scholarly journals Generation of human blastocyst-like structures from pluripotent stem cells

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yong Fan ◽  
Zheying Min ◽  
Samhan Alsolami ◽  
Zhenglai Ma ◽  
E. Zhang ◽  
...  
Keyword(s):  
Early Stage ◽  
Three Dimensional ◽  
Human Embryos ◽  
Blastocyst Development ◽  
Developmental Defects ◽  
Cell Lineages ◽  
Human Blastocyst ◽  
Reliable Model ◽  
Human Embryogenesis

AbstractHuman blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm, all of which are essential for early development and organ formation. However, due to ethical concerns and restricted access to human blastocysts, a comprehensive understanding of early human embryogenesis is still lacking. To bridge this knowledge gap, a reliable model system that recapitulates early stages of human embryogenesis is needed. Here we developed a three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids are similar with human embryos that were cultured for 8 or 10 days in vitro, in terms of embryonic structures, cell lineages and transcriptomic profiles. In conclusion, we developed a scalable system to mimic human blastocyst development, which can potentially facilitate the study of early implantation failure that induced by developmental defects at early stage.

scholarly journals Generation of human blastocyst-like structures from pluripotent stem cells

2021 ◽  
Author(s):  
Yong Fan ◽  
Zhe-Ying Min ◽  
Samhan Alsolami ◽  
Zheng-Lai Ma ◽  
Ke Zhong ◽  
...  
Keyword(s):  
Single Cell ◽  
Developmental Stages ◽  
Three Dimensional ◽  
Cell Lineage ◽  
Human Embryos ◽  
Developmental Potential ◽  
Cell Lineages ◽  
Human Embryogenesis ◽  
Post Implantation

AbstractHuman blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast, and primitive endoderm, all of which are essential for early development and organ formation1,2. However, due to ethical concerns and restricted access to human blastocysts, we lack a comprehensive understanding of early human embryogenesis. To bridge this knowledge gap, we need a reliable model system that recapitulates early stages of human embryogenesis. Here we report a ∼three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids also exhibited the developmental potential to undergo post-implantation morphogenesis in vitro to form structures with a cellular composition and transcriptome signature similar to human embryos that had been cultured in vitro for 8 or 10 days. In conclusion, human EPS-blastoids provide a new experimental platform for studying early developmental stages of the human embryo.HighlightsA method for generating human blastoids from EPS cells.Human blastoids resemble blastocysts in terms of morphology and cell lineage composition.Single-cell transcriptomic analyses reveal EPI, PE, and TE cell lineages in human blastoids.Human blastoids mimic in vitro the morphogenetic events of pre- and early post-implantation stages.

scholarly journals Functional 3D Human Liver Bud Assembled from MSC-Derived Multiple Liver Cell Lineages

2018 ◽  
Vol 28 (5) ◽  
pp. 510-521 ◽  
Author(s):  
Jing Li ◽  
Feiyue Xing ◽  
Feng Chen ◽  
Liumin He ◽  
Kwok-Fai So ◽  
...  
Keyword(s):  
Human Liver ◽  
Early Stage ◽  
Three Dimensional ◽  
Alternative Methods ◽  
Liver Sinusoidal Endothelial Cells ◽  
Medicine Research ◽  
Cell Lineages ◽  
Self Assembled ◽  
Liver Tissues

The severe shortage of donor liver organs requires the development of alternative methods to provide transplantable liver tissues such as stem cell-derived organoids. Despite several studies describing the generation of vascularized and functional liver tissues, none have succeeded in assembling human liver buds containing hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs). Here, we report a reproducible, easy-to-follow, and comprehensive self-assembly protocol to generate three-dimensional (3D) human liver buds from naïve mesenchymal stem cells (MSCs), MSC-derived hepatocytes, and HSC- and LSEC-like cells. By optimizing the ratio between these different cell lineages, the cell mixture self-assembled into 3D human liver buds within 72 h in vitro, and exhibited similar characteristics with early-stage murine liver buds. In a murine model of acute liver failure, the mesenteric transplantation of self-assembled human liver buds effectively rescued animal death, and triggered hepatic ameliorative effects that were better than the ones observed after splenic transplantation of human hepatocytes or naïve MSCs. In addition, transplanted human liver buds underwent maturation during injury alleviation, after which they exhibited a gene expression profile signature similar to the one of adult human livers. Collectively, our protocol provides a promising new approach for the in vitro construction of functional 3D human liver buds from multiple human MSC-derived hepatic cell lineages; this new technique would be useful for clinical transplantation and regenerative medicine research.

scholarly journals Protocol for a developmental landscape of 3D-cultured human pre-gastrulation embryos

2019 ◽  
Author(s):  
Lifeng Xiang ◽  
Yu Yin ◽  
Tianqing Li
Keyword(s):  
Three Dimensional ◽  
Primitive Streak ◽  
Self Organization ◽  
Rna Seq ◽  
Cell Type ◽  
Blastocyst Development ◽  
Blastocyst Culture ◽  
Human Blastocyst ◽  
Human Embryogenesis ◽  
Technological Platforms

Abstract Human embryogenesis is not well understood. Knowledge detailing human pre-gastrulation embryonic development including spatial self-organization and cell type ontogeny remains limited by available two-dimensional technological platforms. Here, we present a three-dimensional (3D) blastocyst-culture system, which enables human blastocyst development through primitive streak anlage (PSA). By the 3D-platform combined with immunofluorescence imaging and single-cell RNA-Seq, we reveal a developmental landscape of human pre-gastrulation embryos. Our protocol allows recording and analysis of embryo developmental landmarks and mechanisms from human blastocysts to pre-gastrulation stage (day 14 post- fertilization).

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

In Vitro Three Dimensional Imaging of Human Carotid Atherosclerotic Plaques Using Ultrasonography

2011 ◽  
Author(s):  
Renate W. Boekhoven ◽  
Marcel C. M. Rutten ◽  
Marc R. H. M. van Sambeek ◽  
Frans N. van de Vosse
Keyword(s):  
Mechanical Properties ◽  
Carotid Artery ◽  
Carotid Endarterectomy ◽  
Early Stage ◽  
Three Dimensional ◽  
Atherosclerotic Plaques ◽  
Unstable Plaques ◽  
Human Carotid ◽  
Selection Of

Ruptured atherosclerotic plaques in the carotid artery are the main cause of stroke (70–80%). To prevent it, carotid endarterectomy is the procedure of choice in patients with a recent symptomatic 70–99% stenosis. Today, the selection of candidates is based on stenosis size only. However, endarterectomy is beneficial for only 1 out of 6 patients [1], the patients with unstable plaques (Fig. 1). Knowledge of mechanical properties of different components in the atherosclerotic arteries is important, because it will allow the identification of plaque stability at an early stage.

scholarly journals Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Marloes I. Hofstee ◽  
Martijn Riool ◽  
Igors Terjajevs ◽  
Keith Thompson ◽  
Martin J. Stoddart ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Cells ◽  
Early Stage ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Maximum Size ◽  
Human Neutrophils ◽  
Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

Glycoprotein 130 promotes human blastocyst development in vitro

2013 ◽  
Vol 99 (6) ◽  
pp. 1592-1599.e3 ◽  
Author(s):  
Fredwell Hambiliki ◽  
Jörg Hanrieder ◽  
Jonas Bergquist ◽  
Julius Hreinsson ◽  
Anneli Stavreus-Evers ◽  
...  
Keyword(s):  
Blastocyst Development ◽  
Human Blastocyst ◽  
Development In Vitro

scholarly journals Systematic Development, Validation and Optimization of a Human Embryo Culture System

2020 ◽  
Vol 1 (1) ◽  
pp. 1-14
Author(s):  
Mitchel C. Schiewe ◽  
Shane Zozula ◽  
Nancy L. Nugent ◽  
John B. Whitney ◽  
Ilene Hatch ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture System ◽  
Age Groups ◽  
Clinical Effectiveness ◽  
Single Step ◽  
Human Embryos ◽  
Blastocyst Development ◽  
Paraffin Oil ◽  
Study Results

Objective: To develop and validate a reliable in vitro culture system for human embryos. Design: Retrospective analyses of a series of four studies were conducted between 2006 and 2010 to assess the effect of incubator type (CO2 box versus Tri-gas minibox), media type, oil type, and hyaluronate supplementation. Optimization of in vitro blastocyst development was verified by assessing our National CDC/ART Surveillance reports between 2010 and 2016. Material and Methods: All patients experienced controlled ovarian hyperstimulation, followed by egg retrieval 35 h post-hCG. Cumulus-oocyte complexes were temporarily cultured in P1 or LG Fert medium plus HSA. Eggs were moved to a more complex media (G-medium or Global®-LG medium) containing a synthetic protein and embryo adhesion supplement (SPS and EAS, respectively; mLG) post-ICSI insemination. Zygotes were assigned to group culture in 25 µl droplets under oil (light mineral oil or paraffin oil; 37 °C) and embryo development was evaluated on Days 3, 5, and 6 and transferred on Day 3 to 5 depending on the number/quality of embryos available and the IVF history of the patient. Transfers were performed under ultrasound guidance, primarily using a Sureview-Wallace catheter, and enriched ET medium containing 500 µg/mL EAS. Results: Pilot study results (Expt. 1) showed that a mLG single-step medium could be effectively used in combination with Sanyo MCO-5 tri-gas (TG) incubators. Once adapted to SCIRS Lab in 2007 (Expt. 2), the latter culture system yielded improved blastocyst production and pregnancy outcomes compared to CO2 in air sequential incubation in P1/Multi-blast medium. In Expt. 3, the mLG/TG system yielded high levels of ≥2BB quality blastocysts (51 to 66%) across all age groups, and greater (p < 0.05) pregnancy success/live birth rates using fewer embryos transferred on Day 5 versus Day 3. After validating its clinical effectiveness, mLG was then prospectively compared to a new generation G-media (1.5 & 2.5; Expt. 4) and determined that the crossover treatment using paraffin oil (Ovoil™) allowed the mLG system to be optimized. Subsequently, a compilation of our Annual CDC/ART reported data over six years verified the overall viability of in vitro cultured and vitrified blastocysts produced in the mLG/TG system. Conclusion: By systematically evaluating and implementing various components of an embryo culture system we were able to optimize blastocyst development over the last decade. Our mLG/TG culture system modified an exceptionally well designed KSOMAA LG medium using endotoxin-free EAS and SPS additives to support cellular membrane wellness under stressful in vitro conditions (e.g., culture, cell biopsy, vitrification). Our use of the mLG/TG culture system has proven to be effective, creating reliably high blastocyst production, implantation, and healthy live births.

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