Embryogenesis occurs within a complex and dynamic cellular environment that influences cell fate decisions. Pluripotent stem cells (PSCs) are a valuable tool for research into disease models as well as a resource for cell therapy due to their capacity to self-renew and differentiate into all cell types. Mimicking aspects of the embryonic microenvironment in vitro impacts the resultant functional cells. The aim of this work was to develop a controlled and scaleable process for the generation of hematopoietic progenitor cells (HPCs) from embryonic stem cells (ESCs). We demonstrated with bioreactor-grown embryoid bodies (EBs) that increased HPC generation can be elicited by decreasing the oxygen tension by a mechanism where vascular endothelial growth factor receptor 2 (VEGFR2) activation is controlled through competition with the ligand decoy VEGFR1. This is important as it demonstrates the inherent responsiveness of the developing hematopoietic system to external forces and influences. We also established a serum-free system that facilitates directed differentiation, determining 5 ng/ml bone morphogenetic protein-4 (BMP4) with 50 ng/ml thrombopoietin (TPO) could generate 292 ± 42 colony forming cells (CFC)/5 x 10^4 cells with early VEGF treatment (25 ng/ml, day 0-5). We also controlled aggregate size influencing relative endogenous and exogenous growth factor signaling and modulating mesodermal differentiation; CFC output was optimal when initialized with 100 cell aggregates. For the first time, we demonstrated efficacy of local growth factor delivery by producing HPCs with gelatin microparticles (MP). Overall, these design components generate HPCs in a controlled and reproducible manner using a serum-free bioprocess that couples size controlled aggregates containing gelatin MPs for localized growth factor release of BMP4 and TPO with hypoxia to induce endogenous VEGF production. These strategies provide a tunable platform for developing cell therapies and high density growth, within a bioreactor system, can be facilitated by hydrogel encapsulation of the aggregates.

Embryogenesis occurs within a complex and dynamic cellular environment that influences cell fate decisions. Pluripotent stem cells (PSCs) are a valuable tool for research into disease models as well as a resource for cell therapy due to their capacity to self-renew and differentiate into all cell types. Mimicking aspects of the embryonic microenvironment in vitro impacts the resultant functional cells. The aim of this work was to develop a controlled and scaleable process for the generation of hematopoietic progenitor cells (HPCs) from embryonic stem cells (ESCs). We demonstrated with bioreactor-grown embryoid bodies (EBs) that increased HPC generation can be elicited by decreasing the oxygen tension by a mechanism where vascular endothelial growth factor receptor 2 (VEGFR2) activation is controlled through competition with the ligand decoy VEGFR1. This is important as it demonstrates the inherent responsiveness of the developing hematopoietic system to external forces and influences. We also established a serum-free system that facilitates directed differentiation, determining 5 ng/ml bone morphogenetic protein-4 (BMP4) with 50 ng/ml thrombopoietin (TPO) could generate 292 ± 42 colony forming cells (CFC)/5 x 10^4 cells with early VEGF treatment (25 ng/ml, day 0-5). We also controlled aggregate size influencing relative endogenous and exogenous growth factor signaling and modulating mesodermal differentiation; CFC output was optimal when initialized with 100 cell aggregates. For the first time, we demonstrated efficacy of local growth factor delivery by producing HPCs with gelatin microparticles (MP). Overall, these design components generate HPCs in a controlled and reproducible manner using a serum-free bioprocess that couples size controlled aggregates containing gelatin MPs for localized growth factor release of BMP4 and TPO with hypoxia to induce endogenous VEGF production. These strategies provide a tunable platform for developing cell therapies and high density growth, within a bioreactor system, can be facilitated by hydrogel encapsulation of the aggregates.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13141号 / 論医博第2141号 / 新制||医||1026(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 横出 正之, 教授 萩原 正敏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

骨形成蛋白 (bone morphogenetic proteins, BMPs) 屬於TGF家族的成員，過去的研究指出BMPs對神經系統的發育及維持非常的重要，並且會參與調控突觸的形成。然而，在哺乳類動物的研究中，BMPs在神經肌肉系統中所調控的生理功能仍未完全了解。本實驗室初步的研究資料顯示BMPs的type II受體 (bone morphogenetic protein type II receptor, BMPRII) 會表現在神經與肌肉接合處 (neuromuscular junction, NMJ) ，而從本論文中的免疫染色實驗結果觀察到骨形成蛋白-4 (BMP4) 會表現在肌肉及許旺細胞上，且BMP4與乙醯膽鹼受體 (acetylcholine receptors, AChRs) 有colocalization的現象。由double nerve ligations的實驗觀察到BMP4會堆積在打結處的兩端，顯示BMP4可能是由肌肉或許旺細胞分泌後送進運動神經元之軸突內運輸，其方向為雙向性運輸，而利用Q-PCR mRNA定量實驗發現BMP4 mRNA在double-ligated之坐骨神經中表現量下降，但在肌肉中表現量則顯著增加。 由上述實驗顯示肌肉細胞為BMP4主要來源之ㄧ，利用NG108-15神經細胞及C2C12肌肉細胞培養，我們發現BMP4 mRNA在C2C12肌小管上有高度表現，相反地在分化後的NG108-15神經細胞上表現量極少，而BMP4的mRNA及protein在C2C12肌肉上的表現量則受到神經衍生蛋白Agrin的調控。此外我們亦發現來自於肌肉的BMP4則會保護分化後的NG108-15神經細胞對抗Glutamate所誘導的細胞死亡反應。綜合這些結果，我們認為BMP4主要來自於運動神經元之周邊的肌肉及許旺細胞，其可能會參與調控運動神經元的存活機制。 / Bone morphogenetic proteins (BMPs), members of the TGF superfamily, have been shown to play important roles in the development of nervous system including neuronal survival and synaptogenesis. However, the physiological functions of BMP signaling at the mammalian neuromuscular system are not well understood. Our preliminary data showed that proteins of the type II bone morphogenetic receptors (BMPRII) were specifically expressed in nerve terminals at neuromuscular junctions. In this study, we found that proteins of bone morphogenetic protein-4 (BMP4) were detected at Schwann cells and colocalized with postsynaptic acetylcholine receptors (AChRs) in skeletal muscle fibers. In double-ligated nerves, BMP4 proteins were accumulated at the proximal and distal portions of the axons, suggesting that Schwann cell- and muscle fiber-derived BMP4 proteins were anterogradely and retrogradely transported by motor neurons. Additionally, BMP4 mRNA was significantly up-regulated in the muscle but down-regulated in ligated sciatic nerves. The physiological functions of BMP4 in the neuromuscular system were further examined in vitro. We found that mRNA of BMP4 was highly expressed in differentiated C2C12 muscle cells, but it was barely detectable in NG108-5 neurons. The expression of BMP4 mRNA and protein in C2C12 muscle cells were upregulated when the motor neuron-derived factor, agrin, was presented in the culture. Moreover, muscle-derived BMP4 could protect NG108-5 neurons from glutamate-induced excitotoxicity. These results together suggest that BMP4 is a peripheral-derived factor that may regulate the survival of motor neurons.

The objective of the study: To characterize stem cells isolated from mouse skeletal muscle and evaluate their regenerative potential to repair injured bone and articular cartilage in a mouse and rat animal model. The aims of the study: 1. Isolate MDSCs from mouse skeletal muscle, to determine their phenotypic characteristics and differentiation potential in vitro. 2. Evaluate osteogenic and chondrogenic regeneration capacity of MDSCs genetically engineered to express bone morphogenetic protein 4 (BMP4). 3. Evaluate osteogenic and chondrogenic differentiation potential of MDSCs-expressing BMP4 in vivo. 4. Investigate the possibility of MDSCs-expressing BMP4 and vascular endothelial growth factor (VEGF) to enhance bone repair. 5. Investigate the possibility of MDSCs-expressing BMP4 and anti-angiogenic factor sFlt1 to enhance articular cartilage repair. The brief overview of the study This investigation addresses important issues regarding: 1) stem cell isolation from skeletal muscle and their specific characteristics that enable us to identify these cells from other populations of stem cells in skeletal muscle; 2) the efficiency of muscle-derived stem cell-mediated bone and cartilage formation in vivo using retroviral vectors to express bone morpho-genetic protein 4 (BMP4); 3) the enhancement of osteogenic and chondrogenic potential of BMP4-secreting muscle-derived stem cells by addition of cells engineered to express vascular endothelial growth factor (VEGF) or its... [to full text] / Skeleto raumuo yra patogus ir lengvai prieinamas kamieninių ląstelių šaltinis. Satelitinės ląstelės, kurios kitaip vadinamos raumenų kamieninėmis ląstelėmis ir yra linkusios diferencijuoti miogenine linkme, dalyvauja raumens regeneracijoje ir gali savarankiškai atsinaujinti. Mūsų laboratorijoje izoliuotos raumeninės kilmės kamieninės ląstelės (RKKL) yra laikomos satelitinių ląstelių pirmtakėmis, tačiau nuo jų skiriasi. RKKL gali diferencijuoti ne tik miogenine linkme, bet ir kitomis linkmėmis (kauline, kremzline, riebalinio audinio, nervų, endotelio ir kraujodaros), ir joms yra būdinga ilgalaikė proliferacija, savarankiškas atsinaujinimas, privilegija imuninės sistemos atžvilgiu ir atsparumas oksidacijos sukeltam stresui. Šio darbo metu atlikti tyrimai atsako į labai svarbius klausimus, susijusius su: 1) RKKL išskyrimu ir jų specifinių savybių, leidžiančių šias ląsteles atskirti nuo kitų skeleto raumenų kamieninių ląstelių populiacijų, nustatymu; 2) genetiškai modifikuotų RKKL sukeliamo kaulų ir kremzlių formavimosi in vivo veiksmingumu, naudojant retroviruso vektorių kaulų morfogenezės baltymo 4 (BMP4) raiškai; 3) RKKL osteogeninio ir chondrogeninio pajėgumo stiprinimu, vienu metu naudojant ląsteles, išskiriančias BMP4, ir ląsteles, išskiriančias kraujagyslių endotelio augimo faktorių (VEGF) arba sFlt1. Šio tyrimo naujumas yra tas, jog iš esmės pirmą kartą parodyta, kad iš išgrynintų pelės skeleto raumenų ląstelių (PP6) populiacijos, išskirtos ląstelių sukibimo su... [toliau žr. visą tekstą]

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20669号 / 医博第4279号 / 新制||医||1024(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 林 康紀, 教授 渡邉 大, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>This dissertation explains why behavioral genetic research can be better informed by using characteristics in the human iris as biomarkers for personality, and is divided into five parts. Part I gives an introduction to the classical twin method and an overview of the findings that have led most developmental researchers to recognize that the normal variation of personality depends on a complex interplay between genetic and environmental factors. Part II highlights empirical findings that during the last twenty years have gradually moved genetic and environmental theory and research to evolve toward one another, and also presents the theory of genetics and experience that currently is used to explain how the interplay between genes and the environment works. Part III explains why, from a developmental perspective, it is of interest to identify candidate genes for personality, and gives a brief overview of genes that have been associated with personality. Problems associated with genetic research on the molecular level and how these apply to personality are also highlighted. Part IV examines molecular research on the iris and the brain, which suggests that genes expressed in the iris could be associated with personality, and explains how the use of iris characteristics can increase power to test candidate genes for personality by taking advantage of the self-organizing properties of the nervous system. The empirical foundation for the questions posed in this dissertation and also the empirical results are presented here. Part V discusses the associations found between iris characteristics and personality, and exemplifies how iris characteristics can be used within the theoretical frameworks presented in parts I, II, III and IV. In other words, Part V explains how iris characteristics – in addition to identify as well as test candidate genes for personality – can be used to investigate how people’s experiences in themselves are influenced by genetic factors.</p>

This dissertation explains why behavioral genetic research can be better informed by using characteristics in the human iris as biomarkers for personality, and is divided into five parts. Part I gives an introduction to the classical twin method and an overview of the findings that have led most developmental researchers to recognize that the normal variation of personality depends on a complex interplay between genetic and environmental factors. Part II highlights empirical findings that during the last twenty years have gradually moved genetic and environmental theory and research to evolve toward one another, and also presents the theory of genetics and experience that currently is used to explain how the interplay between genes and the environment works. Part III explains why, from a developmental perspective, it is of interest to identify candidate genes for personality, and gives a brief overview of genes that have been associated with personality. Problems associated with genetic research on the molecular level and how these apply to personality are also highlighted. Part IV examines molecular research on the iris and the brain, which suggests that genes expressed in the iris could be associated with personality, and explains how the use of iris characteristics can increase power to test candidate genes for personality by taking advantage of the self-organizing properties of the nervous system. The empirical foundation for the questions posed in this dissertation and also the empirical results are presented here. Part V discusses the associations found between iris characteristics and personality, and exemplifies how iris characteristics can be used within the theoretical frameworks presented in parts I, II, III and IV. In other words, Part V explains how iris characteristics – in addition to identify as well as test candidate genes for personality – can be used to investigate how people’s experiences in themselves are influenced by genetic factors.