京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23472号 / 医科博第131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 滝田 順子, 教授 髙折 晃史, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Hematopoietic stem cells (HSCs) lie at the top of the hematopoietic hierarchy and give rise to all mature blood cells. They are tightly regulated not only by cell-intrinsic but also cell-extrinsic mechanisms that allow HSCs to respond to dynamic physiological demands of the body. HSCs build the hematopoietic system during development and maintain homeostasis in adults by changing their properties according to different needs. A niche is the microenvironment where HSCs reside and receive extrinsic regulation. Understanding the niche is crucial for elucidating how HSCs are regulated by extrinsic cues. During mammalian development, HSCs pass through several different niches, among which the liver is the major site for their rapid expansion and maturation. The fundamental question of what cells constitute the fetal liver niche in vivo remains largely elusive. It is also unclear whether and how cell-extrinsic maintenance mechanisms accompany changes in HSC properties during ontogeny. Here, I genetically dissected the cellular components of the HSC niche in the fetal liver by identifying the cellular source of a key cytokine, stem cell factor (SCF). In addition, I found that HSCs switch to depend on thrombopoietin (TPO), another key factor, during ontogeny and uncovered the mechanism by which HSCs gain this dependence.

Hematopoietic stem cells (HSCs) give rise to all blood cells and are characterized by their ability for life-long self-renewal and multilineage differentiation. HSC function is regulated by complex cell-intrinsic and -extrinsic pathways, but these regulatory mechanisms are not completely understood. Recent work has demonstrated that the epitranscriptional modification N6-methyladenosine (m6A) has important roles in the regulation of many physiologic and pathologic processes in various cell types, but it was previously unknown if and how m6A may regulate adult HSC function. In this work, I uncover the role for m6A in HSC regulation, both cell-intrinsically in regulating HSC differentiation and cell–extrinsically by regulating the formation the HSC bone marrow niche.

Thanks to their ability to differentiate into variable cell types and migrate to the site of an injury mesenchymal stem cells (MSC) are broadly used in regenerative medicine. Their relative easy availability together with the property to control the immune system determines them as a cure of autoimmune diseases or a recovery of wounded tissues. Similar features posses Sertoli cells which take place in the seminiferous tubule of testis. Cell culture of testicular stem cells from juvenile male testes of X. tropicalis (XtTSC) was established in supervisor's laboratory. This cell culture showing both MSC's and SeC's properties was transformed to carry red fluorescent protein RFP. The aim of this diploma thesis was to investigate an behavior of transformed XtTSC in living organism, therefore cells were transplanted into the X. tropicalis tadpoles in stage 41. Subsequently, their migration potential was explored. To study of XtTSC's differentiation potential it was necessary to introduce a reliable sectioning techniques for the subsequent immunohistochemical analysis. Based on our experiments, we found that the XtTSC's cell culture contains precursors of SeC and peri-tubular myoid cells, however in vivo these cells turned into the dedifferentiated MSC-like state allowing a strong migration through the...

Mammalian skin is a dynamic organ that constantly undergoes self-renewal during homeostasis and regenerates in response to injury. Crucial for the skin’s self-renewal and regenerative capabilities is the epidermis and its stem cell populations. Here we have interrogated the role of primary cilia and Intraflagellar Transport 20 (Ift20) in epidermal development as well as during homeostasis and wound healing in postnatal, adult skin. Using a transgenic mouse model with fluorescent markers for primary cilia and basal bodies, we characterized epidermal primary cilia during embryonic development as well as in postnatal and adult skin and find that both the Interfollicular Epidermis (IFE) and hair follicles (HFs) are highly ciliated throughout development as well as in postnatal and adult skin. Leveraging this transgenic mouse, we also developed a technique for live imaging of epidermal primary cilia in ex vivo mouse embryos and discovered that epidermal primary cilia undergo ectocytosis, a ciliary mechanism previously only observed in vitro. We also generated a mouse model for targeted ablation of Ift20 in the hair follicle stem cells (HF-SCs) of adult mice. We find that loss of Ift20 in HF-SCs inhibits ciliogenesis, as expected, but strikingly it also inhibits hair regrowth. Closer examination of these mice reveals that Ift20 is crucial in maintaining HF-SC identity. Specifically, ablation of Ift20 in HF-SCs results in loss of SOX9 expression in HF-SCs and results in ectopic expression of the IFE marker KLF5 in HF-SCs. Additionally, ectopic differentiation is observed in HF-SCs following loss of Ift20. Finally, using both in vitro and in vivo models, we also characterize the role of primary cilia and Ift20 in epidermal wound healing. We find that loss of Ift20 slows collective keratinocyte migration in vitro and also slows HF-SC migration in vivo during wound repair. Interestingly our data suggests that Ift20 regulates keratinocyte migration in a primary cilia-independent manner. Instead, we find that Ift20 mediates focal adhesion (FA) turnover during keratinocyte migration. Specifically, Ift20 together with Rab5, regulates recycling of FA integrins and loss of Ift20 inhibits proper return of integrins to the keratinocyte surface. Overall, we demonstrate that the epidermis is highly ciliated throughout development and in postnatal skin. We show that Ift20 is crucial in maintaining HF-SC identity and the telogen to anagen transition in HFs. We finally demonstrate that Ift20 regulates keratinocyte migration independent of its function in ciliogenesis and instead regulates recycling of FA integrins through a Rab5 dependent mechanism.

Orientador: Sigmar de Mello Rode / Coorientador: Lucio Murilo dos Santos / Banca: Antonio Olavo Cardoso Jorge / Banca: Fernando Callera / Resumo: Micro-organismos como fungos do gênero Candida, são habitantes comensais da cavidade bucal; em condições normais, co-existem com a microbiota normal sem provocar doenças. Entretanto, alterações locais ou sistêmicas como imunossupressão, desequilíbrio da microbiota oral, hipossalivação e mucosite, secundárias ao tratamento quimioterápico, predispõem pacientes com câncer a um alto risco de infecções fúngicas orais e sistêmicas. O objetivo desse trabalho foi avaliar a presença de leveduras do gênero Candida na cavidade bucal de pacientes onco-hematológicos submetidos a transplante autólogo de células-tronco hematopoiéticas (TACTH). Foram avaliados 27 pacientes, nos períodos pré, e pós-transplante. As amostras da cavidade bucal foram obtidas pela técnica de enxágüe bucal e semeadas em Chromagar Candida para triagem das cepas isoladas. Após o crescimento, foi extraído o DNA e submetido a identificação molecular (PCR) utilizando iniciadores para os genes ribossomais dessa levedura. Após a amplificação do fragmento esperado, as cepas foram sequenciadas utilizando sequenciador automático.Para análise descritiva e estatística dos resultados obtidos, os dados foram submetidos ao teste de Shapiro-Wilk para avaliação da normalidade. Em seguida, o teste de variância Wilcoxon foi utilizado. A significância adotada foi de 5%. Candida spp foi encontrada em 40,74% (11 pacientes), sendo que 2 (18,18%) possuíam mais de uma espécie, dos 9 pacientes colonizados por apenas uma espécie, 7 eram port... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Microorganisms such as fungi of the genus Candida, are commensal inhabitants of the oral cavity; under normal conditions co-exist with normal microflora without causing disease. However, local or systemic changes such as immunosuppression, imbalance in the oral microbiota, hyposalivation and mucositis, secondary to chemotherapy, predispose cancer patients at high risk of oral and systemic fungal infections. The aim of this project is to evaluate the presence of Candida species in the oral cavity of hematological malignances patients undergoing autologous transplantation of hematopoietic stem cells (HSCT). We evaluated 27 patients in preand post-transplant. Samples of the oral cavity were obtained by oral rinse technique and plated on Chromagar Candida for screening of the strains. After growth, the DNA was extracted and subjected to molecular identification (PCR) using primers for the ribosomal genes in this yeast. After amplification of the expected fragment, the strains were sequenced using the sequencer automático. For descriptive and statistical analysis of the results, the data will be submitted to the Shapiro-Wilk test for evaluation of normality. Then the variance Wilcoxon test was used. The significance of 5% was adopted. Candida spp was found in 40.74% (11pacientes), and 2 (18.18%) had more than one species, of the 9 patients colonized by only one species, 7 patients had Candida albicans (77.77%) and 1 Candida dubliniensis (11.11%) and 1 C. krusei (11.11%). Mixed settlements, in the other two patients were composed of C. albicans + C. glabrata; C. albicans + C. dubliniensis. In the second collection (C2), 9 (81.81%) of Candida carriers remained colonized, including by non albicans species. The homology identification of species standardized strains was 85 99%. Within the conditions of this study it was possible to determine with ...(Resumo completo, clicar acesso eletrônico abaixo) / Mestre

In patients with aplastic anaemia that respond to immunosuppressive therapy, quantitative, morphological and functional haematologic derangement have been reported. To explain these findings, abnormalities in the marrow stroma or the stem cell have been postulated. To define the relative contribution of each of the latter, the integrity of the bone marrow from sixteen patients that responded to anti-lymphocyte globulin and high dose methyl prednisolone was compared to normal individuals. Bone marrow mononuclear cells were divided into two fractions. From the first, stroma was cultured in aMEM containing 12.5% of both horse and foetal calf serum and 10-5 M hydrocortisone at 37° C in 5% CO2 in 90% humidity. The medium was changed weekly. Upon confluence, these stromal layers were studied morphologically and with cytospin preparations stained with Sudan black, 0 red oil, alkaline and acid phosphatases. The remainder was monocyte and lymphocyte depleted, CD 34+ progenitors were selected with paramagnetic beads and the population morphologically and immunophenotypically defined. To determine the functional status, control or patient CD 34+ progenitors, were suspended for two hours on normal or aplastic stroma for adherence to take place. The non-adhesive fraction was decanted by standardised washing and cultured for fourteen days in the presence of PHA-conditioned medium in the CFU-gm assay. Strama-adherent progenitors were covered with 0.3% agar and cultured for five days. Aggregates with more than twenty cells were scored (CFU-bl). The remaining CD 34+ cells were cultured in the mixed colony assay with combinations of recombinant cytokines belonging to the G protein super-family and the tyrosine kinase group in dose response studies. Light density cells from patients with treated aplasia contained significantly fewer CD 34+ cells than those present in the control suspensions (mean 0.65%, SD 0.35% vs 1.62%, SD 1.4%; p= 0.002). Normal and aplastic stroma became confluent at three and four weeks. There was no difference on the morphology or the cytochemical stains between the two groups. Functionally, aplastic bone marrow stroma supported CFU-bl formation no differently from normal layers. However, CD 34+ precursors from the patients cultured on control stroma resulted in significantly fewer CFU-bl (p= 0.0002,) and CFU-gm (p= 0.0009). This work provides original evidence supporting the reduced clonogenicity of the corresponding populations of CFU-bl from patients with aplasia is unrelated to attachment to the stroma, but intrinsic to the CD 34+ cells. Moreover, this study shows for the first time that exposure of these progenitors to growth factors belonging to the G protein and tyrosine kinase receptor families have defective responses, correctable only at supra physiological concentrations, while effects on combinations containing c-kit ligand, appear preserved. Following immunosuppressive therapy, the bone marrow is repopulated by a hypoproliferative progenitor cell population which responds suboptimally to physiological cytokine stimulation. This suggests that abnormal interactions between receptors and their ligands or alterations in the signal transduction for cell division by the cytokines belonging to the G superfamily lead to suboptimal growth.

No description available.

Induced pluripotent stem cells (iPSCs) are an attractive cell source for various applications in regenerative medicine and cell-based therapies given their unique capability to differentiate into any cell type of the human body. However, human iPSCs are highly vulnerable to cryopreservation with post-thaw survival rates of 40-60%; this is due to cryoinjury resulting from ice recrystallization when using conventional slow cooling protocols. Ice recrystallization is a process where the growth of large ice crystals occurs at the expense of small ice crystals. Ice recrystallization inhibitors (IRIs) are designed to inhibit the growth of intracellular ice crystals, increasing post-thaw viability. In this study, we tested a panel of four IRIs to determine if the inhibition of ice recrystallization can decrease cellular damage during freezing and improve viability post-thaw of iPSC colonies. We supplemented commercially available and serum-free cryopreservation medium mFreSR, routinely used for the cryopreservation of iPSCs, with a class of N-aryl-D-ß-gluconamide IRIs. A 2-fold increase in post-thaw viability was observed, in a dose dependent response, for N-(4-methoxyphenyl)-D-gluconamide (PMA) at 15 mM, N-(2-fluorophenyl)-D-gluconamide (2FA) at 10 mM, and N-(4-chlorophenyl)-D-gluconamide (4ClA) at 0.5 mM over mFreSR controls. After testing the panel of four IRIs, 2FA frozen iPSCs showed an increase in cell viability, proliferation, and recovery. The addition of ROCK inhibitor (RI), commonly used to increase iPSC viability post thaw, further enhanced the survival of the iPSCs frozen in the presence of 2FA and is used routinely in research. This additive effect increased cell recovery and colony formation post thaw, resulting in increased proliferation with no adverse effects on iPSC pluripotency or differentiation capabilities. The development of improved cryopreservation strategies for iPSCs is key to establishing master clonal cell banks and limiting cell selection pressures, all while maintaining high post-thaw viability and function. This will help ensure sufficient supplies of high-quality iPSC required to meet the cell demands for cell and regenerative based therapies. Since iPSCs hold promise as a potentially unlimited cell source for a plethora of cell-based therapies, improving cryopreservation is essential to the successful deployment of iPSC-derived therapeutic cell products in the future.

Skeletal muscles have a remarkable capacity to repair and regenerate in response to injury by virtue of their unique population of resident muscle stem cells (MuSCs). Recently, several studies have reported that mitochondria are important regulators of fate and function in various types of stem cells including MuSCs. Furthermore, emerging evidence has shown that accumulation of dysfunctional mitochondria leads to stem cell aging, premature commitment and impaired self-renewal. Preliminary evidence from publicly available transcriptomics datasets processed by our lab showed that Phosphatase and tensin homolog (PTEN)-induced putative kinase 1(PINK1) and Parkin/PARK2 genes, two key regulators of mitophagy are expressed in quiescent MuSCs and are transiently down-regulated as MuSCs activate. This led us to hypothesize that maintenance of an optimally functioning population of mitochondria through mitophagy would be important for self-renewal and muscle repair. In vitro single myofiber cultures isolated from mitophagy reporter mice (mito-QC mice), show that mitophagy is active in quiescent MuSCs and is transiently decreased upon MuSCs activation. We also show that mitophagy is re-activated in differentiating and self-renewing MuSCs. To further study muscle regeneration, we used a cardiotoxin (CTX) injury model of the Tibialis anterior (TA) muscle in mouse models harboring a knockout (KO) of PINK1 and Parkin. We show that loss of PINK1 in vivo promotes commitment of MuSCs in response to acute injury and ultimately leads to depletion of the MuSC pool and impaired muscle regeneration compared to wild type (WT) mice following repetitive injuries. Similarly, loss of Parkin in MuSCs in vivo impaired their self-renewal capacity. Consistent with these results, in vitro single myofiber cultures isolated from PINK1-deficient mice showed increased MuSCs commitment and impaired self-renewal. In vitro preliminary results from MuSCs-specific KO of Parkin revealed altered lineage progression, differentiation and self-renewal of MuSCs. Together, these findings suggest that PINK1/Parkin-dependent mitophagy acts as an important mitochondrial quality control mechanism which could be required for regulating MuSCs fate and function during muscle regeneration.