No description available.

Myocardial infarction (MI) frequently occurs in patients with diabetes resulting in higher mortality and morbidity than non-diabetic patients. We and others have shown that bone marrow-derived endothelial progenitor cells (BM-EPCs) promote cardiac neovascularization and attenuate ischemic injury in animal models. Lately, emerging evidence supports that exosomes (Exo), a family of extracellular vesicles, mediate stem cell therapy by carrying cell-specific biological cargo and by inducing signaling via transferring of bioactive molecules to target cells. Despite promising results of stem cells/Exo in preclinical studies, autologous cell-based therapies yielded modest clinical results, suggesting cellular/Exo reparative function may be compromised by the presence of comorbid diseases including complications associated with diabetes. Recent studies suggest that epigenetic mechanisms, such as histone and DNA modifications for gene silencing, promote diabetes-induced vascular complication. Therefore, we hypothesized that diabetic EPCs produce exosomes of altered and dysfunctional content that compromise their reparative function in ischemic heart disease via epigenetic alterations. We collected EPC-Exo from non-diabetic (db/+) and diabetic (db/db) mice and examined their reparative effect in vitro and on permanent left anterior descending (LAD) coronary artery ligation and ischemia/reperfusion (I/R) myocardial ischemic injuries in vivo. Our data demonstrated that compared to non-diabetic EPC-Exo, diabetic EPC-Exo promoted neonatal rat cardiomyocyte cell apoptosis under hypoxic stress and repressed endothelial tube formation and cell survival. In vivo studies revealed that non-diabetic EPC-Exo treatments improved cardiac function and remodeling while diabetic EPC-Exo significantly depressed cardiac function, reduced capillary density, increased fibrosis in the permanent LAD ligation MI injury. Moreover, in the I/R MI model, we found that non-diabetic EPC-Exo mediated cardio-protection was lost compared with diabetic-EPC-Exo, and diabetic-EPC-Exo increased immune cell infiltration, infarcted area, and plasma cardiac troponin-I. Mechanistically, histone 3 lysine 9 acetylation (H3K9Ac), a gene activating histone modification, expression was decreased in mouse cardiac endothelial cells (MCECs) treated with db/db EPC-Exo compared with db/+ EPC-Exo, suggesting diabetic EPC-Exo inhibits endothelial cell gene expression. The H3K9Ac chromatin immunoprecipitation sequencing (ChIP-Seq) results further revealed that diabetic EPC-Exo reduced H3K9Ac level on angiogenic, cell survival, and proliferative genes in MCECs. Moreover, we found that a small molecular inhibitor of HDACs, valproic acid (VPA), effectively prevented diabetic EPC-Exo-medicated H3K9Ac reduction, indicating VPA may rescue the beneficial gene expression and cell function. Taken together, our results provide evidence that diabetic EPC-Exo reparative function is impaired in the ischemic heart and this may be through HDACs-mediated H3K9Ac downregulation leading to inhibition of beneficial genes in recipient cardiac endothelial cells. Reversing diabetic EPC-Exo function by treating with HDAC inhibitors may provide a new path for autologous exosome therapy for myocardial repair in diabetic patients. However, questions still remain on what the content change of stem cell-derived exosome under diabetic condition is.Emerging evidence support a key role of variety of stem /progenitor cell-secreted Exo as a pivotal paracrine entity to mitigate cardiovascular injury. Beside EPC-, cortical bone stem cell (CBSC)-, and cardiac stem/progenitor cell (CPC)- derived Exo are adequate to enhance cardiac repair and regeneration after injury. As widely acknowledged, the comorbidities such as hyperglycemia is a characteristic of diabetes and a major driving factor in CVD. The functional role of stem/progenitor cell- derived Exo and molecular signature of their secreted Exo cargo under hyperglycemic conditions remain elusive. Therefore, we hypothesize that hyperglycemic stress causes transcriptome changes in stem/progenitor cell- derived Exo that may compromise their reparative function. To identify the content change in Exo under hyperglycemia, we performed an unbiased Exo transcriptome signatures from 3 different aforementioned stem/progenitor cells by next generation exosome RNA sequencing (RNA-seq). The results indicated that the size and number of Exo were not changed from 3 stem/progenitor cells between normal and high glucose groups. Furthermore, analysis revealed differential expression of variety of RNA species in Exo and the portions of different RNA were change under hyperglycemia. Specifically, we identified 241 common-dysregulated mRNAs, 21 ncRNAs and 16 miRNAs in three stem cell-derived Exo. Based on mRNA data, Gene Ontology (GO) revealed that potential function of common mRNAs mostly involved in metabolism and transcriptional regulation. We also provided the detail information of these non-annotated ncRNAs and the potential mRNA targets by miRNA-mRNA prediction. This study not only provides potential candidates for individual stem cell types but also identifies common genes in response to hyperglycemia. These reference data are critical for future biological studies and application of stem/progenitor cell-derived Exo in ischemic heart or other diseases to prevent the adverse effects of hyperglycemia-induced stem/progenitor cell- derived Exo dysfunction. / Biomedical Sciences / Accompanied by five Microsoft Excel files: 1) Supplementary Table 1 2) Supplementary Table 2 3) Supplementary Table 3 4) Supplementary Table 4 5) Supplementary Table 5

Sight is the most powerful of all human senses. For the vast majority of people on Earth, the loss of that sense would be unimaginable. Without assistive technology, it would separate them from their ability to work, their ability to travel, and their ability to interact with their loved ones. And yet, this extraordinary process, carefully refined by billions of years of evolution, is threatened for millions of people all over the world from a wide array of diseases of the retina. Many of these diseases arise from malnutrition and infection and are being rapidly eradicated. However, many dozens more result from convoluted permutations of genetics, age, and diet that threaten blindness for millions more with little hope of treatment, even with the best of modern medicine. As our life expectancies extend and our population ages, these diseases will only become more prevalent. In humanity's ever-present pursuit of medicine and knowledge to improve our quality of life, cutting-edge treatments offer promise that one day soon, even these diseases may be eradicated. One key technology capable of treating these devastating illnesses, on the precipice of being translated to real-world clinical treatments, is pluripotent stem cell-derived therapies. Patient-specific pluripotent stem cells, meaning pluripotent stem cells sourced directly from the patient, have a wealth of applications ranging from drug identification to disease modeling to implantation and regeneration. This research has been developed and advanced remarkably in the approximately two decades since the early isolation of pluripotent stem cells. Naturally, this advancement has predominantly been focused on cell and molecular biology. However, this focus has left significant research questions to be answered from engineering perspectives across a wide latitude of sub-disciplines. This dissertation explores three independent avenues of engineering principles as they relate to improving 2D and 3D retinal tissues derived from pluripotent stem cells in materials, devices, and computation. The first aim explores how plant protein-based nanofibrous scaffolds can marry the advantages and minimize the disadvantages of synthetic and animal-derived scaffolds for the culture of 2D retinal pigment epithelium (RPE) constructs. The second aim describes the development and testing of a novel, perfusing rotating wall vessel (RWV) bioreactor to support culture of 3D retinal organoids. Finally, the third aim performs a meta-analysis of published RNA-Seq datasets to determine the precise mechanisms by which bioreactors support organoid growth and extrapolate how these conclusions can support future experiments. / Bioengineering

Articular cartilage is a smooth, white connective tissue that covers and protects the ends of long bones to allow for a smooth, frictionless surface on which to glide for easy movement. Once the tissue is damaged, articular cartilage lacks a direct blood supply, which results in a limited ability to repair itself. This study explores the effect of the growth factor BMP-13 on the chondroinduction of primary human bone marrow-derived mesenchymal stem cells. The results demonstrate the limited ability of BMP-13 to exert a strong chondroinductive effect on human bone marrow-derived MSCs. However, the results do indicate that BMP-13 has the ability to sustain chondroinduction to a certain extent for up to 18 days following initiation by 3 days of exposure to TGF-β3. Results are encouraging for future work that involves growth factor influence on MSCs in articular cartilage tissue engineering.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23569号 / 医博第4783号 / 新制||医||1054(附属図書館) / 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 篠原 隆司, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Lysine-specific Demethylase 1B (KDM1B) is a chromatin regulator which functions as a histone eraser through the removal of the post-translational modifications mono and dimethylation of histone 3 on lysine 4 (H3K4me1/2). This process is enhanced by the formation of a complex with Nuclear Protein Glyoxylate Reductase (NPAC). NPAC resolves the sequestration of the nucleosome histone tail to allow robust demethylation of H3K4me2 by KDM1B, during transcriptional elongation by RNA polymerase 2 (RNAP II). KDM1B is involved in many crucial processes during development. Its physiological functions include the establishment of maternal genomic imprints, reset of the epigenome during somatic cell reprogramming, and regulation of brown adipogenic differentiation. In light of this, the role of KDM1B in human embryonic stem cells (hESCs) is examined through CRISPR/Cas9-editing to further dissect its biological functions during embryogenesis. CRISPR-induced knockouts of KDM1B exhibited similar cell proliferation rate and expression of OCT4 and NANOG pluripotency markers to wildtype cells. Furthermore, KDM1B-/- clones were able to maintain their pluripotency potential by differentiating to all germ layers in teratoma and embryoid body formation assays. In addition, RNA-seq of KDM1B-/- clones showed enrichment of mesoderm lineage-related gene ontology (GO) terms in the downregulated differentially expressed genes. Thus, KDM1B is believed to be dispensable during the pluripotent stage of the cell but proved fundamental during later stages of development.

The human bone marrow (BM) is one of the most complex and critical tissues in the adult, functioning as the site for blood and immune cell production in homeostasis, injury, and disease. The marrow acts as an incredibly diverse stem cell niche, containing stromal and blood cells that help support the maintenance and differentiation capacity of hematopoietic stem and progenitor cells (HSPCs). The cell-cell and cell-matrix interactions within the niche help alter the marrow to trigger blood cell production in response to injury, as well as harbor downstream changes that may persist in the hematopoietic system during disease, such as in cancer metastasis or leukemias of the BM. As the development of engineered human tissue models including organs-on-a-chip (OoC) have emerged over the past decade, there has been an increased relevance of using human BM models to study human- and patient-specific immune interactions in vitro. In this dissertation, we have developed patient-specific bioengineering technologies to model the BM, as well as those to study multi-organ interactions, for a host of translational applications of injury and disease. In Chapters 1 and 2, we introduce a number of concepts in bioengineering and stem cell biology for studying human organ functions outside of the body. In Chapter 3, we describe the tools that are critical for modeling individual organ functions (healthy human BM) and immune cells, as well as when combining multiple OoC systems together. In Chapter 4, we apply these tools for disease modeling, in studying the complex interactions in either acute leukemia development or metastatic colonization of the BM. In Chapter 5, we use our human-specific engineered models for studies of acute and systemic injury, including the effects of cosmic radiation on human tissue function. To tie together the tissue engineered tools developed in this thesis, we described in Chapter 6 the utility of scientific outreach and social media in the widespread dissemination of tissue engineering and stem cell principles to the broader scientific community and general public. Collectively, this dissertation provides a unique look at the use of engineered human tissue systems to model human blood and immune interactions using bioengineering tools, with applications in disease modeling of primary and metastatic cancers, as well as in acute and systemic injuries.

No description available.

Cardiovascular disease is the leading cause of death in the United States. Millions of patients suffer each year from endothelial dysfunction and/or debilitating myocardial damage resulting in decreased quality of life and increased risk of death or disablement. Current pharmacological approaches are only partly effective at treating cardiovascular disease, and hence, better strategies are needed to provide significant improvements in treatment options. Cardiac stem/progenitor cells have the potential to regenerate myocardial tissue and repair damaged heart muscle. There are many different types of cardiac progenitor cells, and each may have certain unique properties and characteristics that would likely be useful for particular clinical applications. A current challenge in the field is to identify, isolate, and test specific cardiac stem/progenitor cell populations for their ability to repair/regenerate myocardial tissue. Our laboratory has discovered a new type of cardiac progenitor cell that expresses the enzyme, Phenylethanolamine-n-methyltransferase (Pnmt). My initial studies focused on identification of Pnmt+ cells based on knock-in of a nuclear-localized Enhanced Green Fluorescent Protein (nEGFP) reporter gene into exon 1 of the Pnmt gene in a stable recombinant Pnmt-nEGFP mouse embryonic stem cell (mESC) line. These cells were differentiated into cardiomyocytes, and I identified nEGFP+ cells using fluorescence, immunofluorescence, and phase-contrast microscopy techniques. Our results showed that only about 0.025% ( 1 per 4000) of the cardiac-differentiating stem cells expressed the nEGFP+ marker. Because of the relative rarity of these cells, optimization of isolation methods proved initially challenging. To overcome this technical barrier, I used a surrogate cell culture system to establish the methods of isolation based on expression of either a fluorescent cell marker (EGFP), or a unique cell surface receptor represented by an inactivated (truncated) version of the human low-affinity nerve growth factor receptor (LNGFR). Plasmid DNA containing these reporter genes was transiently transfected into a permissive cell line (RS1), and reporter gene expression was used to identify and isolate transfected from non-transfected cells using either Fluorescence-Activated Cell Sorting (FACS) or Magnetic-Activated Cell Sorting (MACS) methods. The main objective of the study was to establish the isolation techniques based on the expression of reporter genes (EGFP and LNGFR) in RS1 cells. Following transfection, EGFP+ cells were successfully isolated via FACS as verified by flow cytometric and microscopic analyses, which showed that approximately 96% of the isolated cells were indeed EGFP+. Despite the relative purity of the isolated cell population, however, their viability in culture following FACS was substantially compromised ( 50% attrition). In contrast, MACS enabled efficient isolation of LNGFR+ cells, and the vast majority of these ( 90%) retained viability in culture following MACS. The LNGFR expression was verified using RT-PCR. Further, MACS methods enabled isolation of marked cells in about 5-7 mins, whereas it took 2-4 hours to using FACS to perform similar isolations from the same amount of starting material (10^6 cells). In addition, MACS is a more economical method in that it does not require the use of an expensive laser-based instrument to perform the sorting. These results suggest that MACS was a more efficient, gentle, and feasible technique than FACS for isolation of reporter-tagged mammalian cells. Consequently, future studies aimed at isolation of Pnmt+ cardiac progenitor cells will thus primarily focus on MACS methods.

No description available.