Project description:A human blastoid, which is an artificial human blastocyst, and has a potential as great tool to investigate fundamentals during the development and establish in vitro models to study the pregnancy failure and birth deficiency, without the use of a human embryo. In 2021, although some methods to generate blastoids have been reported, they used human naïve pluripotent stem cells, which often show genomic instability during in vitro culture. Here, we introduce the simple, robust and scalable method to generate human blastoids from more stable human primed embryonic stem cells. By using non-cell-adhesive hydrogel, hESC aggregates received the chemophysical cellular environment, and forming the asymmetric blastoid structure with the cellular distribution like a human blastocyst. The obtained blastoids also showed the capability the implantation in vitro. This model will enable to elucidate the underlying mechanisms of human pre- and post-implantation process, leading to assisted reproductive technology.
Project description:A human blastoid, which is an artificial human blastocyst, and has a potential as great tool to investigate fundamentals during the development and establish in vitro models to study the pregnancy failure and birth deficiency, without the use of a human embryo. In 2021, although some methods to generate blastoids have been reported, they used human naïve pluripotent stem cells, which often show genomic instability during in vitro culture. Here, we introduce the simple, robust and scalable method to generate human blastoids from more stable human primed embryonic stem cells. By using non-cell-adhesive hydrogel, hESC aggregates received the chemophysical cellular environment, and forming the asymmetric blastoid structure with the cellular distribution like a human blastocyst. The obtained blastoids also showed the capability the implantation in vitro. This model will enable to elucidate the underlying mechanisms of human pre- and post-implantation process, leading to assisted reproductive technology.
Project description:A human blastoid, which is an artificial human blastocyst, and has a potential as great tool to investigate fundamentals during the development and establish in vitro models to study the pregnancy failure and birth deficiency, without the use of a human embryo. In 2021, although some methods to generate blastoids have been reported, they used human naïve pluripotent stem cells, which often show genomic instability during in vitro culture. Here, we introduce the simple, robust and scalable method to generate human blastoids from more stable human primed pluripotent stem cells. By using non-cell-adhesive hydrogel, hPSC aggregates received the chemophysical cellular environment, and forming the asymmetric blastoid structure with the cellular distribution like a human blastocyst. The obtained blastoids also showed the capability the implantation in vitro. This model will enable to elucidate the underlying mechanisms of human pre- and post-implantation process, leading to assisted reproductive technology.
Project description:<p>Human embryonic stem cells (hESCs) can self-renew infinitely or differentiate into the 3 germ layer lineages<em> in vitro</em> with certain cues, holding great promise to model early human embryo development <em>ex vivo</em>. It is wildly accepted that hESCs take advantage of both glycolysis and mitochondrial respiration to favor the naïve pluripotency, while prefer glycolysis to support the primed pluripotency. Thus, the function of mitochondrial respiration in primed hESCs has been underestimated for a long time, and has not been fully understood yet. Herein, we report that the adenosine triphosphate (ATP) production rate is comparable between mitochondrial respiration and glycolysis, suggesting that mitoATP may serve as one of the major sources of the ATP pool in primed hESCs. To further reveal the function of mitochondrial respiration, we deployed inducible CRISPRi method to inhibit OGDH expression with high efficiency, resulting in the TCA cycle blockade as well as the diminishment of mitochondrial respiration activity and total ATP level. Of note, OGDH deficiency led to the exit of primed pluripotency accompanied by cell death. Furthermore, the treatment with small molecule inhibitors to block electron transport chain (ETC) can phenocopy the loss-of-function of OGDH in hESCs. Therefore, genetic and pharmacological perturbations on mitochondrial respiration can impair the stemness of primed hESCs. Collectively, the current study unveils that OGDH acts as a key regulator to fine-tune the abundance of TCA cycle metabolites to ensure the mitochondrial respiration activity in primed hESCs, and highlights the pivotal roles of mitochondrial respiration in terms of ATP production and the maintenance of primed pluripotency. Our findings may provide insights into the linkage between pluripotent states and energy metabolism in hESCs.</p>
Project description:We did bulk and single cell RNA sequencing of blastocysts, blastoids, trophoblast stem cells (TSC) and embryonic stem cells (ESC). The goal of these experiment is to describe the transformations of the transcriptome occurring within cells (TSC, ESC) upon formation of a blastoid. E3.25 and E3.5 blastocysts are used as controls. To this end, we first did RNA sequencing of intact structures (E3.25 and E3.5 blastocysts, blastoids, and parental cell lines). In a different series of experiments, we micro-dissected blastocyst, blastoid or trophosphere structures into single cells, and sequenced their mRNAs, to infer cell identity and transcriptome variations.