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Establishment of 3D culture protocols for the maintenance and expansion of human pluripotent stem cell aggregates in a low scale platform and in the DASbox® Mini-Bioreactor SystemHernandez-Bautista, Carlos Alberto 27 July 2022 (has links)
The human Embryonic Stem Cells (hESCs) and human induced Pluripotent Stem Cells (hiPSCs) have offered numerous advantages including but not limited to model diseases, high-throughput drug screening, and regenerative purposes. However, the employment of monolayer cultures has not been sufficient to mimic the in vivo stem cells niche. Thus, three-dimensional suspension cultures have helped us to advance our knowledge and ease the development of the human organs’ counterparts, commonly referred as organoids. Currently, the challenge is the generation of homogenous and reproducible human Pluripotent Stem Cell (hPSC) aggregates, the basic cellular unit to derive organoids. To date, the Ultra-Low Attachment (ULA) 6-well plates have been routinary used for the hPSC aggregates formation, which mainly relies on the inhibition of the Rho-associated kinase (ROCK) pathway resulting in the enhancement of cell survival coming from cryopreserved stocks or when passaging. However, little is known in this regard when analyzing the aggregate formation of hPSCs with two widely used compounds: RevitaCellTM Supplement and Y27632. Importantly, due to the high demand required from the regenerative medicine, I aimed to upscale the hPSC aggregates production in the DASbox® Mini-Bioreactor System. In this thesis, I established protocols for the hPSC aggregates formation by using two different types of media in two platforms being the ULA 6-well plates and the DASbox® Mini-Bioreactor System. In addition, I demonstrated that monolayer confluence cultures before single cell inoculations are paramount for the formation of bona fide hPSC aggregates in healthy and X aneuploid hiPSCs, precisely two hESCs and five hiPSCs.
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Towards the in vitro production of haematopoietic stem cells : lessons from the early human embryoEasterbrook, Jennifer Elizabeth January 2018 (has links)
The production of fully functional haematopoietic stem cells (HSCs) for clinical transplantation is a highly sought after goal in the field of regenerative medicine. Given their capacity for extensive self-renewal and differentiation into any cell type, human pluripotent stem cells (hPSCs) provide a potentially limitless source of haematopoietic cells in vitro for clinical application. However, to date, fully functional HSCs have not been produced from hPSCs without the overexpression of transcription factors. In this study I first investigated the production of HSCs and haematopoietic progenitor cells (HPCs) in an established clinical-grade haematopoietic differentiation protocol. I demonstrated the efficient and reproducible production of HPCs but showed that the strategy did not produce fully functional HSCs that could repopulate the haematopoietic system of immune-deficient mice. Modification of the protocol by manipulation of the hedgehog signalling pathway and co-aggregation with OP9 stromal cells did not provide any significant enhancement of HPC production. To gain the required knowledge with which to improve our current protocol, I therefore switched my focus towards studying the development of HSCs in the early human embryo. It has been shown that HSCs first emerge from the ventral wall of the dorsal aorta in the aorta-gonad-mesonephros (AGM) region of the human embryo but the precise location and the mechanisms underpinning this process remain unknown. In this study, I established a culture system to map the spatio-temporal distribution of HSCs and to investigate the presence of HSC precursors. I showed that embryonic HSCs emerge predominantly around and above the vitelline artery entry point in the dorsal aorta and can be maintained in our explant culture system. I then performed RNA-sequencing of cells derived from AGM sub-regions, and this identified molecular signatures which could potentially underlie the ventral polarity of HSC emergence in the AGM. To elucidate the role of the stromal compartment in this unique haematopoietic niche, I derived stromal cell lines from the human AGM region and showed they were capable of supporting haematopoiesis in vitro. This work has provided some important insights into the mechanisms regulating HSC development in the human AGM region and identified interesting candidate molecules for future testing in differentiation protocols. This knowledge brings us a step closer to the successful in vitro production of HSCs for clinical use.
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Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture / 3D培養を用いたヒト多能性幹細胞から内胚葉系譜の発生に沿ったインスリン産生細胞への誘導Takeuchi, Hiroki 23 July 2014 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18511号 / 医博第3931号 / 新制||医||1006(附属図書館) / 31397 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 上本 伸二, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Building a better Placode: Modeling Neural Plate Border interactions with hPSCsBlair, Joel 05 October 2021 (has links)
No description available.
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NON-CODING RNA REGULATORS INDUCE HUMAN CARDIOMYOCYTE PROLIFERATIONYibo Xu (8520990) 21 June 2022 (has links)
Adult mammalian <a></a><a>cardiomyocytes </a>(CMs, or heart muscle cells) have little, if any, ability to proliferate in response to injury, and after myocardial infarction this defect underlies the poor regenerative ability of human hearts. In contrast, early stage of CMs (such as fetal CMs) still have some ability to proliferate, and we seek to identify novel gene regulators as potential therapeutic targets for heart regeneration. Here we use human pluripotent stem cells (hPSCs) as an in vitro human model to investigate the roles of emerging long non-coding RNAs (lncRNAs), with the lengths of over 200 nucleotides are able to be transcribed but not translated into protein, for heart regeneration. With public available RNA-sequencing data, we identified several human genes, including lncRNAs, that are highly enriched in fetal CMs. We generated targeted gene knockout hPSC lines using CRISPR/Cas9-mediated genome editing and will use them to study the roles of selected genes in regulating CM proliferation. To identify more therapeutic targets, we also generated a fluorescence ubiquitination cell cycle indicator (FUCCI) reporter cell line that express either green (indicating dividing cells) or red fluorescence (indicating non-dividing cells), on which we’ll perform unbiased genome-wide screening to identity genes that regulate CM proliferation. High-throughput chemical screening will also be performed on FUCCI reporter lines to identify potential therapeutic drugs for heart regeneration.
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