acase@tulane.edu / Cancer’s response to mechanical vibration via high-intensity focused ultrasound and disruptive chemical agents (Mechanochemical Disruption) was examined in vitro and in vivo. We demonstrated that mechanochemical disruption of cellular structures induced phenotypic alterations in surviving tumor cells that prevented cancer progression. Mechanochemical disruption inhibited uncontrolled proliferation, tumorigenicity, metastatic development, and re-sensitized multiple cancer types to chemical treatment via alterations in protein expression and impediment of pro-survival signaling. Our study identified a novel curative therapeutic approach that can prevent the development of aggressive cancer phenotypes. / 1 / hakm murad

Direct in vivo cellular reprogramming offers the potential for local neural replacement to promote post-stroke neural regeneration and recovery. Pericytes are perivascular cells involved in blood-brain barrier maintenance under physiological conditions but are reprogrammed into multipotent induced neural progenitor cells (i-NPCs) in response to ischemia. The atypical protein kinase C (aPKC)-CREB binding protein (CBP) pathway regulates ischemia-activated pericyte (a-pericyte) reprogramming and neuronal differentiation. Our previous work showed that the pathway inhibitor compound C (CpdC) facilitated a- pericyte reprogramming into i-NPCs in culture, and that monoacylglycerol lipase (Mgll) inhibitor JZL184 was able to promote NPC differentiation to generate newborn neurons by mimicking pathway activation. In this regard, we propose to use acute CpdC treatment followed by chronic JZL184 treatment to enhance reprogramming of a-pericytes into i-NPCs and subsequently promote their neuronal differentiation, ultimately improving post-stroke functional recovery. Using the endothelin-1 (ET-1)/L-NAME ischemic stroke model in a pericyte lineage tracing transgenic mouse line, I characterized the ability of a-pericytes in the ischemic lesion site to generate neural (i-NPC, newborn neurons) and non-neural (microglia and fibroblasts) cell types. The CpdC+JZL184 treatment had early effects on enhancing a- pericyte reprogramming efficiency to produce i-NPCs at 7 days post-stroke and promoting their differentiation to generate neuroblasts at 14 days post-stroke. However, it did not affect stroke volume and produced minimal alterations to the pattern of post-stroke motor function recovery. Interestingly, I discovered a novel role of tamoxifen treatment prior to stroke in regulating reprogramming of a-pericytes and efficacy of compound C treatment. These studies inform the necessity of optimization of drug delivery for better control over the timing and duration to directly target in vivo i-NPC reprogramming and reveal a novel pharmacological paradigm to control the aPKC-CBP pathway.

Bronchopulmonary dysplasia (BPD), the chronic lung disease that develops in premature babies following mechanical ventilation and oxygen exposure, is the most common complication of extreme prematurity. Currently, there is no cure for BPD. Increasing evidence indicates early-onset emphysema and pulmonary vascular disease in survivors with BPD (Aukland et al., 2006; Wong et al., 2008), suggesting an irreversible arrest in lung growth and/or premature lung aging resulting in life-long health problems (J. Sucre et al., 2021). Transient in vivo cellular reprogramming through the activation of the Yamanaka reprogramming factors Oct4, Sox2, Klf4, c-Myc (OSKM), ameliorate cellular and physiological hallmarks of aging and to promote tissue regeneration and improve organ function after injury. (Chen et al., 2021a; Hishida et al., 2022b; Lu et al., 2020) This thesis focuses on determining if transient in vivo cellular reprogramming can regenerate an established lung injury in a BPD mouse model. Two strategies, (a) Adeno-Associated virus (AAV) mediated transient overexpression of the OSK factors and (b) using a transgenic reprogrammable mouse line to overexpress the OSKM factors were employed to test the efficiency of in vivo cellular reprogramming in regenerating the lungs. Both the strategies, under the conditions tested, did not regenerate established lung injury in a BPD mouse model but the feasibility of both these strategies was established here laying a foundation for the next phase of the study.

The derivation of pluripotent stem cells (now termed induced pluripotent stem cells, iPSC) from mature somatic cells was a finding of seminal importance to fundamental cell biology. Thus established iPSC technology has been predicted to advance fields that previously relied on the ethically disputed use of embryonic stem cells. Being pluripotent (able to differentiate into every cell type present in the human body) and sharing most other characteristics with embryonic stem cells, but being much readier obtainable and their derivation free from ethical restraints, human induced pluripotent stem cells (hiPSC) provide access to cell types and insights into cell processes previously unattainable to researches. For this thesis, a hiPSC line was established from a skin biopsy donated by a Down’s syndrome patient. Most of what is known today about the molecular neurobiology behind this disease has been gathered from mice models or human post mortem studies, but this has a limited extrapolation potential to early human brain development in DS patients, as Down’s syndrome is an inherently human disease whose defining phenotype is established early during embryonic development. Having access to human pluripotent cells able to recapitulate the events of early neurogenesis is thus invaluable to the understanding of the mechanisms of this disorder. In parallel, work has been performed on optimizing iPSC reprogramming protocol. By exchanging one of the transcription factors used for reprogramming with a reporter gene, genomic integration of reprogramming factors has become possible to be traced visually, enabling more efficient selection of reprogrammed iPSC colonies.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23611号 / 医科博第134号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 小川 誠司, 教授 山中 伸弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Stem cells have the ability to self-renew and to differentiate into a variety of cell types. Stem cells can be found naturally in the body, can be derived from the inner cell mass of blastocysts, or can be made by dedifferentiation of adult cells. Regenerative medicine aims to utilize the potential of stem cells to treat disease and injury. The ability to create stem cell lines from a patient's own tissues allows for transplantation without immunosuppressive therapy as well as patient-specific disease modeling and drug testing. The objective of this study was to use cellular dedifferentiation to create in vitro cell lines with which to study regenerative medicine. First, we used siRNA targeted against myogenin to induce the dedifferentiation of murine C2C12 myotubes into myoblasts. Timelapse photography, immunofluorescence, and western blot analysis support successful dedifferentiation into myoblasts. However, the inability to separate the myotubes and myoblasts prior to siRNA treatment confounded the results. This system has the potential to be used to study mechanisms behind muscle cell regeneration and wound healing, but a better method for separating out the myoblasts needs to be developed before this will be achievable. Second, we used a doxycycline-inducible lentiviral vector encoding the transcription factors Oct4, Sox2, cMyc, and Klf4 to create a line of naive-like porcine induced pluripotent stem cells (iPSCs). This reprogramming vector was verified first in murine cells, the system in which it was developed. Successful production of both murine and porcine iPSC lines was achieved. Both showed alkaline phosphatase activity, immunofluorescence for pluripotency marker (Oct4, Sox2, and Nanog) expression, PCR for upregulation of endogenous pluripotency factors (Oct4, Sox2, cMyc, Klf4, and Nanog), and the ability to form embryoid bodies that expressed markers of all three germ layers. Additionally, we were able to create secondary porcine iPSC lines by exposing cellular outgrowths from embryoid bodies to doxycycline to initiate more efficient production of porcine iPSCs. The secondary porcine iPSCs were similar to the primary porcine iPSCs in their morphology, behavior, alkaline phosphatase expression, and Nanog expression with immunofluorescence. The porcine iPSCs were dependent on doxycycline to maintain pluripotency, indicating that they are not fully reprogrammed. Despite this dependence on doxycyline, this system can be used in the future to study the process of reprogramming, to develop directed differentiation protocols, and to model diseases. / Master of Science / Stem cells have the ability to self-renew and to differentiate into a variety of cell types. Stem cells can be found naturally in the body, can be derived from the inner cell mass of blastocysts (the stage of development just prior to implantation), or can be made by dedifferentiating, or reprogramming, adult cells into stem cells. Regenerative medicine aims to utilize the potential of stem cells to treat disease and injury. The ability to create stem cell lines from a patient’s own tissues allows for transplantation without immunosuppressive therapy as well as patient-specific disease modeling and drug testing. The objective of this study was to use cellular dedifferentiation to create cell lines in the laboratory with which to study regenerative medicine. First, we knocked down the expression of myogenin, a key factor in muscle cell development, to induce the dedifferentiation of mouse myotubes (adult muscle cells) into myoblasts (progenitor cells). Various methods of analysis supported successful dedifferentiation into myoblasts, but the inability to completely separate myotubes and myoblasts prior to myogenin knockdown confounded the results. With better separation of the cells, this system has the potential to be used to study mechanisms behind muscle cell regeneration and wound healing. Second, we used a viral vector encoding reprogramming factors to create both mouse and pig induced pluripotent stem cells (iPSCs) from skin cells. Pluripotent cells have the ability to differentiate into any cell type in the body, except for the placenta. Multiple pluripotency assays indicated that both the mouse and pig iPSCs were truly pluripotent. Additionally, we were able to differentiate the iPSCs into adult cells, then reprogram those back into “secondary” iPSCs. The production of secondary iPSCs is much more efficient compared to the initial creation of the primary iPSCs, which increases the usefulness of these cells for future experiments. Unfortunately, the porcine iPSCs were dependent on the reprogramming vector to maintain pluripotency. This indicates that these cells are not fully reprogrammed. Despite this, the system can still be used in the future to study the process of reprogramming, to develop cellular differentiation protocols, and to model diseases.

A geração de células-tronco pluripotentes induzidas (iPS) a partir de células somáticas demonstrou que células adultas de mamíferos podem ser reprogramadas a um estágio de pluripotência através da inserção de fatores de transcrição embrionários. Esta descoberta tem levantado questões fundamentais sobre os mecanismos, que através destes fatores de transcrição, influenciam epigeneticamente as células e seus potenciais de diferenciação após a reprogramação e um normal desenvolvimento. Componentes lipídicos e lipoprotéicos afetam vários aspectos no comportamento celular durante sua manutenção e diferenciação, podendo afetar diretamente fatores essenciais em processos de reprogramação celular, manutenção da pluripotência e perfil epigenético das células. Nesse sentido, esta tese propôs o estudo da composição lipídica com diferentes abordagens entre células iPS, células-tronco embrionárias (H1) e células fibroblastos (BJ). Foram produzidas três linhagens de células pluripotentes induzidas no modelo humano que foram caracterizadas quanto 1a sua pluripotência e utilizadas, juntamente às linhagens H1 e BJ como modelos para o estudo da composição lipídica proposto. Foram identificadas e estudadas um total de 44 espécies lipídicas das classes PC, PE, PI, SM e PS, e discutidas frente a reprogramação celular e manutenção da pluripotência. Foi identificado um padrão de composição fosfolipídica distinta entre células pluripotentes e não pluripotentes, e especulamos que a presença dessas espécies parecem ter um envolvimento fundamental para a manutenção da pluripotência. Este padrão, mostrou pela análise de componente principal, que durante o processo de reprogramação, alterações na composição lipídica ocorrem de forma com que a pluripotência surge durante a reprogramação, evidenciando alterações lipídicas particulares do estádio da pluripotência, sugerindo uma ligação entre estas alterações na composição lipídica com as alterações metabólicas da própria reprogramação celular. O estudo da quantificação de fosfolipídios entre linhagens celulares pluripotentes e não pluripotentes evidenciaram que existe uma diferença fosfolipídica entre estas linhagens, observamos que as linhagens iPS e H1, do ponto de vista das classes observadas e os fosfolipídios quantificados, são similares entre si e diferentes de células não pluripotentes. É evidente que estas moléculas lipídicas, individualmente, não são capazes de modular processos como a reprogramação celular, entretanto, é de extrema importância o entendimento das mesmas dentro da reprogramação celular e manutenção da pluripotência. Nossos dados sugerem que a composição lipídica de células pluripotentes tem importante papel para o desenvolvimento e evolução do processo de reprogramação celular e o entendimento da manutenção da pluripotência / The generation of induced pluripotent stem cells (iPS) from adult somatic cells has shown that mammalian cells can be reprogrammed to a pluripotent state by the insertion of embryonic transcription factors. This finding has raised questions about the fundamental mechanisms through which these transcription factors epigenetically influence cells, their potential of differentiation after reprogramming and normal development. Lipid and lipoprotein components affect numerous aspects of cell behavior during its maintenance and differentiation, which can directly affect main factors in cell reprogramming processes, maintenance of pluripotency and epigenetic profile of the cells. Thus, this thesis proposed to study, with different approaches, the lipid composition of iPS cells, embryonic stem cells (H1) and fibroblast cells (BJ). Three induced pluripotent cell lines were produced in the human model. They were characterized regarding their pluripotency and used along with H1 and BJ cell lines, as models for the proposed lipid composition study. A total of 44 species of lipid from the classes PC, PE, PI, PS and SM have been identified, studied and discussed regarding cellular reprogramming and maintenance of pluripotency. A different phospholipid composition pattern was observed between pluripotent and non-pluripotent cells, and it is speculated that the presence of these species appears to have a major involvement on the maintenance of pluripotency. This array showed, by the principal component analysis, that during the reprogramming process changes in the lipid composition occur, so that pluripotency takes place during reprogramming, highlighting lipid changes particular of the pluripotency state, suggesting a connection between these changes in lipid composition and the metabolic changes of cell reprogramming. The study of the quantitation of phospholipids from pluripotent and non-pluripotent cell lines indicated a phospholipid difference between these cell lines when considering the observed classes and quantified phospholipid. It was eminent that iPS lines and H1 are similar and differ from non-pluripotent cells. It is clear that these lipid molecules are not individually capable of modulating processes such as cell reprogramming, however, it is extremely important to understand them within cellular reprogramming and maintenance of pluripotency. Our data suggests that the lipid composition of pluripotent cells has important role in the development and evolution of cellular reprogramming process and the understanding the maintenance of pluripotency

Current usage of human embryonic stem cells (hES) and induced pluripotent stem cells (iPS) in clinical therapies and personalized medicine are limited as a result of ethical, technical and medical problems that arise from isolation and generation of these cells. Isolation of hES cells faces ethical problems associated with their derivation from human pre-implantation embryos. The most controversial aspect of hES cell isolation targets the generation of autologous hES cell lines which requires the transfer of a somatic-cell nucleus from the patient to an enucleated oocyte. While already established embryonic stem cell lines from IVF embryos can be used in a similar manner, lack of genetic identity can cause therapy rejection from the host, and prevent their use in personalized medicine. Induced pluripotent stem cells on the other hand, are generated from somatic cells that have been reprogrammed in vitro to behave like stem cells. While these cells can potentially be used for personalized medicine without the risk of rejection by the host system, derivation methods prevent their therapeutic use. The most efficient method used to generate iPS cells involves usage of viral particles which can result in viral DNA being integrated in the host cell’s genome and render these cells non-compliant for clinical therapies. Other methods not involving viral particles exist as well, but the reprogramming efficiency is too low and technical problems with generating large enough numbers of cells prevent these methods from being feasible approaches for clinical therapies. Direct reprogramming of a differentiated cell into a developmentally more plastic cell would offer alternatives to applications in regenerative medicine that currently depend on either embryonic stem cells (ES), adult stem cells or iPS cells. We hypothesize that Xenopus laevis egg cytoplasmic extract contains critical factors needed for reprogramming that may allow for non-viral, chemically defined derivation of human induced pluripotent/multipotent cells which can be maintained by addition of exogenous FGF2. In this thesis we investigated a new method for generation of multipotent cells through determining the ability of select fractions of Xenopus laevis egg extract to induce multipotency in already differentiated cells. We were able to identify select fractions from the extract that in combination with exogenously added FGF2 can reprogram and maintain the reprogrammed cells in an undifferentiated state. The findings of this work also determined that Xenopus laevis egg extract mRNA is required for achieving full reprogramming. The body of work presented in this thesis showed the ability of FGF2 isoforms to bind and activate select FGF receptor tyrosine kinases, act as extracellular mitogenic factors to support growth of hES cells in an undifferentiated state as well bind to nuclear DNA and affect expression of endogenous genes. Moreover, we showed that all FGF2 isoforms can induce expression of stem cell specific proteins in human dermal fibroblasts as well as extend lifespan of human dermal fibroblasts in vitro. In this work we identified HECW1, the gene coding for E3 ubiquitin ligase NEDL1, as a novel nuclear target for all FGF2 isoforms and showed that overexpression of recombinant FGF2 isoforms in human dermal fibroblasts can down regulate expression of HECW1 gene.

<p>The success of gene medicine ultimately depends on the efficient intracellular delivery and sustained expression of nucleic acid therapeutics, yet nonviral gene delivery performed with cationic polymer carriers has been chronically hindered by the slow release of nucleic acid payloads at their targets, as well as the transient nature of exogenous transgene expression. Polymer-nucleic acid nanocomplexes made with passive gene carriers using traditional bulk methods have proven inadequate for most translational applications. The objective of this work is to improve nonviral gene delivery through the selection, formulation, and application of improved nanoparticles. </p><p> After screening a number of number of cationic polymer delivery systems ranging from natural to synthetic, high molecular weight to low, binary and ternary, we identified a bioreducible linear poly(amido amine) able to give sustained, robust expression of both DNA and RNA through serial dosing. We next turned our attention to the process of nanocomplex assembly. Traditional assembly via bulk mixing is poorly controlled, and the poor quality of these nanocomplexes is a significant impediment to both the establishment of robust structure-function relationships and the advancement of nonviral gene delivery. So, we developed an emulsion-based microfluidic nanomanufacturing platform to better control the self-assembly process, and thus the physical properties of nanocomplexes. Confined mixing within picoliter droplets generates self-assembled nanocomplexes that are more uniform and more effective. This microfluidic nanomanufacturing approach possesses broad utility in the production of polymer-nucleic acid nanocomplexes; we demonstrated that its benefits extend to multiple gene carriers, a range of nucleic acid payloads, and translationally relevant cell types. Then, we applied the improved nanomanufactured particles to begin to address an unmet clinical need, namely the lack of a safe and ethical source of cells to treat neurodegenerative diseases. Nonviral cellular reprogramming strategies eliminate the integration of viral DNA sequences and represent a potentially safer alternative to viral transdifferentiation methods to generate therapeutic cells. Using nanomanufactured polymer-nucleic acid nanocomplexes, we improved the efficiency of the nonviral cellular reprogramming of fibroblasts directly to functional induced neuronal cells. </p><p> Nonviral gene therapy will continue to demand more sophisticated delivery systems to continue to progress. Microfluidic nanomanufacturing represents a reproducible and scalable platform to synthesize more uniform and effective nanocomplexes that not only improves their functional performance, but may also help establish clearer structure-function relationships that will inform future gene carrier design. Complementing the innovative chemical and biological approaches to create multifunctional nanoparticles, this study indicates that microfluidic nanomanufacturing can serve as a parallel physical strategy to both optimize the properties of polymer-nucleic acid nanocomplexes and improve their performance in applications with important clinical implications.</p> / Dissertation

A geração de células-tronco pluripotentes induzidas (iPS) a partir de células somáticas demonstrou que células adultas de mamíferos podem ser reprogramadas a um estágio de pluripotência através da inserção de fatores de transcrição embrionários. Esta descoberta tem levantado questões fundamentais sobre os mecanismos, que através destes fatores de transcrição, influenciam epigeneticamente as células e seus potenciais de diferenciação após a reprogramação e um normal desenvolvimento. Componentes lipídicos e lipoprotéicos afetam vários aspectos no comportamento celular durante sua manutenção e diferenciação, podendo afetar diretamente fatores essenciais em processos de reprogramação celular, manutenção da pluripotência e perfil epigenético das células. Nesse sentido, esta tese propôs o estudo da composição lipídica com diferentes abordagens entre células iPS, células-tronco embrionárias (H1) e células fibroblastos (BJ). Foram produzidas três linhagens de células pluripotentes induzidas no modelo humano que foram caracterizadas quanto 1a sua pluripotência e utilizadas, juntamente às linhagens H1 e BJ como modelos para o estudo da composição lipídica proposto. Foram identificadas e estudadas um total de 44 espécies lipídicas das classes PC, PE, PI, SM e PS, e discutidas frente a reprogramação celular e manutenção da pluripotência. Foi identificado um padrão de composição fosfolipídica distinta entre células pluripotentes e não pluripotentes, e especulamos que a presença dessas espécies parecem ter um envolvimento fundamental para a manutenção da pluripotência. Este padrão, mostrou pela análise de componente principal, que durante o processo de reprogramação, alterações na composição lipídica ocorrem de forma com que a pluripotência surge durante a reprogramação, evidenciando alterações lipídicas particulares do estádio da pluripotência, sugerindo uma ligação entre estas alterações na composição lipídica com as alterações metabólicas da própria reprogramação celular. O estudo da quantificação de fosfolipídios entre linhagens celulares pluripotentes e não pluripotentes evidenciaram que existe uma diferença fosfolipídica entre estas linhagens, observamos que as linhagens iPS e H1, do ponto de vista das classes observadas e os fosfolipídios quantificados, são similares entre si e diferentes de células não pluripotentes. É evidente que estas moléculas lipídicas, individualmente, não são capazes de modular processos como a reprogramação celular, entretanto, é de extrema importância o entendimento das mesmas dentro da reprogramação celular e manutenção da pluripotência. Nossos dados sugerem que a composição lipídica de células pluripotentes tem importante papel para o desenvolvimento e evolução do processo de reprogramação celular e o entendimento da manutenção da pluripotência / The generation of induced pluripotent stem cells (iPS) from adult somatic cells has shown that mammalian cells can be reprogrammed to a pluripotent state by the insertion of embryonic transcription factors. This finding has raised questions about the fundamental mechanisms through which these transcription factors epigenetically influence cells, their potential of differentiation after reprogramming and normal development. Lipid and lipoprotein components affect numerous aspects of cell behavior during its maintenance and differentiation, which can directly affect main factors in cell reprogramming processes, maintenance of pluripotency and epigenetic profile of the cells. Thus, this thesis proposed to study, with different approaches, the lipid composition of iPS cells, embryonic stem cells (H1) and fibroblast cells (BJ). Three induced pluripotent cell lines were produced in the human model. They were characterized regarding their pluripotency and used along with H1 and BJ cell lines, as models for the proposed lipid composition study. A total of 44 species of lipid from the classes PC, PE, PI, PS and SM have been identified, studied and discussed regarding cellular reprogramming and maintenance of pluripotency. A different phospholipid composition pattern was observed between pluripotent and non-pluripotent cells, and it is speculated that the presence of these species appears to have a major involvement on the maintenance of pluripotency. This array showed, by the principal component analysis, that during the reprogramming process changes in the lipid composition occur, so that pluripotency takes place during reprogramming, highlighting lipid changes particular of the pluripotency state, suggesting a connection between these changes in lipid composition and the metabolic changes of cell reprogramming. The study of the quantitation of phospholipids from pluripotent and non-pluripotent cell lines indicated a phospholipid difference between these cell lines when considering the observed classes and quantified phospholipid. It was eminent that iPS lines and H1 are similar and differ from non-pluripotent cells. It is clear that these lipid molecules are not individually capable of modulating processes such as cell reprogramming, however, it is extremely important to understand them within cellular reprogramming and maintenance of pluripotency. Our data suggests that the lipid composition of pluripotent cells has important role in the development and evolution of cellular reprogramming process and the understanding the maintenance of pluripotency