Preserving viability of frozen gametes and reproductive tissues is crucial to understand and overcome species-specificities in respect to the diversity in cryobiological properties and requirements among cell types and tissues. The use of different animal models to study ovarian tissue cryopreservation will help to uncover several important factors related to germ cells preservation. Horses (Equus ferus caballus) have been proven to be an excellent model for reproductive biology studies with implications for humans. White-tailed deer (Odocoileus virginianus) are one of the most abundant wild species in the United States, but little information about their reproductive features are known. Therefore, five studies were conducted in this Dissertation with the following general objectives: (i) to develop ovarian tissue cryopreservation techniques for horses and white-tailed deer species; and (ii) to determine the effects of ovarian tissue cryopreservation techniques on morphological and molecular mechanisms related to folliculogenesis in horse and white-tailed deer species. In study one, equine ovarian tissue was used to determine the ideal ovarian fragment size for better cooling resistance under storage at 4°C. In study two, equine ovarian tissues were used to determine the toxicity effect of cryoprotective agents on ovarian tissue pre- and post-cryopreservation. In study three, equine ovarian tissues were used to compare slow-freezing versus vitrification; and to determine the best cryoprotective agents for each cryopreservation method. In study four, white-tailed deer reproductive tracts were used to characterize the age effect on reproductive features. In study five, white-tailed deer ovarian tissue was used to compare slow-freezing versus vitrification methods to preserve preantral follicles under in vitro culture. The main findings of the horse studies were: (i) equine ovarian tissue can be stored at 4°C for up to 24 h when biopsy ovarian fragments are used; (ii) ethylene glycol seems to be a less harmful cryoprotectant agent to equine preantral follicles; and (iii) both slow-freezing and vitrification methods similarly preserved the follicle morphology after time of culture. The main findings of the white-tailed deer studies were: (i) aging caused quantitative and qualitative effects on the ovarian reserve of white-tailed deer; (ii) fresh ovarian tissue can be cultured for up to seven days preserving the tissue integrity; and (iii) fragments cryopreserved by vitrification had higher follicle viability during in vitro culture than by the slow-freezing method. In conclusion, this work demonstrated the viability to cryopreserve equine and white-tailed deer ovarian tissue. Furthermore, the frozen-thawed equine and white-tailed deer ovarian tissue can be cultured for up to seven days.

The incidence of colorectal cancer (CRC) is increasing and the prognosis for patients with advanced or metastatic disease is relatively poor. Immunotherapies hold great promise, but deploying them effectively in CRC patients will require further knowledge of the complex cellular and molecular interactions that occur between intestinal tumours and the host immune system. The objective of this study is to understand the mechanisms by which lack of immune cell regulation in the gut can drive the formation of colon adenocarcinomas. In addition, this work aims to identify new mechanisms involved in progression to metastatic disease. Using mouse model systems, we found that aberrant activity of Treg cells deficient in IL-10 can promote inflammation-driven CRC. IL-10 deficient Tregs have increased capacity to drive tumourigenesis compared to their CD4⁺ effector T cell counterparts. RNA sequencing revealed specific upregulation of several genes, including a newly-described cytokine, in tumour-promoting Tregs. We explored cytokine regulation and the tumour microenvironment, and show that the inflammatory cytokine IL-6 and TGFÎ2 are necessary for tumour formation in this model. Moreover, disease is associated with a marked stromal cell signature that is induced as a consequence of Treg deficiency in IL-10 production. Gp38⁺ stromal cells are dominant producers of IL-6, and potent ECM modellers. Furthermore, tumours driven by IL-10 deficient Tregs express high amounts of the pro-mesenchymal transcription factor Sox4. Using combined in vitro and in vivo analyses, we confirm that Sox4 is involved in tumour growth and characterise its expression in CRC patients. Collectively, our findings suggest that Tregs and stromal cells act together to foster a microenvironment that promotes disease progression, notably through the expression of Sox4 in tumour cells. These findings open an exciting avenue to explore the phenotype of tumour-promoting Tregs and to study Sox4 function in metastatic disease.

Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Mark L. Weiss / The traditional cell culture method after isolation from the body involves growing cells in 2 dimensions on plastic culture plate. However, the natural structure and physiology is 3 dimensions. To mimic in vivo environment, there has an increasing interest to find the way to maintain physiological properties. Here, we describe culturing human umbilical cord mesenchymal stromal cells (HUC-MSCs_in 3D setting using human platelet lysate gel. This gel is a fibrin-based structure like a blood clot. The preparation step of human platelet lysate (HPL) is by freeze- thaw cycles in order to release factors important for cells to grow and expand. Using of HPL to substitute for fetal bovine serum reduces potential cross contamination between species and xenogenicity. To maintain HPL media as a liquid, we add the anticoagulant heparin. Without adding anticoagulant, the gel will form. The aim of this study is to retrieve HUC-MSCs from HPL gel using Nattokinase, to characterize HUC-MSCs following the International Society for Cell Therapy’s MSC criteria, and to test a 3D invasion model with HPL-gel based structure. The result shows that using 1.75% Nattokinase at 60 minutes can recover the cells without reducing cell number and viability. After Nattokinase treatment, cells are able to attach to plastic and to increase in number. Moreover, they are able to differentiate into fat, bone, and cartilage no different from cells grown in 2D culture. However, to test surface markers by flow cytometry, all MSC markers are positive except CD 105. They are also positive of cell surface markers that should be negative. When seeded back to 2D culture for an additional passage, the MSCs meet ISCT criteria the same as control.

Orientador: Sandra Helena Penha de Oliveira / Banca: Gustavo Pompermaier Garlet / Banca: Willian Fernando Zambuzzi / Resumo: Células-tronco mesenquimais (CTMs) obtidas a partir da medula óssea são capazes de se diferenciarem, sobretudo, em condrócitos, adipócitos e osteoblastos. Durante a osteogênese in vitro, alguns parâmetros são utilizados para caracterizar este processo, tais como atividade da fosfatase alcalina (FAL), mineralização e expressão de proteínas associadas à osteoblastos. Ratos espontaneamente hipertensos (SHR) são um modelo animal de hipertensão essencial humana e desenvolvem hipertensão após 4 semanas de idade. Esta linhagem apresenta alterações significativas no metabolismo ósseo. O objetivo do presente estudo foi investigar se, o genótipo hipertensivo poderia interferir na diferenciação osteoblástica das CTMs de ratos SHR e qual mecanismo está alterado quando comparadas com a linhagem progenitora, ratos Wistar. Para isso, nós obtivemos CTMs da medula óssea de ratos Wistar e SHR com 4 semanas de idade, sem a hipertensão estabelecida, afim de avaliar somente o possível efeito do genótipo hipertensivo na diferenciação osteogênica in vitro. Nós induzimos, ou não, a diferenciação osteogênica in vitro por meio da utilização dos indutores osteogênicos: ácido ascórbico, β-glicerofosfato e dexametasona. Os resultados demonstraram que, CTMs indiferenciadas de SHR (SHRC) demonstraram taxa de proliferação aumentada em comparação a CTMs, na mesma condição, de Wistar (WC), e após a indução da osteogênica, a taxa de proliferação apresentou uma diminuição acentuada no grupo SHR (SHRMO) do que no grupo Wistar na mesma condição (WMO). Embora não fora observada diferença significativa na atividade da FAL entre SHRMO e WOM no 7° dia, a mineralização e a diferenciação osteoblástica foram menores no grupo SHRMO no mesmo período experimental. Os fatores de transcrição Osterix e β-catenina parecem estar envolvidos na diferenciação reduzida no grupo SHRMO... / Abstract: Mesenchymal stem cells (MSCs) from bone marrow are able to differentiate mainly into chondrocytes, adipocytes and osteoblasts. During in vitro osteogenesis, some parameters are used to characterize this process, such as the activity of alkaline phosphatase (ALP), mineralization and osteoblast-associated proteins expression. Spontaneously hypertensive rats (SHR) is an animal model of human essential hypertension. This animals developing hypertension after 4 weeks of age. This strain shows significant changes in bone metabolism. The aim of this study was to investigate whether the hypertensive genotype could influence the osteoblastic differentiation of MSCs from SHR and which mechanism are altered when compared to the parental strain, Wistar rats. For that, we have obtained bone marrow MSCs from Wistar and SHR rats at 4 weeks of age, without hypertension established in order to evaluate only the possible effect of hypertensive genotype on osteogenic differentiation in vitro. We induced or non-osteogenic differentiation in vitro using osteogenic inducers: ascorbic acid, dexamethasone and β-glycerophosphate. The results demonstrate that undifferentiated MSCs SHR (SHRC) showed increased proliferation rate compared to MSCs, in the same condition Wistar (WC) and after osteogenic induction, proliferation rate showed a marked decrease in SHR (SHRMO) than in Wistar group in the same condition (WMO). Although it was not observed significant difference in ALP activity between WMO and SHRMO on day 7, mineralization and osteoblast differentiation were lower on group SHRMO in the same experimental period. The transcription factors Osterix and β-catenin appear to be involved in reduced differentiation in SHRMO group because they showed lower expression in this experimental group. Furthermore, the decreased... / Mestre

Abstract Secreted signalling molecules are important for the regulation of developmental cell responses. In the developing kidney, signalling occurs between epithelial ureteric bud and metanephric mesenchyme and in between their derivatives. Wnt6 gene activity was localized to the ureteric bud and newly formed branches of the ureteric tree during early stages of kidney development. In a classic organ culture system, Wnt6 signalling induced the activation of marker genes for early nephrogenesis. The metanephric mesenchymes isolated from the Wnt4 deficient embryos were also induced, and the Wnt4 gene became activated in the presence of a Wnt6 signalling source. We propose that Wnt-6 is involved as a metanephric inducer and controls nephrogenesis. Wnt4 is essential for nephrogenesis in mouse and we indicate an additional role for Wnt4 in the control of periureteric stromal differentiation. A failure in vascular development was also found. Bmp4 expression in the medullar stroma of the Wnt4-deficient kidneys was absent concomitantly with a loss of expression of the smooth muscle marker, α-SMA. In vitro Wnt4 signalling induced Bmp4 expression and local α-SMA production. Hence, we conclude that lack of Wnt4 signalling leads to a loss of the periureteric smooth muscle cells, and Wnt4 may locally regulate this cell population in normal kidneys via regulation of Bmp4 signalling. The pluripotent neural crest cells are proposed to play regulatory roles in the early metanephros. Here, the use of transgenic animals allowed visualisation of the lumbo-sacral neural crest (NC) cells in close proximity to the early metanephros. The NC cells, however, disappeared in most part of the kidney by E12.5. The Splotch embryos lack the NCs from the early urogenital region. A developmental defect in the kidneys of Splotch embryos was not observed in vivo or in vitro. The results suggest that the neural crest is not essential for early embryonic kidney development. In sum, the work presented indicates an important role for Wnt6 in the induction of kidney tubules in vitro, for Wnt4 in the specification of kidney smooth muscle cells and for endothelial development in kidney. The neural crest cells apparently have no active morphogenetic role in early kidney development.

Medulloblastoma (MB), a cancer of the cerebellum, is the most common solid tumor affecting children. In the cerebellum, Sonic Hedgehog (Shh) drives the proliferative expansion of granule neuron progenitors (GNP). These cells are located in the external granule layer (EGL) and are the cells of origin of Shh-MB. We recently identified Norrie Disease Pseudoglioma (Ndp) as a novel downstream target of Hh signaling in the developing retina. Ndp encodes an X-linked cysteine-rich secreted protein called Norrin, which is best known for its role in angiogenesis and blood brain barrier (BBB) maintenance in the developing retina and cerebellum, respectively. Norrin mediates this effect by binding to its receptor Frizzled4 (Fzd4) and co-receptors LRP5/6 and Tpsan12 to activate the canonical, β-catenin-dependent Wnt signaling pathway in endothelial cells (ECs). We detected the expression of Ndp and all required receptors in mouse GNPs and MB samples. To investigate a potential role for Ndp in Hh-driven MB, we genetically and pharmacologically inactivated Ndp/Fzd4 signaling in Ptch+/- mice (a mouse model for human Gorlin syndrome), which dramatically increased the incidence and reduced the latency of MB. This accelerated rate of tumorigenesis was caused by an increase in the number of preneoplastic lesions (PNLs), the precursor lesions to MB, and a faster conversion of these lesions to MB. We showed that Ndp mediates this increase in tumorigenesis by signaling through endothelial cell receptor Fzd4 to alter the GNP stroma, which is characterised by 5 major alterations: 1) activated angiogenic program, 2) open BBB, 3) aberrant deposition of extracellular matrix, 4) aberrant lymphocyte recruitment and 5) reduction in meningeal lymphatic vasculature. We propose that these stromal alterations are associated with a pro-tumor microenvironment that promotes DNA damage in GNPs and leads to enhanced lesion formation and progression towards MB. This research highlights 1) an unanticipated role for Ndp/Fzd4 signaling in Shh-MB initiation and progression, 2) a role for stromal signaling in the regulation of MB development and 3) a previously undescribed role for Ndp signaling in maintaining meningeal cerebellum lymphatic vessels.

Acute myeloid leukemia (AML) often remains refractory to current chemotherapy and transplantation approaches despite many advances in our understanding of mechanisms in leukemogenesis. The bone marrow “niche” or microenvironment, however, may be permissive to leukemia development and studying interactions between the microenvironment and leukemia cells may provide new insight for therapeutic advances. Mesenchymal stem cells (MSCs) are central to the development and maintenance of the bone marrow niche and have been shown to have important functional alterations derived from patients with different hematological disorders. The extent to which MSCs derived from AML patients are altered remains unclear. The aim of this study was to detect changes occurring in MSCs obtained from human bone marrow in patients with AML by comparing their function and gene expression pattern with normal age-matched controls. MSCs expanded from patients diagnosed with acute leukemia were observed to have heterogeneous morphological characteristics compared to the healthy controls. Immunohistochemistry and flow data confirmed the typical cell surface immunophenotype of CD90+ CD105+ CD73+ CD34- CD45-, although MSCs from two patients with AML revealed reduced surface expression of CD105 and CD90 antigens respectively. Differentiation assays demonstrated the potential of MSCs from AML patients and healthy donors to differentiate into bone, fat and cartilage. However, the ability of MSCs from AML samples to support hematopoietic function of CD34+ progenitors was found to be impaired while the key hematopoietic genes were found to be differentially expressed on AML-MSCs compared to nMSCs. These studies indicate that there exist differences in the biologic profile of MSCs from AML patients compared to MSCs derived from healthy donors. The results described in the thesis provide a formulation for additional studies that may allow us to identify new targets for improved treatment of AML.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22319号 / 医博第4560号 / 新制||医||1041(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 濵﨑 洋子, 教授 稲垣 暢也, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Cigarette smoking (CS) continues to be a significant modifiable factor contributing to a variety of diseases including cardiovascular, pulmonary and renal pathologies. It was suggested that smoking have detrimental effect of the body’s progenitor cells of bone marrow and peripheral organs. Since the concept of cell therapy that utilizes adipose stem/stromal cells (ASC) is gaining momentum it becomes critical to assess the therapeutic activities of the progenitors isolated from smokers. This study has revealed that CS negatively impacts the vasculogenic potential of ASC, in vitro, as well as weakening their therapeutic activity in vivo when tested in mouse model of hindlimb ischemia. We hypothesized that the decrease in vasculogenic activity of ASC is attributed to a higher level of expression of an angiostatic factor Activin A by ASC from CS donors. These findings clearly suggest that smokers should be evaluated for potential exclusion from early clinical trials of autologous cell therapies, or assessed as a separate cohort. The donor’s health status should be considered when choosing between autologous vs allogeneic cell therapies. We then examined the effect of CS on development of kidney pathology in mice. CS exposure led to decrease in kidney weights, capillary rarefaction, and cortical blood perfusion, and in parallel led to increase in kidney fibrosis and iron deposition. Interestingly, infusion of healthy ASC to the mice following CSexposure reversed CS-induced damages. This strongly support the notion that ASC-based therapy may provide rejuvenation effect. In the other subset of studies, we hypothesized that CS-induced lung emphysematous changes are preceded by suppression of bone marrow (BM) hematopoietic progenitor cells (HPC). We have revealed that intermittent BM mobilization with AMD3100 may mitigate the CS-induced myelo-suppression and deterioration of lung function and morphology. We observed that treatment of mice with AMD3100, while exposed to CS, preserves HPC at the levels of healthy control mice. Furthermore, AMD3100 treatment preserved lung parenchyma from pathological changes. These data suggest that while CS has a myelo-suppressive effect, administration of AMD3100 preserved BM-HPC and ameliorated lung damage.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18160号 / 医博第3880号 / 新制||医||1003(附属図書館) / 31018 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 生田 宏一, 教授 宮地 良樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM