Background. Clear cell sarcoma of the kidney (CCSK) is known for its propensity to metastasize to bone, but it also spreads to other sites including the brain. This study was undertaken to describe the treatment and outcomes of patients with recurrent CCSK involving the brain. Methods. A retrospective records review was conducted on eight patients with CCSK who developed brain metastases after complete responses to initial therapy. Results. The recurrences occurred at a median of 24.5 months after initial diagnosis (range, 12-53 months). At the time of recurrence, patients were treated with multimodal therapy including biopsy or resection, radiation therapy, and chemotherapy. All patients received a variable number of courses of ifosfamide, carboplatin, and etoposide (ICE), with or without other agents. Four patients received high-dose chemotherapy with autologous stem cell rescue. One patient died from complications of bacteremia 8 weeks after starting chemotherapy. The other seven patients achieved a complete response after either surgery or ICE chemotherapy. Of these, six patients were alive without disease with a median follow-up of 30 months from the time of recurrence (range, 24 to 71 months). All six survivors received radiation therapy and four had gross total resections. Three survivors received high-dose chemotherapy with stem cell rescue. Conclusion. Patients with recurrent CCSK involving the brain can have durable survival after recurrence. ICE chemotherapy, together with radiation therapy and surgery, provides a reasonable salvage regimen for recurrent CCSK. It is unclear whether high-dose chemotherapy confers a benefit compared to conventional-dose chemotherapy.

Indiana University-Purdue University Indianapolis (IUPUI) / Prostate inflammation is a common health concern as an important risk factor for prostate cancer and Benign Prostatic Hyperplasia (BPH). Inflammation induces epithelial apoptosis and epithelial hyperplasia, suggesting that inflammation promotes the tissue repair and regeneration process. Progenitor cells are critical in maintaining epithelial homeostasis in adult tissues. However, the roles of prostate progenitor cells, especially during prostate inflammation, are understudied. I proposed that prostate epithelial progenitor cells (PEPCs) are involved in inflammation-induced epithelial hyperplasia, and are driven by regulation from inflammatory pathways. Here, we showed that sphere formation ability of prostate epithelial cells is increased by inflammation. We identified that a population of prostate progenitor cells, named prostate epithelial progenitor cells, were expanded by inflammation under the regulation of IL-1/insulin-like growth factor 1 (IGF-1) signaling pathway, a previously identified critical regulation pathway of inflammation-induced epithelial hyperplasia. The expansion of PEPCs also correlated with the intensity of inflammation. We then identified that survivin was upregulated in prostate epithelial cells by inflammation and was mainly co-localized with proliferation markers in prostate epithelial cells. This upregulation depended on IL-1/IGF-1 signaling. In vivo treatment with the survivin inhibitor LQZ-7F reduced both survivin expression and proliferation in prostate epithelial cells during inflammation. Using our label retaining strategy, we compared the survivin expression pattern in two prostate regeneration models. We discovered that different populations of progenitor cells may be involved in different regeneration processes. We identified that survivin was expressed in a specific population of reactivated cells that respond to the inflammatory environment and was independent of the known slow-cycling stem cells found in the prostate epithelium. In summary, I have identified that PEPCs are involved in epithelial hyperplasia and are dependent on survivin signaling. My work defines how survivin serves as a key regulator of epithelial hyperplasia in an inflammatory environment.

The ability for hematopoietic stem cells (HSCs) to regenerate the vascular and blood systems following injury suggests great potential for future therapies. Unfortunately, the various signaling pathways that regulate the regeneration of the adult HSC population in the bone marrow are not clearly understood. One of the proposed regulators for the regeneration of the hematopoietic system is an extracellular secreted protein R Spondin-2 (Rspo2), also known as roof plate-specific spondin-2. The novel interaction between the Rspo2 protein and c-kit+sca-1+lineage– (KSL) HSCs shows an increased number of KSL and of more differentiated hematopoietic stem and progenitor cells (HSPCs) in vivo during both the steady and injured states. To determine the most efficient concentration of Rspo2 for such an interaction to occur, various doses of Rspo2 recombinant protein are plated with the KSL cells. They are then examined through flow cytometry and colony forming cell (CFC) assay. Rspo2 is widely known to interact with the canonical Wnt3a protein to activate the beta-catenin pathway. However, when various concentrations of Rspo2 recombinant proteins are plated with the Wnt3a protein, the results show the opposite effect of plating the cells only with the Rspo2 protein. The overall increase in the total number of cells and KSL cells was concluded to be not significant. This study nonetheless provides the scientific community with a greater foundation for the usage of Rspo2 concentration for future experiments.

Stomata act as small valves that help in the regulation of gas exchange in plants. These valves arise from epidermal stem cells that asymmetrically divide to become meristemoids, which are precursor cells that eventually give rise to guard cells. Our lab used microarray-based gene expression profiling to identify genes that might play a role in maintaining or controlling meristemoid behavior. One gene, At5g60880, encodes a small protein that plays a role in stomata patterning. The sequence of this protein provides little information about its putative function. In order to gain insight into the function of this protein, we obtained mutant plants (SALK_86936) and characterized their phenotype. These plants exhibited abnormal asymmetric divisions resulting in clustered stomata. Since nothing is known about this protein, we determined the At5g60880 gene expression pattern by making transgenic plants carrying a fusion between the native At5g60880 promoter and green fluorescent protein (GFP). Confocal laser scanning microscopy was used to visualize the expression pattern of GFP in developing leaves. At5g60880 appears to be expressed in most epidermal cells. To better understand the possible activity of this protein, we also created transgenic plants used to deduce the protein subcellular localization. These plants constitutively express a translational fusion between the At5g60880 protein and GFP driven by the CaMV 35S promoter. Preliminary results reveal cortical and cytoplasmic localization of this fusion protein. Understanding the function of this novel protein will ultimately contribute to our knowledge of stomata patterning mechanisms, and more generally stern cell differentiation in plants.

Skeletal muscle stem cells, known as satellite cells (SC), are essential for skeletal muscle regeneration/repair and have been linked to muscle hypertrophy in humans. There is a consensus within the literature that SC activate and proliferate in response to external stimuli, such as mechanical damage or exercise. However, the effect of nutritional supplementation in conjunction with exercise on SC function is not fully understood. This may, in part, be due to varying responses of individuals to specific nutritional ingredients. Therefore, this study examined the efficacy of a multicomponent supplement containing whey protein isolate, leucine, creatine monohydrate, calcium citrate, and vitamin D, all of which have independently been shown to confer beneficial effects to skeletal muscle mass or function. Accordingly, when considering individual variability, a multicomponent nutritional strategy, when combined with resistance exercise, may be more likely to produce an augmented response compared to isolated supplements. Healthy, young males and females (18-26 y; ± 0.55) were randomly assigned to a multi-ingredient supplement (MIS)(n=13, 7M) or collagen peptide (CP)(n = 13, 6M) group. Participants performed a whole-body linear resistance-training program 4 times a week for 10-weeks. Skeletal muscle biopsies were obtained from the vastus lateralis pre and post 10 weeks of resistance training. Additionally, biopsies were obtained following an acute bout of damaging eccentric exercise prior to and following the 10 weeks of training. Thus, this design provided a resting and an acute damage response (48-hours post damage) before and after 10-weeks of resistance exercise and supplementation. Training resulted in an 83% and 40% increase in the basal SC population for mixed fibres in the MIS and CP group (P < 0.05), respectively, with no group differences. No effect of time or group was found for acute SC proliferation. However, when collapsing groups, a 635% increase was observed in the relative delta SC activation following 10 weeks (P < 0.05). Also, similar increases were observed in both groups for myonuclear accretion and myonuclear domain (P < 0.05). The MIS group had a 16% larger increase in type II CSA compared to the CP group (P < 0.05). Irrespective of supplementation, our findings suggest 10-weeks of resistance exercise is capable of increasing the basal SC population, SC activation, myonuclear accretion, and myonuclear domain. Furthermore, consuming a MIS lead to superior increases in type II CSA, compared to the CP group. / Thesis / Master of Science (MSc) / Skeletal muscle stem cells, known as satellite cells (SC), are essential for skeletal muscle regeneration/repair and have been linked to muscle hypertrophy in humans. SC activate and proliferate in response to external stimuli, such as mechanical damage or exercise. However, the effect of nutritional supplementation in conjunction with exercise on SC biology is not fully understood. This may, in part, be due to varying responses by individuals to specific nutritional ingredients. In this regard, multi-ingredient supplementation (MIS) has been implemented with the intention of eliciting a maximal adaptive response across all participants. Therefore, this study examined the efficacy of a multicomponent supplement containing whey protein isolate, leucine, creatine monohydrate, calcium citrate, and vitamin D, all of which have independently been shown to confer beneficial effects for skeletal muscle mass or function. Our findings suggest 10-weeks of resistance exercise is capable of increasing the basal SC population, SC activation, myonuclear accretion, and myonuclear domain. Nutritional Supplementation had no further effect. Importantly, however, consuming a MIS lead to greater increases in type II CSA, when compared to a control supplement.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20285号 / 医科博第76号 / 新制||医科||5(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 篠原 隆司, 教授 浅野 雅秀, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20618号 / 医博第4267号 / 新制||医||1023(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 髙折 晃史, 教授 羽賀 博典, 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

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23794号 / 医博第4840号 / 新制||医||1058(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 河本 宏, 教授 小川 誠司, 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

The future of human embryonic stem cell (hESC) research with regards to their applicability in a therapeutic setting, relies on the development and standardisation of consistent and robust methods to demonstrate their defining characteristics; their pluripotent ability to form all three germ layers and their capacity for self-renewal. Although much research has been carried out to investigate new methods of culturing hESCs, many of these studies have not robustly concluded the impact of prolonged culture on genetic and genomic stability nor have they examined in any comparative detail the impact of the culture conditions such as differences in feeders used or the media composition in which the stem cells are cultured in. The aim of this thesis therefore was to investigate and evaluate methods for improving the uniform and robust culture and characterisation of hESCs over prolonged periods in culture. Four hESC lines ( RH5, HUES9, SHEF1 and NCL5) were chosen on the basis that they had not previously been well characterised and therefore could potentially benefit the wider stem cell community by increasing diversity, rather than continue to use the already small subset of well publicised lines. The RH5, HUES9, SHEF1 and NCL5 cells were subjected to long term passaging using recombinant enzyme TrypLE™ Express, on human feeders, mouse feeders and feeder free matrix Matrigel in combination with defined media mTeSR1, for uniform scale up. Changes in characteristic stem cell surface markers were compared using two techniques; flow cytometry and quantitative in situ fluorescence microscopy. Genomic stability was assessed by real time PCR. Chromosomal integrity was monitored using array genomic hybridisation (aCGH). Array genomic hybridisation analysis of cells cultured for 20 passages by enzymatic passaging revealed changes in copy number variations in all the stem cell lines. Aberrations on chromosomes 12, 17 and 20, appeared most commonly as a result of long term culture. Although no significant differences were seen between hESCs cultured on mouse and human feeders, cultures on Matrigel showed fewer detected chromosomal aberrations. Expression of cell surface stemness markers SSEA3, SSEA4, TRA1-60 and TRA1-81 were maintained by hESC cultured on all matrices and confirmed by the use of flow cytometry and high throughput quantitative immunofluorescence imaging using the TissueFaxs™ cell analysis microscopy system. In depth imaging revealed subtle but important differences in the way in which hESCs attach and proliferate on different matrices. Genetic profiling of each of the stem cell lines using Taqman Low density array cards to assess the expression of 96 genes by Real Time PCR, demonstrated the continued expression of stemness genes 21 at late passage, and low level expression of differentiation genes, inherent to particular stem cell lines. Although both mouse and human feeders and Matrigel support the undifferentiated growth of hESCs, subtle differences from the hESCs were seen as a result of their use, most obviously, changes in morphology and how they proliferate. This was further explored in the stem cell line NCL5, as it demonstrated a readiness to adapt to new matrices, better chromosomal stability and higher expression of cell surface markers compared with the other hESC lines. Using in vitro differentiation assays to all three germ layers, NCL5 cultured to late passage (p+20) on human feeder iMRC5, mouse feeder iMEF and feeder free matrix Matrigel, demonstrated the ability to differentiate to ectoderm, endoderm and mesoderm progenitors after induction using three 7 day flat based directed differentiation protocols. Altered differentiation patterns were detected by Real Time PCR and TissueFaxs™ imaging and quantitative analysis, as a consequence of the prolonged culture on the specific matrices used. Such key findings allude to the strong influences of microenvironment and will help to improve the standardisation of in vitro differentiation assays. From these studies, chromosomal changes had no impact on NCL5 stem cell lines‘ ability to form progenitors, however small genetic instabilities may still play a role in terminal differentiation of germ lineage specific cell types. The findings of the programme of work described has led to the successful culture methods and characterisation testing validated in this project being incorporated into routine culture and banking of research grade hESCs at the UK Stem Cell Bank. These protocols will now be made more widely available and should assist stem cell researchers in adopting the most suitable and optimum conditions for culturing stem cells in the undifferentiated and stable state. With the huge surge in stem cell research over the past decade, the development of robust characterisation and culture methods will undoubtedly have significant impact on the exploitation of these cells for regenerative medicine and to assist with this a future aim of the stem cell bank will be to standardise methodologies for clinical grade banking.