The discovery of induced pluripotent stem cells (iPSCs) circumvents the ethical concerns of human embryonic stem cells, which fasten the stem cell research. After that, various types of transfection methods have emerged to induce cellular reprogrammig, in order to generate iPSCs in an effective and safe manner. Out of all the integrating and non-integrating ways, message RNA (mRNA) reprogramming is found to be one of the most efficient and safe methods, which lays also the basis of my project. Combining with the expertise of microfabrication, we tested the mRNA reprogramming in a microfluidic chip system and found out that the cellular reprogramming was improved greatly (~50folds) comparing to the standard cell culture conditions, like the multi-well plate of petri dishes. Thus, by making the fully use of microfluidics-based mRNA reprogramming, we explored the reprogramming process itself and later the applications of iPSCs. The first chapter contains brief but fundamental background information for this work. It tells us how the iPSCs have been discovered and the advantages of non- integrating method, mainly on the mRNA transfection ways. Also, we introduce illustrate how the human embryo development in vivo and the demanding in vitro 3D organoids models to study embryogenesis. Basic principle of microfabrication is also introduced. The second chapter talks about the main aim of my whole project. The thrid chapter gives detalied information to all the experiment materials and protocols that I have been used in this project. The fourth result chapter is the core chapter. Here I listed the results obtained until now. In the this charpter, to anwser the first question of exploring the reprogramming process and identify the intermediate stages, cell populations, gene trajactory and possible signaling pathway, we make use of highy efficient reprogramming in microfluidics by mRNA transfection and temporal multi-omics. I We combined secretome analysis with single-cell transcriptomics to reveal the functional extrinsic protein communication channels between reprogramming sub- populations and the reshaping of a favorable extracellular environment. We moved forward with the help of the microfluidic culture setup by objectively identifying the reprogramming subpopulation trajectories and relationships based on an integrative secreted proteome and scRNA-seq study. The former found several secreted cytokines, growth factors, and extracellular matrix(ECM)-related proteins that were truly present in the extracellular space during reprogramming and helped to construct an environmental signaling system that resembled the early embryonic basal lamina. Two primary pathways during reprogramming were found by scRNA-seq. One of them was entirely committed to secretory activity and the other one was committed to cellular reprogram. Previous investigations may have overlooked the importance of the extracellular environment by failing to recognize immature hiPSCs as a secretome target due to their low abundance or limited secretory activity. On the basis that cellular reprogramming is a transcriptional factores driven processm our findings combine also the idea that human cellular reprogramming is dependent on extracellular context and cell populations, in this work, we followed an unbiased approach that supported the idea that the route to pluripotency can be broadened by cell-non-autonomous mechanisms. Paracrine signalling is established by highly regulated dynamics with multi-factorial contribution. We identified the HGF/MET/STAT3 axis as a significant facilitator of reprogramming that functions through HGF accumulation in the constrained environment of microfluidics but requires exogenous supply in standard dishes to increase efficiency. We showed the use of HGF for gain of function during reprogramming in a conventional culture system, but this efficiency was amenable to further enhancement when multifactorial contributions were used.

The discovery of induced pluripotent stem cells (iPSCs) circumvents the ethical concerns of human embryonic stem cells, which fasten the stem cell research. After that, various types of transfection methods have emerged to induce cellular reprogrammig, in order to generate iPSCs in an effective and safe manner. Out of all the integrating and non-integrating ways, message RNA (mRNA) reprogramming is found to be one of the most efficient and safe methods, which lays also the basis of my project. Combining with the expertise of microfabrication, we tested the mRNA reprogramming in a microfluidic chip system and found out that the cellular reprogramming was improved greatly (~50folds) comparing to the standard cell culture conditions, like the multi-well plate of petri dishes. Thus, by making the fully use of microfluidics-based mRNA reprogramming, we explored the reprogramming process itself and later the applications of iPSCs. The first chapter contains brief but fundamental background information for this work. It tells us how the iPSCs have been discovered and the advantages of non- integrating method, mainly on the mRNA transfection ways. Also, we introduce illustrate how the human embryo development in vivo and the demanding in vitro 3D organoids models to study embryogenesis. Basic principle of microfabrication is also introduced. The second chapter talks about the main aim of my whole project. The thrid chapter gives detalied information to all the experiment materials and protocols that I have been used in this project. The fourth result chapter is the core chapter. Here I listed the results obtained until now. In the this charpter, to anwser the first question of exploring the reprogramming process and identify the intermediate stages, cell populations, gene trajactory and possible signaling pathway, we make use of highy efficient reprogramming in microfluidics by mRNA transfection and temporal multi-omics. I We combined secretome analysis with single-cell transcriptomics to reveal the functional extrinsic protein communication channels between reprogramming sub- populations and the reshaping of a favorable extracellular environment. We moved forward with the help of the microfluidic culture setup by objectively identifying the reprogramming subpopulation trajectories and relationships based on an integrative secreted proteome and scRNA-seq study. The former found several secreted cytokines, growth factors, and extracellular matrix(ECM)-related proteins that were truly present in the extracellular space during reprogramming and helped to construct an environmental signaling system that resembled the early embryonic basal lamina. Two primary pathways during reprogramming were found by scRNA-seq. One of them was entirely committed to secretory activity and the other one was committed to cellular reprogram. Previous investigations may have overlooked the importance of the extracellular environment by failing to recognize immature hiPSCs as a secretome target due to their low abundance or limited secretory activity. On the basis that cellular reprogramming is a transcriptional factores driven processm our findings combine also the idea that human cellular reprogramming is dependent on extracellular context and cell populations, in this work, we followed an unbiased approach that supported the idea that the route to pluripotency can be broadened by cell-non-autonomous mechanisms. Paracrine signalling is established by highly regulated dynamics with multi-factorial contribution. We identified the HGF/MET/STAT3 axis as a significant facilitator of reprogramming that functions through HGF accumulation in the constrained environment of microfluidics but requires exogenous supply in standard dishes to increase efficiency. We showed the use of HGF for gain of function during reprogramming in a conventional culture system, but this efficiency was amenable to further enhancement when multifactorial contributions were used.

EXPLORE CELLULAR REPROGRAMMING PROCESS AND MICROFLUIDICS-BASED NASCENT IPSCS FOR INVESTIGATING HUMAN DEVELOPMENT IN VITRO / Qin, Wei. - (2023 Jan 26).

EXPLORE CELLULAR REPROGRAMMING PROCESS AND MICROFLUIDICS-BASED NASCENT IPSCS FOR INVESTIGATING HUMAN DEVELOPMENT IN VITRO

QIN, WEI
2023

Abstract

The discovery of induced pluripotent stem cells (iPSCs) circumvents the ethical concerns of human embryonic stem cells, which fasten the stem cell research. After that, various types of transfection methods have emerged to induce cellular reprogrammig, in order to generate iPSCs in an effective and safe manner. Out of all the integrating and non-integrating ways, message RNA (mRNA) reprogramming is found to be one of the most efficient and safe methods, which lays also the basis of my project. Combining with the expertise of microfabrication, we tested the mRNA reprogramming in a microfluidic chip system and found out that the cellular reprogramming was improved greatly (~50folds) comparing to the standard cell culture conditions, like the multi-well plate of petri dishes. Thus, by making the fully use of microfluidics-based mRNA reprogramming, we explored the reprogramming process itself and later the applications of iPSCs. The first chapter contains brief but fundamental background information for this work. It tells us how the iPSCs have been discovered and the advantages of non- integrating method, mainly on the mRNA transfection ways. Also, we introduce illustrate how the human embryo development in vivo and the demanding in vitro 3D organoids models to study embryogenesis. Basic principle of microfabrication is also introduced. The second chapter talks about the main aim of my whole project. The thrid chapter gives detalied information to all the experiment materials and protocols that I have been used in this project. The fourth result chapter is the core chapter. Here I listed the results obtained until now. In the this charpter, to anwser the first question of exploring the reprogramming process and identify the intermediate stages, cell populations, gene trajactory and possible signaling pathway, we make use of highy efficient reprogramming in microfluidics by mRNA transfection and temporal multi-omics. I We combined secretome analysis with single-cell transcriptomics to reveal the functional extrinsic protein communication channels between reprogramming sub- populations and the reshaping of a favorable extracellular environment. We moved forward with the help of the microfluidic culture setup by objectively identifying the reprogramming subpopulation trajectories and relationships based on an integrative secreted proteome and scRNA-seq study. The former found several secreted cytokines, growth factors, and extracellular matrix(ECM)-related proteins that were truly present in the extracellular space during reprogramming and helped to construct an environmental signaling system that resembled the early embryonic basal lamina. Two primary pathways during reprogramming were found by scRNA-seq. One of them was entirely committed to secretory activity and the other one was committed to cellular reprogram. Previous investigations may have overlooked the importance of the extracellular environment by failing to recognize immature hiPSCs as a secretome target due to their low abundance or limited secretory activity. On the basis that cellular reprogramming is a transcriptional factores driven processm our findings combine also the idea that human cellular reprogramming is dependent on extracellular context and cell populations, in this work, we followed an unbiased approach that supported the idea that the route to pluripotency can be broadened by cell-non-autonomous mechanisms. Paracrine signalling is established by highly regulated dynamics with multi-factorial contribution. We identified the HGF/MET/STAT3 axis as a significant facilitator of reprogramming that functions through HGF accumulation in the constrained environment of microfluidics but requires exogenous supply in standard dishes to increase efficiency. We showed the use of HGF for gain of function during reprogramming in a conventional culture system, but this efficiency was amenable to further enhancement when multifactorial contributions were used.
EXPLORE CELLULAR REPROGRAMMING PROCESS AND MICROFLUIDICS-BASED NASCENT IPSCS FOR INVESTIGATING HUMAN DEVELOPMENT IN VITRO
26-gen-2023
The discovery of induced pluripotent stem cells (iPSCs) circumvents the ethical concerns of human embryonic stem cells, which fasten the stem cell research. After that, various types of transfection methods have emerged to induce cellular reprogrammig, in order to generate iPSCs in an effective and safe manner. Out of all the integrating and non-integrating ways, message RNA (mRNA) reprogramming is found to be one of the most efficient and safe methods, which lays also the basis of my project. Combining with the expertise of microfabrication, we tested the mRNA reprogramming in a microfluidic chip system and found out that the cellular reprogramming was improved greatly (~50folds) comparing to the standard cell culture conditions, like the multi-well plate of petri dishes. Thus, by making the fully use of microfluidics-based mRNA reprogramming, we explored the reprogramming process itself and later the applications of iPSCs. The first chapter contains brief but fundamental background information for this work. It tells us how the iPSCs have been discovered and the advantages of non- integrating method, mainly on the mRNA transfection ways. Also, we introduce illustrate how the human embryo development in vivo and the demanding in vitro 3D organoids models to study embryogenesis. Basic principle of microfabrication is also introduced. The second chapter talks about the main aim of my whole project. The thrid chapter gives detalied information to all the experiment materials and protocols that I have been used in this project. The fourth result chapter is the core chapter. Here I listed the results obtained until now. In the this charpter, to anwser the first question of exploring the reprogramming process and identify the intermediate stages, cell populations, gene trajactory and possible signaling pathway, we make use of highy efficient reprogramming in microfluidics by mRNA transfection and temporal multi-omics. I We combined secretome analysis with single-cell transcriptomics to reveal the functional extrinsic protein communication channels between reprogramming sub- populations and the reshaping of a favorable extracellular environment. We moved forward with the help of the microfluidic culture setup by objectively identifying the reprogramming subpopulation trajectories and relationships based on an integrative secreted proteome and scRNA-seq study. The former found several secreted cytokines, growth factors, and extracellular matrix(ECM)-related proteins that were truly present in the extracellular space during reprogramming and helped to construct an environmental signaling system that resembled the early embryonic basal lamina. Two primary pathways during reprogramming were found by scRNA-seq. One of them was entirely committed to secretory activity and the other one was committed to cellular reprogram. Previous investigations may have overlooked the importance of the extracellular environment by failing to recognize immature hiPSCs as a secretome target due to their low abundance or limited secretory activity. On the basis that cellular reprogramming is a transcriptional factores driven processm our findings combine also the idea that human cellular reprogramming is dependent on extracellular context and cell populations, in this work, we followed an unbiased approach that supported the idea that the route to pluripotency can be broadened by cell-non-autonomous mechanisms. Paracrine signalling is established by highly regulated dynamics with multi-factorial contribution. We identified the HGF/MET/STAT3 axis as a significant facilitator of reprogramming that functions through HGF accumulation in the constrained environment of microfluidics but requires exogenous supply in standard dishes to increase efficiency. We showed the use of HGF for gain of function during reprogramming in a conventional culture system, but this efficiency was amenable to further enhancement when multifactorial contributions were used.
EXPLORE CELLULAR REPROGRAMMING PROCESS AND MICROFLUIDICS-BASED NASCENT IPSCS FOR INVESTIGATING HUMAN DEVELOPMENT IN VITRO / Qin, Wei. - (2023 Jan 26).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3471271
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