Peripheral artery disease (PAD) and the consecutive build-up of an atherosclerotic plaque restricting blood flow to the lower limbs lead to critical limb ischaemia, one of the most common circulation problems in the world. Although a small number of interventions (such as surgery or revascularization treatments) are available, patients with this condition are often too ill for these procedures, giving a poor prognosis for the disease. Several strategies to promote neovascularization using different stem cell populations with angiogenic potential have been proposed as plausible therapies. Perivascular cells (PCs), key structural components of the wall of small and large blood vessels have numerous advantages over other cell types since they are highly abundant, easy to obtain from the stromal vascular fraction (SVF) of human adipose tissue (an ethically approved source) and have mesenchymal and angiogenic properties. The work described in this thesis addressed the hypothesis that PCs isolated from human white adipose tissue would promote the recovery of blood flow in an ischaemic hindlimb by increasing blood vessel number and blood perfusion to the foot. To investigate whether PCs from human white adipose tissue could rapidly increase neovascularization and, therefore, be used as a possible therapeutic treatment for PAD and critical limb ischaemia, the initial aim was to validate, characterise and demonstrate the properties of the murine equivalent of these cells, in order to establish a direct link between the injected cells and the ones natively found in the mouse. This was then followed by the use of murine models of angiogenesis to determine whether transplanted human PCs stimulate angiogenesis in vivo. Initial studies using immunohistochemistry, fluorescence-activated cell sorting (FACS) and in vitro mesodermal differentiation demonstrated that perivascular cells (namely pericytes and adventitial cells) are present in multiple mouse organs, can be sorted to purity, and have mesenchymal stem cell (MSC) properties. These cells had similar characteristics to their human counterparts, thus validating the mouse as a suitable model for determining whether transplanted human PCs could stimulate angiogenesis. Using in vitro and two in vivo (sponge implantation and hindlimb ischaemia) models, it was shown that human PCs have angiogenic properties being capable of tube formation and interaction with endothelial cells, as well as promoting angiogenesis within sponges. Contrary to expectations, PCs did not increase blood perfusion to the mouse ischaemic hindlimb, despite increasing microcirculation within the skeletal muscle and myofibre regeneration. This work showed that PCs obtained from human adipose tissue have important therapeutic implications in promoting angiogenesis and skeletal muscle regeneration but failed to increase arteriogenesis which is the key mechanism allowing the restoration of blood perfusion.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17816号 / 医博第3814号 / 新制||医||999(附属図書館) / 30631 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 一山 智, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Neural crest stem cells (NCSCs) are embryonic multipotent cells that give rise to a wide range of cell types that include those forming the peripheral neural cells and the mesodermal cells of the face including the facial bones. In neonatal and adult skin, skin-derived precursor cells (SKPs) are multipotent dermal precursors that share similarities with NCSCs and can differentiate into peripheral neural and mesodermal cells, such as adipocytes. Based on the similarities between SKPs and NCSCs, I asked, in this thesis, whether rodent or human SKPs can differentiate into skeletal mesodermal cell types by determining their ability to differentiate into osteoblasts and chondrocytes. In culture, rodent and human SKPs differentiated into alkaline phosphatase-, osteopontin- and type-I collagen-positive osteoblasts that produced mineral deposits and into type-II collagen expressing chondrocytes. Clonal analysis showed that SKPs are multipotent for the osteogenic and chondrogenic lineages. To ask whether SKPs can generate these cells in vivo, genetically-tagged naïve rat SKPs were transplanted into a tibia bone fracture model. Six weeks post-transplantation, SKP-derived osteoblasts and osteocytes were present in the newly formed bone, showing their osteogenic differentiation in vivo. At three weeks post-transplantation, some of the injected cells differentiated into hypertrophic chondrocytes in the callus and others into perivascular cells in areas just outside the callus. To test whether it is the local environment that dictates the phenotype of transplanted SKPs, GFP-tagged undifferentiated rat SKPs were injected into the hypodermis of the skin, an adipogenic environment. Four weeks post-transplantation, SKPs differentiated into adipocytes, but not in inappropriate cell types. These results further the known differentiation potential of SKPs, show that local environment of a bone fracture or the hypodermis of the skin is sufficient to induce the differentiation of undifferentiated SKPs into appropriate cell types and suggest the use of SKPs as source of mesodermal precursor cells for cell therapy.

Neural crest stem cells (NCSCs) are embryonic multipotent cells that give rise to a wide range of cell types that include those forming the peripheral neural cells and the mesodermal cells of the face including the facial bones. In neonatal and adult skin, skin-derived precursor cells (SKPs) are multipotent dermal precursors that share similarities with NCSCs and can differentiate into peripheral neural and mesodermal cells, such as adipocytes. Based on the similarities between SKPs and NCSCs, I asked, in this thesis, whether rodent or human SKPs can differentiate into skeletal mesodermal cell types by determining their ability to differentiate into osteoblasts and chondrocytes. In culture, rodent and human SKPs differentiated into alkaline phosphatase-, osteopontin- and type-I collagen-positive osteoblasts that produced mineral deposits and into type-II collagen expressing chondrocytes. Clonal analysis showed that SKPs are multipotent for the osteogenic and chondrogenic lineages. To ask whether SKPs can generate these cells in vivo, genetically-tagged naïve rat SKPs were transplanted into a tibia bone fracture model. Six weeks post-transplantation, SKP-derived osteoblasts and osteocytes were present in the newly formed bone, showing their osteogenic differentiation in vivo. At three weeks post-transplantation, some of the injected cells differentiated into hypertrophic chondrocytes in the callus and others into perivascular cells in areas just outside the callus. To test whether it is the local environment that dictates the phenotype of transplanted SKPs, GFP-tagged undifferentiated rat SKPs were injected into the hypodermis of the skin, an adipogenic environment. Four weeks post-transplantation, SKPs differentiated into adipocytes, but not in inappropriate cell types. These results further the known differentiation potential of SKPs, show that local environment of a bone fracture or the hypodermis of the skin is sufficient to induce the differentiation of undifferentiated SKPs into appropriate cell types and suggest the use of SKPs as source of mesodermal precursor cells for cell therapy.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年1月31日 匠原龍太郎氏の博士論文として提出された

<p>Transforming growth factor β-activated kinase 1 (TAK1), a hub kinase at the convergence of multiple signaling pathways, is critical to the development of the central nervous system and has been found to play a role in cell death and apoptosis. TAK1 may have the potential to elucidate mechanisms of cell cycle and neurodegeneration. The Belecky-Adams laboratory has aimed to study TAK1 and its potential roles in cell cycle by studying its role in chick retinal development as well as its possible implication in the progression of diabetic retinopathy (DR). Chapter 3 includes studies that explore TAK1 in a study in chick retinal development and TAK1 in in vitro studies in retinal microglia. Using the embryonic chick, immunohistochemistry for the activated form of TAK1 (pTAK1) showed localization of pTAK1 in differentiated and progenitor cells of the retina. Using an inhibitor or TAK1 activite, (5Z)-7-Oxozeaenol, in chick eye development showed an increase in progenitor cells and a decrease in differentiated cells. This study in chick suggests TAK1 may be a critical player in the regulation of the cell cycle during retinal development. Results from experimentation in chick led to studying the potential role of TAK1 in inflammation and neurodegeneration. TAK1 has previously been implicated in cell death and apoptosis suggesting that TAK1 may be a critical player in inflammatory pathways. TAK1 has been implicated in the regulation of inflammatory factors in different parts of the CNS but has not yet been studied specifically in retina or in specific retinal cells [3, 4]. Chapter 2 includes studies from the Belecky-Adams laboratory of in vitro work with retinal microglia. Retinal microglia were treated with activators and the translocation to the nucleus of a downstream factor of TAK1 was determined: NF-kB. Treatment of retinal microglia in the presence of activators with TAKinib, an inhibitor of TAK1 activation, revealed that TAK1 inhibition reduces the activation of downstream NF-kB. Together this data suggests that TAK1 may be implicated in various systems of the body and further studies on its mechanisms may help elucidate potential therapeutic roles of the kinase.</p>