The lymphatic vasculature is composed of lymphatic endothelial cells (LECs) that coalesce into a branched hierarchy of small capillaries and larger collecting vessels that regulate interstitial fluids, lipid uptake and immunity. Few studies have focused on pulmonary lymphatic system. To fill these critical knowledge gaps, we interrogated the fetal maturation program of lymphatic endothelium, and we provide evidence that CSF1R-lineage progenitors contribute to LECs in the lung during a temporally defined period in early postnatal life. The pulmonary lymphatic system is required for fluid clearance and air breathing at birth, suggesting a prenatal maturation program. To interrogate this, we developed a cell sorting strategy to enrich pulmonary LECs by their unique cell surface immunophenotype (CD45-, EPCAM-, CD31+, VEGFR3+, PDPN+, LYVE1+) for transcriptional profiling. These experiments highlighted the coordinate down-regulation of genes involved in “cell cycle”, and “mRNA processing” along with coordinate upregulation of “complement/coagulation cascade”, “lipid metabolism”, and “angiogenesis” genes from embryonic day E16.5 to E18.5. The most significantly enriched gene set corresponded to the “interferon-alpha/beta signaling” pathway which was confirmed with qRT-PCR and in-situ hybridization. These data provide the first description of the transcriptional landscape of fetal pulmonary LEC maturation. During development, all LECs are thought to originate from embryonic veins, however multiple studies have suggested a myeloid origin for a subset of LECs. A relationship between myeloid cells and the pulmonary LECs has not been elucidated. Here, we used myeloid-specific inducible CSF1R-CreERtdTomato lineage tracing mice and identified rare, single cells that co-expressed CSF1R- CreERtdTomato and Prox1, the master lymphatic regulator, in the postnatal day 3 lung. This process was temporally restricted to the early postnatal period. Lineage tracing with additional myeloid-Cre mice (CSF1R-iCre and CX3CR1-Cre) also showed contribution to postnatal LECs. To determine the biological significance of CSF1R-derived LECs to postnatal lung biology, we performed conditional Prox1 loss of function experiments. CSF1R-CreER mediated deletion of Prox1 resulted in lymphatic hypoplasia, edematous foci and clotting. These findings suggest that early postnatal CSF1R+ progenitors contribute to the pulmonary lymphatic endothelium and that vascular clotting may result from lymphatic malformation/dysfunction. / 2021-06-14T00:00:00Z

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19620号 / 医博第4127号 / 新制||医||1015(附属図書館) / 32656 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 森田 智視, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic stem (HSC) and progenitor (HSPC) cells reside in low oxygen (~1- 4%, low O2) bone marrow niches which provide critical signals for maintenance, selfrenewal, and differentiation. Exposure of HSC/HSPCs to air (~21%) for less than 10 minutes irreversibly diminishes numbers of phenotypic and functional stem cells, a phenomenon termed extra physiologic oxygen stress/shock. Yet, most studies harvest and analyze HSC/HSPCs in air and often in fixed cells, leaving endogenous signaling mechanisms unidentified. To better understand the endogenous mechanisms regulating HSCs and HSPCs, we generated the first low O2 landscape of phenotypic/functional/signaling alterations in live, low O2 harvested/sorted HSC/HSPCs utilizing novel technology. HSC (LSKCD150+) and HSC/HSPC (LSK) expression, frequency, and stem cell maintenance retention were enhanced in low O2 relative to historic data and our air data. Transcriptomics uncovered low O2 differential pathway regulation of HSC/HSPCs and HSCs with analysis identifying low O2 enrichment of genes/pathways including Ca2+ ion binding, altered sodium hydrogen (Na+/H+) activity, viral entry, and transmembrane receptor activity in both HSCs and HSPCs. In exploring the low O2 landscape, we investigated differential low O2 regulation of Ca2+ and SARS-CoV-2 related pathways/mechanisms in HSCs and HSPCs. Differential Ca2+ regulation was observed in our transcriptional/proteomic analysis corroborated by phenotypic/functional data demonstrating increases in low O2 of cytosolic and mitochondrial Ca2+ flux, ABC Transporter (ABCG2) and Na+/H+ (NHE1) expression, discovery of a novel low O2 Ca2+ high HSPC population that enhances HSC maintenance compared to Ca2+ low populations and blunting of this population and subsequent enhanced stem cell maintenance upon NHE1 inhibition (Cariporide). Multi-omics analyses also identified enhancements in COVID19-related pathways in low O2 that corresponded with enhanced expression of SARS-CoV-2 receptors/co-receptors, SARS-CoV-2 spike protein (SP) binding, and expansion of SP-bound HSC/HSPCs in low O2 compared to air, as well as enhanced stem cell maintenance of SP-bound, versus unbound, cells in low O2. Together, these data presented show low O2 harvest/retention of HSC/HSPCs enhances stem cell maintenance, which could be utilized to improve HSC expansion, and leads to differential pathway/signaling regulation of various biological pathways in HSC/HSPCs including Ca2+ and SARS-CoV-2/viral infection that results in phenotypic and functional consequences. / 2024-11-01

Nephrons develop from a specialized group of mesenchyme cells known as the nephron progenitors. Nephron progenitors can very dynamic as they can self-renew, migrate, and change their cell morphology. These alterations are essential for orientating and organizing select cells for progression through various stages of nephrogenesis. However, the underlying mechanisms that drive these dynamic morphological changes are not fully understood. Shroom3 is an actin-binding protein that regulates cell shape changes by modulating the actin cytoskeleton. In mice and humans, mutations in Shroom3 are associated with poor nephron function and chronic kidney disease. Despite these findings, the underlying mechanisms of Shroom3 function and how genetic mutations contribute to abnormal nephron formation are unclear. Here, we investigated functional roles for Shroom3 in the nephron progenitor population by analyzing E13.5 and E18.5 Wildtype and Shroom3 deficient mice (termed Shroom3-/-). First, using in-situ hybridization (ISH) and immunofluorescence (IF), we confirm Shroom3 expression in select nephron progenitors. Next, we demonstrated abnormal cell shape and abnormal nephron progenitor cell clustering using H&E staining and Pax2 immunofluorescence. We showed a reduction in nephron progenitor cell numbers and decreased cell length in E13.5 Shroom3-/- kidneys. Using markers of cell orientation, we discovered altered cell orientation in some but not all nephron progenitor cells. While analyzing the cell cytoskeleton, we also demonstrated the abnormal distribution of F-actin in Shroom-/- nephron progenitors. Lastly, immunofluorescence and transmission electron microscopy analysis of Shroom3-/- nephron progenitors confirmed the abnormal shape and reduced filopodia-like thin actin-based membrane protrusions. Our findings conclude that Shroom3 is essential for maintaining and regulating nephron progenitor cell morphology. Taken together, these findings could help explain why Shroom3 mutations are highly associated with kidney disease. / Thesis / Master of Science in Medical Sciences (MSMS)

No description available.

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.

Alzheimer's disease (AD) leads to neurodegeneration resulting in cognitive and physical impairments. AD is denoted by accumulation of intracellular neurofibrillary tangles, known as tau, and extracellular plaques of the amyloid beta protein (Aβ). Aβ results from the proteolytic cleavage of the amyloid precursor protein (APP) by β- and gamma-secretases in the amyloidogenic pathway. Although, Aβ has been widely studied for neurodegeneration, the role of APP in both, the healthy and diseased conditions, has not yet been entirely understood. The function that APP has in neural stem cell (NSC) proliferation, differentiation, and migration during adult neurogenesis has been previously studied. Additionally, APP has be shown to be overexpressed after neural damage resulted from conditions, such as AD and traumatic brain injury (TBI). In this study, the role of APP in in vitro damaged neural tissue cells was further investigated by evaluating neural progenitor cell proliferation, migration, and differentiation after a scratch assay. For these purposes, induced pluripotent stem (iPS) cells from AD patients were differentiated into neural progenitor cells to model the disease conditions and later treated with Phenserine to reduce their levels of APP expression. The results suggested that APP may enhance neural progenitor cell proliferation and glial differentiation while inhibiting neural progenitor cell migration and neuronal cell specialization after neural tissue damage.