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Cryopreservation of human embryonic stem cells and hepatocytesChen, Shi January 2013 (has links)
No description available.
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Experimental studies on the development of haemopoietic tissueMoore, Malcolm A. S. January 1967 (has links)
No description available.
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Characterization of Primary Cilia and Intraflagellar Transport 20 in the EpidermisSu, Steven January 2020 (has links)
Mammalian skin is a dynamic organ that constantly undergoes self-renewal during homeostasis and regenerates in response to injury. Crucial for the skin’s self-renewal and regenerative capabilities is the epidermis and its stem cell populations. Here we have interrogated the role of primary cilia and Intraflagellar Transport 20 (Ift20) in epidermal development as well as during homeostasis and wound healing in postnatal, adult skin. Using a transgenic mouse model with fluorescent markers for primary cilia and basal bodies, we characterized epidermal primary cilia during embryonic development as well as in postnatal and adult skin and find that both the Interfollicular Epidermis (IFE) and hair follicles (HFs) are highly ciliated throughout development as well as in postnatal and adult skin. Leveraging this transgenic mouse, we also developed a technique for live imaging of epidermal primary cilia in ex vivo mouse embryos and discovered that epidermal primary cilia undergo ectocytosis, a ciliary mechanism previously only observed in vitro. We also generated a mouse model for targeted ablation of Ift20 in the hair follicle stem cells (HF-SCs) of adult mice. We find that loss of Ift20 in HF-SCs inhibits ciliogenesis, as expected, but strikingly it also inhibits hair regrowth. Closer examination of these mice reveals that Ift20 is crucial in maintaining HF-SC identity. Specifically, ablation of Ift20 in HF-SCs results in loss of SOX9 expression in HF-SCs and results in ectopic expression of the IFE marker KLF5 in HF-SCs. Additionally, ectopic differentiation is observed in HF-SCs following loss of Ift20. Finally, using both in vitro and in vivo models, we also characterize the role of primary cilia and Ift20 in epidermal wound healing. We find that loss of Ift20 slows collective keratinocyte migration in vitro and also slows HF-SC migration in vivo during wound repair. Interestingly our data suggests that Ift20 regulates keratinocyte migration in a primary cilia-independent manner. Instead, we find that Ift20 mediates focal adhesion (FA) turnover during keratinocyte migration. Specifically, Ift20 together with Rab5, regulates recycling of FA integrins and loss of Ift20 inhibits proper return of integrins to the keratinocyte surface. Overall, we demonstrate that the epidermis is highly ciliated throughout development and in postnatal skin. We show that Ift20 is crucial in maintaining HF-SC identity and the telogen to anagen transition in HFs. We finally demonstrate that Ift20 regulates keratinocyte migration independent of its function in ciliogenesis and instead regulates recycling of FA integrins through a Rab5 dependent mechanism.
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Zc3h13: A Master Regulator of Epitranscriptomic Landscape during Early DevelopmentChirathivat, Napon January 2021 (has links)
Mouse epiblast stem cells (EpiSC) are pluripotent cells derived of the epiblast of post-implantation blastocysts that can self-renew indefinitely in culture, display lineage-restricted differentiation, and appear to closely resemble human embryonic stem cells (ESC). Despite significant advances in the last decade, the precise molecular mechanisms and many master regulator (MR) genes underlying stem cell self-renewal, pluripotency, interactions with surrounding cells, and lineage-specific differentiation still remain elusive. The goal of this thesis is to address these gaps of knowledge using a systematic approach to identify novel MR genes and functionally validate them using genetically modified mouse models.In order to elucidate MR genes that control understudied biological processes, previous work in the Shen lab have computationally reconstructed the regulatory network of EpiSC and interrogated the EpiSC interactome with pluripotency signatures of EpiSC lines. One MR gene of interest from the previous analysis is ZC3H13, which encodes a protein that has been previously shown to be a crucial for N6-methyladenosine modification in RNA (m⁶A). This suggests a novel connection between m⁶A epitranscriptional modifications and primed state pluripotency.
In my thesis research, I have shown that Zc3h13 is essential for proper trophoblast lineage differentiation and the importance of m6A modifications in early embryonic development. Using two Zc3h13 knockout mouse lines, I have found that Zc3h13 null embryos are embryonic lethal at the peri-implantation stage due to a failure to implant into the uterus. In vitro outgrowth analysis revealed a lack of trophoblast giant cells in Zc3h13 null outgrowths, and the lack of enlarged nuclei in the Zc3h13 null outgrowth suggests a failure in endoreduplication. Immunofluorescence analysis of Zc3h13 null blastocysts showed that the trophectoderm cells of Zc3h13 null blastocyst expressed trophectoderm specific factors at abnormal levels, indicating a severe dysregulation of the trophectoderm regulatory network.
To elucidate the effects of Zc3h13 knockout on pluripotency, I also performed a detailed immunofluorescence analysis of Zc3h13 null inner cell mass (ICM), which expressed pluripotency factors at normal levels. However, Zc3h13 null blastocysts were less efficient at generating ESC lines and the Zc3h13 KO ESC generated were morphologically abnormal. Dot blot and mass spectrometry analysis showed that Zc3h13 KO ESC had a dramatically lower level of m⁶A modification, suggesting a connection between m6A epitranscriptional modification and endoreduplication. Interestingly, chimera and teratoma analysis showed that while Zc3h13 KO ESC can contribute to derivatives of the three primary lineages, Zc3h13 KO ESC has a bias towards neuroectoderm differentiation.
In this thesis, I have shown the importance of m6A transcriptional regulation in trophoblast giant cell differentiation. Taken together, my studies can help further the understanding of the biological functions of m⁶A modifications as well as the relationship between transcriptional regulation and cell fate transition. My work highlights another level of gene regulation through epitranscriptional modification and the importance of the epitranscriptomic landscape in cell fate transition and development.
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Bioactive factors secreted by differentiating embryonic stem cellsNgangan, Alyssa V. 07 July 2011 (has links)
Current therapeutic strategies to stimulate endogenous angiogenic processes within injured tissue areas are typically based on introducing exogenous pro-angiogenic molecules or cell populations. Stem cell transplantation for angiogenic therapy aims to deliver populations of cells that secrete angiogenic factors and/or engraft in the new branching vasculature within the damaged tissue. Utilizing stem or progenitor cells has been shown to induce a rather robust angiogenic response despite minimal repopulation of the host vasculature, suggesting that stem cells may provide paracrine factors that transiently induce endogenous angiogenesis of tissues undergoing regeneration. Early differentiating embryonic stem cell (ESC) aggregates, referred to as embryoid bodies (EBs), can undergo vasculogenic differentiation, and also produce extracellular matrix and growth factors that induce proliferation, differentiation, and tissue morphogenesis. Taken together, the ESC extracellular environment may be an effective means by which to manipulate cell behavior. Thus, the objective of this project was to harness morphogens derived from ESCs undergoing differentiation and analyze their bioactive potential. To examine the expression of extracellular factors within EBs, gene expression arrays in conjunction with a variety of analytical tools were utilized to gain an understanding of the importance of extracellular factors in ESC differentiation. Furthermore, the soluble fraction of secreted factors contained within EB-conditioned media was compared to the matrix-associated factors produced by EBs, which led to the development of novel ESC-derived matrices via mechanical acellularization methods. Acellular embryonic stem cell-derived matrices demonstrated the retention of bioactive factors that impacted aspects of angiogenesis. In conclusion, extracellular factors were modulated in response to the progression of EB differentiation and can further be harnessed via acellularization techniques, in order to deliver bioactive ESC-secreted factors in a cell-free manner.
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Federal regulation of human embryonic stem cell research.Crocker, Catherine L. Franzini, Luisa, Schroder, Gene D. January 2008 (has links)
Source: Masters Abstracts International, Volume: 47-02, page: 0981. Adviser: Luisa Franzini. Includes bibliographical references.
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How to regulate embryo research? : a procedural approachChampon, Benoit January 2003 (has links)
Over the past few years, embryo research has been a widely discussed topic. New techniques such as embryo stem cell research or therapeutic cloning are considered by scientists to be very promising. Nevertheless, opponents of these experimentations warn against the commodification of human life forms and argue that the moral status of embryos should protect them from being destroyed purely for research. / Legislations on this topic have been enacted in most Western countries, though they are still much criticised. Is there an adequate way of regulating embryo research? Our argument suggests that consensus can only be procedurally obtained. That is, we believe that only legislative assemblies should have authority to take a position on this controversial topic, which is subject to moral disagreement, and as such, judges should only have a minor role.
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Genome stability in the preimplantation embryoZuccaro, Michael V. January 2021 (has links)
The mammalian zygote and resulting embryo is the starting point of life, and thus must overcome continuous insult from DNA stress and damage while maintaining genome stability and integrity. This thesis examines genome stability in the context of chromosome changes, both in the context of ploidy and whole genome duplications as well as double-strand DNA breakage and chromosome loss.
Regarding the ploidy portion of this work, while possible to derive and maintain, mammalian haploid stem cells undergo spontaneous, irreversible diploidization. Here, we investigated the mechanisms driving diploidization using human and mouse embryos, and human embryonic stem cells experimental systems. We demonstrate that diploidization occurs early in development and is often unproductive, with diploidized cells failing to contribute to the developing embryo. Diploidization involves delayed mitotic progression, incomplete alignment of chromosomes, and occurs through mitotic slippage or failed cytokinesis after exit from mitosis without formation of a midbody. Diploidization is associated with DNA damage and aneuploidies, with an upstream component being a decreased nuclear to cytoplasmic ratio. Increasing this ratio in haploid mouse embryos improves developmental outcomes and decreasing this ratio in diploids results in poor outcomes. A sensor of the nuclear to cytoplasmic ratio, CHK1, is required for haploid maintenance as inhibition increases binucleation and diploidization in haploid human embryonic stem cells. Thus, we demonstrate the earliest upstream driver of diploidization as being the nuclear-cytoplasmic ratio in haploid mammalian cells, rather than the actual haploid state.
Regarding the double-strand DNA breakage portion of this work, the preferred mechanism by which human embryos repair double-strand breaks was investigated. Utilizing allele-specific CRISPR-Cas9 cleavage, we show that human embryos repair double-strand breaks primarily through non-homologous end joining. In embryos left unrepaired or misrepaired, partial or whole chromosome loss occurs, which can be easily overlooked and misinterpreted with common on-target analyses such as PCR. Off-target Cas-9 activity recapitulated findings on an entirely separate chromosome, confirming the preference of the human embryo for non-homologous end joining and microhomology-mediated end joining, as well as chromosome loss where repair was unsuccessful.
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Engineering microenvironmental cues for guiding stem cell fatePark, Ji Sun January 2020 (has links)
Injury, aging, and congenital disabilities of the muscular and neural systems impose a significant burden on patients and their families. Due to the tissue’s limited regenerative capacity, effective treatment interventions for restoring progressive damage is still lacking. Cell replacement therapy is primarily limited by the restricted supply of viable donor cells and variable graft survival. For addressing these limitations, we propose new strategies to obtain a target cell of interest from an autologous cell source. Herein, we engineer cell fate decisions by 1) harnessing host microenvironment and the CRISPR/dCas9-mediated transcriptional activation system to promote myogenesis of human endothelial progenitor cells (EPCs); and 2) employing substrate-mediated biophysical cues with soluble factors (biochemical cues) to drive cell commitment to neuronal lineages.
For the first strategy, we hypothesized that therapeutic cells could be obtained in situ by employing the CRISPR/dCas9 system to engineer cell fate in the host tissue. Using this system, we transactivated MYOD1, a master regulator for myogenesis, to directly reprogram primary EPCs to skeletal myoblasts (SkMs). EPCs were chosen as a cell source for their easy accessibility, high proliferation, and potential contribution to regenerate vasculature and musculature tissue. The early myogenic commitment of EPCs was confirmed in vitro by MYOD1 expression, which yielded a 230-fold higher induction than the original EPCs. These cells were then transplanted for assessing their therapeutic efficacy in myotoxin-induced muscle injury model in immunodeficient mice. A one-month post-injury study resulted in the integration of induced SkMs to the injured host tissue, promotion of neoangiogenesis, and reduction in fibrotic scar formation. These findings indicate that CRISPR/Cas9-mediated target gene activation can be achieved in situ to accelerate muscle regeneration after myotoxin-induced damage.
For the second strategy, we utilized both soluble and insoluble factors to convert the cell fate of neural stem/progenitor and somatic cells to various neuronal lineages, including motor neurons (MNs) and dopaminergic (DA) neurons. For soluble factors, cells were exposed to various biochemical factors, inspired by the neuronal niche environs during the natural developmental process. For insoluble factors, the conductive graphene substrate was used to support the endogenous electrical signal between neurons for enhancing the neuronal phenotypes and their functionality. We postulated that exposing the cells to these collective stimuli in vitro can alter their intrinsic signaling pathway to tailor their fate to neuronal lineages. To test the hypothesis, neural stem/progenitor and somatic cells were cultured on various substrates with or without electroactive graphene and aligned patterns. After two weeks to one month of cell fate induction in the chemically defined conditions, our results implied that cell adhesion, survival, neurite outgrowth, and maturation were facilitated on the electroactive substrates with aligned patterns compared to the control platforms.
Taken together, our results in this dissertation demonstrate the feasibility of tailoring the donor cell fates within or across the germ layers. We achieved this by employing a transcriptional gene activation system and tunable microenvironmental cues elicited by soluble (chemical and growth factors) and insoluble (physical cues from the substrate) factors. Utilizing such strategies hold great promise for elucidating the optimal conditions to guide cell fate to target lineages. This work provides a rational basis for establishing a robust protocol and an in vitro culture platform to module cell fate decisions that could help realize the autologous cell-based therapy for muscular and neurodegenerative diseases.
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How to regulate embryo research? : a procedural approachChampon, Benoit January 2003 (has links)
No description available.
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