Yuping Ge ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Retinoic Acid ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Spermatogonial Stem Cell ◽  
Mouse Escs ◽  
Positive Ratio ◽  
Low Efficiency

Spermatogenic lineage has been directly generated in spermatogonial stem cell (SSC) conditions from human pluripotent stem cells (PSCs). However, it remains unknown whether mouse embryonic stem cells (ESCs) can directly differentiate into advanced male germ cell lineage in the same conditions. Here, we showed rather low efficiency of germ-like cell generation from mouse ESCs in SSC conditions. Interestingly, addition of retinoic acid (RA) into SSC conditions enabled efficient differentiation of mouse ESCs into germ-like cells, as shown by the activation of spermatogenesis-associated genes such as Mvh, Dazl, Prdm14, Stella, Scp1, Scp3, Stra8 and Rec8. In contrast, for cells cultured in control medium, the activation of the above genes barely occurred. In addition, RA with SSC conditions yielded colonies of Acrosin-expressing cells and the positive ratio reached a peak at day 6. Our work thus establishes a simple and cost-efficient approach for male germ like cell differentiation from mouse PSCs and may propose a useful strategy for studying spermatogenesis in vitro.

scholarly journals Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

2020 ◽  
Author(s):  
Jesse V Veenvliet ◽  
Adriano Bolondi ◽  
Helene Kretzmer ◽  
Leah Haut ◽  
Manuela Scholze-Wittler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Neural Tube ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
High Level ◽  
Post Implantation

AbstractPost-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes inaccessible to deep analysis in vivo. Mouse embryonic stem cells (mESCs) can form aggregates reflecting the post-occipital embryo (gastruloids), but lacking proper morphogenesis. Here we show that embedding of aggregates derived from mESCs in an extracellular matrix compound results in Trunk-Like-Structures (TLS) with a high level of organization comprising a neural tube and somites. Comparative single-cell RNA-seq analysis demonstrates that TLS execute gene-regulatory programs in an embryo-like order, and generate primordial germ cell like cells (PGCLCs). TLS lacking Tbx6 form ectopic neural tubes, mirroring the embryonic mutant phenotype. ESC-derived trunk-like structures thus constitute a novel powerful in vitro platform for investigating lineage decisions and morphogenetic processes shaping the post-implantation embryo.One sentence summaryA platform for generating trunk-like-structures with precursors of spinal cord, bone and muscle from stem cells in a dish

Lineage restriction of the myogenic conversion factor myf-5 in the brain

Development ◽  
1995 ◽  
Vol 121 (12) ◽  
pp. 4077-4083 ◽  
Author(s):  
S. Tajbakhsh ◽  
M.E. Buckingham
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Conversion Factor ◽  
Embryonic Stem ◽  
Posterior Hypothalamus ◽  
Myogenic Lineage ◽  
Myogenic Factor ◽  
The Brain

myf-5 is one of four transcription factors belonging to the MyoD family that play key roles in skeletal muscle determination and differentiation. We have shown earlier by gene targeting nlacZ into the murine myf-5 locus that myf-5 expression in the developing mouse embryo is closely associated with the restriction of precursor muscle cells to the myogenic lineage. We now identify unexpected expression of this myogenic factor in subdomains of the brain. myf-5 expression begins to be detected at embryonic day 8 (E8) in the mesencephalon and coincides with the appearance of the first differentiated neurons; expression in the secondary prosencephalon initiates at E10 and is confined to the ventral domain of prosomere p4, later becoming restricted to the posterior hypothalamus. This expression is observed throughout embryogenesis. No other member of the MyoD family is detected in these regions, consistent with the lack of myogenic conversion. Furthermore, embryonic stem cells expressing the myf-5/nlacZ allele yield both skeletal muscle and neuronal cells when differentiated in vitro. These observations raise questions about the role of myf-5 in neurogenesis as well as myogenesis, and introduce a new lineage marker for the developing brain.

scholarly journals Bioengineered embryoids mimic post-implantation development in vitro

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mehmet U. Girgin ◽  
Nicolas Broguiere ◽  
Sylke Hoehnel ◽  
Nathalie Brandenberg ◽  
Bastien Mercier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem ◽  
Cell Aggregation ◽  
In Vitro Models ◽  
Solid Matrix ◽  
Trophoblast Stem Cell ◽  
Development In Vitro ◽  
Signaling Interactions ◽  
Post Implantation

AbstractThe difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.

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