Inflammatory arthritis (IA) is a debilitating disease that is characterized by joint destruction. This destruction is caused by osteoclasts (OCLs) degrading bone within the synovium, however the exact cellular origins of these synovial OCLs is not well understood. We hypothesize that the synovial OCLs seen in IA are independently derived from two contributing cell lineages: the canonical source of OCL which are Hematopoietic Stem Cell (HSC) derived monocytes and the newly described Erythro-Myeloid Progenitor (EMP). To explore the contribution of these two lineages to synovial OCL, we used Cx3cr1CreERT2;Rosa26LSL-tdTomato mice to label EMP-derived cells, and Flt3Cre;Rosa26LSL-YFP to label HSC-derived cells. Using immunofluorescent histology, we found that synovial OCLs formed under arthritic conditions derive from both HSC and EMP-progenitors, suggesting the possibility that regulatory mechanisms unique to each developmental lineage promote OCL differentiation in arthritic joints. In support of these observations, in IA we detected two populations of mononuclear cells, as possible osteoclast precursors, that express both the OCL marker TRAP and the monocytic/macrophage marker CD68. These mononuclear TRAP+ CD68+ cell populations are found within the inflamed synovium and in bone periosteal surfaces of arthritic joints, and are mostly EMP derived, however, the HSC lineage significantly contributes to osteoclast formation, as suggested by our lineage-tracing strategy. We are currently investigating the dynamics of these cell populations during the early, peak, and resolution stages in an acute murine model of IA. To better understand regulatory differences between OCL derived from each of these lineages, we plan to isolate EMP- and HSC-derived OCLs directly from joints to study their precise phenotype, cytokine differentiation requirements, resorptive capacity and transcriptional activity, by flow cytometry, in vitro cell cultures and single cell RNA sequencing, respectively.

Endocannabinoids modulate a diverse array of functions including progenitor cell proliferation in the central nervous system, and odorant detection and food intake in the mammalian central olfactory system and larval Xenopus laevis peripheral olfactory system. However, the presence and role of endocannabinoids in the peripheral olfactory epithelium have not been examined in mammals. We found the presence of cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2) receptor protein and mRNA in the olfactory epithelium. Using either immunohistochemistry or calcium imaging we localized CB1 receptors on neurons, glia-like sustentacular cells, microvillous cells and progenitor-like basal cells. To examine the role of endocannabinoids, CB1- and CB2- receptor-deficient (CB1-/-/CB2-/-) mice were used. The endocannabinoid 2-arachidonylglycerol (2-AG) was present at high levels in both C57BL/6 wildtype and CB1-/-/CB2-/- mice. 2-AG synthetic and degradative enzymes are expressed in wildtype mice. A small but significant decrease in basal cell and olfactory sensory neuron numbers was observed in CB1-/-/CB2-/- mice compared to wildtype mice. The decrease in olfactory sensory neurons did not translate to impairment in olfactory-mediated behaviors assessed by the buried food test and habituation/dishabituation test. Collectively, these data indicate the presence of an endocannabinoid system in the mouse olfactory epithelium. However, unlike in tadpoles, endocannabinoids do not modulate olfaction. Further investigation on the role of endocannabinoids in progenitor cell function in the olfactory epithelium is warranted.

BACKGROUND: Human mesenchymal progenitor cells are multipotent cells that can be harvested from various adult and fetal tissues. They exhibit the potential to differentiate into several cell lineages, most notably osteogenic, chondrogenic, and adipogenic lineages. Conditions such as osteoporosis, metabolic disease, and arthritis are examples of dysfunction of tissues formed by the mesenchyme. The inability of these conditions to be healed by the body’s own mechanisms has raised considerable interest in the potential of using mesenchymal progenitor cells as a therapeutic intervention. This concept opens the possibility of harvesting mesenchymal progenitor cells from an individual, growing them into the desired tissue, and implanting them back into the individual. Treatment of this nature is much less invasive than current methods, overcomes rejection by the immune system, and could potentially demonstrate better outcomes in individuals suffering from degenerative disease of the mesenchyme. AIMS/OBJECTIVES: The aims of this study were to determine and to characterize the differentiation of human adipose-derived mesenchymal progenitor cells into osteocytes, chondrocytes, and adipocytes. The differentiation capacity of the mesenchymal progenitor cells was evaluated through cell staining, immunofluorescence, and RNA sequencing. METHODS: Subcutaneous adipose tissue was collected from patients undergoing elective panniculectomies. The abdominal panniculus was liposuctioned, and small explants of fat were embedded in Matrigel. Mesenchymal progenitor cells were extracted from the explants and plated for differentiation into osteogenic, chondrogenic, and adipogenic lineages. Control cells were grown in parallel in basal media to confirm differentiation. Dye staining for differentiation was performed with Alizarin Red S, Alcian Blue, and Oil Red O, and immunofluorescence staining was performed to indicate lineage-specific markers for differentiation. RNA sequencing was also completed on the different cell lineages. RESULTS: Human adipose-derived mesenchymal progenitor cells displayed the capacity to differentiate into osteogenic, chondrogenic, and adipogenic lineages as evidenced by dye staining. Osteogenic differentiation was confirmed with Alizarin Red S staining of calcium deposits in the differentiated cells, whereas staining in the control resulted in no calcium deposits. Alcian Blue staining confirmed chondrogenic differentiation as glycoproteins secreted by the differentiated cells were evident and different in morphology compared with the control cells. Oil Red O staining indicated adipogenic differentiation by showing lipid droplets in the differentiated cells and no lipid droplets in the control. RNA sequencing provided support that lineage differentiation was successful. Immunofluorescence staining further proved that differentiated cells expressed lineage-specific proteins and demonstrated morphological differences. CONCLUSIONS: This study demonstrates that mesenchymal progenitor cells harvested from human adipose tissue have the potential to differentiate into adipogenic, chondrogenic, and osteogenic cell lineages when induced with differentiation media. The differentiation of these cells can be assessed with dye staining, RNA sequencing, and immunofluorescence staining methods. Further studies should be done to investigate the potential of mesenchymal progenitor cells for therapeutic interventions in the treatment of various illnesses related to the mesenchyme.

Indiana University-Purdue University Indianapolis (IUPUI) / Fracture repair is costly and difficult to treat. One of the main causations of nonunion is a lack of essential blood supply. The needed blood is supplied by the growth of new blood vessels, a process known as angiogenesis, that invade the damaged tissue early in the healing process. We proposed using bone tissue engineering as an effective therapy. This therapy uses stem cells to aid in tissue regeneration. Endothelial progenitor cells (EPCs) were selected due to their ability to form tube-like networks in vitro. EPCs were isolated from murine bone marrow and lung tissue. We tested EPC’s tube forming, proliferative, and wound migration ability in vitro. To test their ability in vivo we created a femoral fracture in young and old mice. EPCs were seeded to the fracture site upon a collagen scaffold. The in vitro studies displayed that the bone marrow and lung-derived endothelial cells presented EPC traits. In the mouse fracture model bone marrow, endothelial cells did not significantly improve the healing process. In the future, we want to improve our cell extraction and purification method, as well as test a new stem cell delivery biomaterial. We also want to select and use a growth factor (GF) that can help to promote bone regeneration in tandem with the EPCs.

No description available.

Obesity is a major global health concern due to its steadily increasing rates and significant contribution to numerous diseases, including cancer. Ovarian cancer specifically, is associated with a 30% increased risk with obesity, although the mechanisms for this are unknown. Waist-to-hip ratio has been especially associated with ovarian cancer, suggesting that visceral fat may be the greatest contributor. Here, we investigated individual visceral fat depots as independent contributors to cancer progression, specifically focusing on adipose tissue-derived stem and progenitor cells, which have previously been shown to be recruited by cancer cells and participate in cancer progression. We confirmed that ovarian cancer tumor burden was indeed significantly increased in mice on a high fat as compared to low fat diet. To further investigate mechanisms, we examined changes in progenitor populations that occurred in intra-abdominal parametrial (pmWAT), retroperitoneal (rpWAT), and omental (omWAT) white adipose tissue (WAT) depots with cancer presence. The greatest tumor burden was evident in omWAT, which also displayed an increase in CD45- cells but a decrease in adipose progenitor cells (APC) and endothelial progenitor cells, suggesting that there was an increase in stromal cells, but that the stem cells were pushed towards differentiation. PmWAT and rpWAT showed remarkably stable progenitor populations. However, a tumor from pmWAT had a significant presence of CD45- cells, actually matching that of its surrounding tissue and differing from the omWAT tumors, indicating that microenvironment has a major influence on tumor stromal cells. We also found that with high fat diet, many cancer-associated changes were exacerbated, such as an increased inflammatory response in all tissues and further decreases in APCs in omWAT. In vitro studies further confirmed that ovarian cancer cells and SVF cells were able to directly interact. Additionally, SVF cells were able to increase the proliferation, mobility, and invasiveness of cancer cells. Conversely, co-culturing also enhanced the proliferation and mobility of SVF cells, providing further evidence that SVF cells may be recruited by cancer cells and that their relationship may be bilateral. Thus, this study provides a good foundation for examining the cellular contributions of adipose tissue to cancer. By further characterizing the mechanism for the association between obesity and cancer development, we could find novel targets to decrease the progress of cancer development in at-risk obese individuals. / Ph. D.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13159号 / 論医博第2146号 / 新制||医||1029(附属図書館) / (主査)教授 河本 宏, 教授 前川 平, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

There is increasing evidence to suggest that fetal events can predetermine reproductive health and general wellbeing in adulthood, a process termed 'fetal programming'. This refers to the association between altered fetal growth/development and health disorders in adulthood e.g. the metabolic syndrome, which is linked to low male testosterone levels. Studies from both Europe and the USA have shown that adult male testosterone levels have been declining, independent of age. As low testosterone levels in aging men are associated with increased morbidity and mortality, this highlights the importance of investigating how testosterone levels are determined or potentially ‘programmed’ during fetal development. Evidence from human and rodent studies have shown that reduced fetal androgen exposure results in lower adult testosterone levels, although the mechanism(s) is unknown, to date. One way to explain how a fetal insult (e.g. androgen deficiency) could affect (testosterone producing) adult Leydig cells in adulthood, is if their progenitor cells were present during fetal life and were thus affected by such an insult. This hypothesis has been unexplored to date, due to the lack of a unifying/defining marker for adult Leydig progenitor cells. An earlier study promoted the hypothesis for the studies in this thesis, namely that chicken ovalbumin upstream promoter transcription factor-II (COUP-TFII) might constitute such a marker, as inducible knockout of COUP-TFII in pre-pubertal male mice results in failure of adult Leydig cells to develop. Therefore, the hypothesis which was explored in this thesis was that 'fetal programming' of COUP-TFII+ adult Leydig progenitor cells prior to their differentiation into adult Leydig cells, would explain how fetal events could predetermine adult testosterone levels. To investigate whether adult Leydig cells (ALC) develop from COUP-TFII+ interstitial cells, firstly an adult Leydig cell ablation/regeneration model was used, which involved a single injection of ethane dimethane sulphonate (EDS). This identified that in rats, ALC derive from COUP-TFII+ interstitial cells which do not express any other phenotypical adult Leydig or interstitial cell markers prior to differentiation. Secondly, COUP-TFII+ adult Leydig progenitor cells are abundant in the fetal testis and conserved across species, including man. Thirdly, fetal interstitial cells which differentiated into ALC, as evident from an ALC lineage tracer model, also expressed COUP-TFII. Overall, these findings suggest that the COUP-TFII+ interstitial cells which differentiate into ALC are 'adult Leydig progenitor cells'. The findings from this thesis also show that the identified adult Leydig progenitor cells express the androgen receptor (AR) in fetal life. Furthermore, experimental reduction of androgen action in fetal life in transgenic mice (AR knockout) or chemical manipulations to reduce fetal testosterone levels (di(n-butyl) phthalate; DBP exposure) resulted in a similar reduction (~40%) in progenitor cell numbers from birth through to adulthood. A parallel reduction of adult Leydig cell numbers across postnatal development was found in mice, but not rats, but as a result of altered fetal androgen action, both models showed evidence for compensated adult Leydig cell failure. This is defined as normal/low testosterone and elevated luteinising hormone (LH) levels. Cell-selective knockout of AR in peritubular myoid (PTM) cells (PTM-ARKO) or Sertoli cells (SC-ARKO) did not affect the numerical development of adult Leydig progenitor cells. To manipulate testicular testosterone action in postnatal life, rats were exposed to a potent AR antagonist, flutamide, which reduced the number of adult Leydig progenitor cells but did not affect ALC number/function. However, the combination of fetal DBP+postnatal flutamide exposure reduced adult Leydig progenitor cells and resulted in compensated ALC failure. Overall, these studies highlight the importance of fetal androgens for the normal development of adult Leydig progenitor cells and for the subsequent development of normally functioning adult Leydig cells. As fetal deficits in androgen exposure resulted in adult Leydig cell dysfunction, this thesis also investigated three separate models to determine whether increased fetal androgen exposure could increase/enhance adult Leydig progenitor cell development, resulting in a 'gain of adult Leydig cell function'. In the first model to increase fetal androgen exposure, pregnant dams injected with testosterone propionate (TP; 20mg/kg/day e14-21.5) were discarded, due to confounding factors including fetal growth restriction and aromatisation of TP. The second model utilised dihydrotestosterone (DHT; 10mg/kg/day), administered to pregnant dams, but there were no effects found in adulthood to male offspring. It was concluded that the administered dose was not sufficient to increase intratesticular testosterone levels in the fetus. The third model utilised an inducible nitric oxide synthase knockout (iNOS-/-) mouse model, for which previous evidence showed increased testis weight, Leydig and Sertoli cell number (~50%), and normal testosterone but low LH levels in adulthood. Stereological quantification showed an increase in the number of adult Leydig progenitor cells in postnatal, but not fetal life, which resulted in the conclusion that the observed changes were a consequence of postnatal effects. Finally, a potential mechanism to explain how DBP-induced androgen deficiency in fetal life, could result in adult Leydig cell dysfunction in adulthood was investigated. Analysis of testicular genes in adulthood, involved in the steroidogenic pathway, showed a reduction in 3b-hsd and StAR. The reduced StAR expression was associated with increased repressive histone methylation (H3K27me3) in its proximal promoter region, as demonstrated by a chromatin immunoprecipitation (ChIP) assay, qPCR, and densitometrical analysis. Accordingly, adult Leydig cells were shown to express increased H3K27me3 by immunohistochemistry, a change also evident in adult Leydig progenitor cells in the fetal testis. This would provide a potential mechanism to explain how fetal events can 'programme' adult Leydig cell testosterone production, namely via an epigenetic change to adult Leydig progenitor cells. In summary, the results in this thesis show how fetal events, including androgen action on progenitor cells, can potentially programme adult Leydig cell function and thus determine testosterone levels. As testosterone is crucial to man, the findings reported in this thesis may have important implications for the general health and longevity of man.

Thesis (DScMedSc (Medical Sciences)--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: See full text for abstract / AFRIKAANSE OPSOMMING: Geen opsomming was ingehandig met tesis

Doctor of Philosophy / Department of Animal Sciences and Industry / John M. Gonzalez / This series of studies focuses on the impact of intra-litter variation on fetal myogenesis, and the ability of porcine progenitor cells to respond to growth promoting stimuli. In study 1, the smallest (SM), median (ME), and largest (LG) male fetuses from each litter were selected for muscle morphometric analysis from gilts at d-60 ± 2 and 95 ± 2 of gestation. On d-60 and 95 of gestation LG fetuses had greater whole muscle cross-sectional area (CSA) than ME and SM fetuses, and ME fetuses had greater whole muscle CSA than SM fetuses. Indicating that SM and ME fetuses are on a delayed trajectory for myogenesis compared to LG fetuses. At d-60 the advanced trajectory of LG compared to ME fetuses was due to increased development of secondary muscle fibers; whereas, the advanced myogenic development of LG and ME fetuses compared to SM fetuses was due to the presence of fewer primary and secondary muscle fibers. At d-95 of gestation the advanced myogenic development of LG and ME was due to increased hypertrophy of secondary muscle fibers. For study 2, porcine fetal myoblasts (PFM) were isolated from SM, ME, and LG fetuses from d-60 ± 2 of gestation fetuses and for study 3, porcine satellite cells (PSC) were isolated from the piglet nearest the average body weight of the litter. Both myogenic cell types were utilized to evaluate effects of porcine plasma on proliferation, differentiation, and indications of protein synthesis. For the proliferation assay, cells were exposed to one of three treatments: high serum which consisted high-glucose Dulbecco's Modified Eagle Medium supplemented with 10% (vol/vol) fetal bovine serum, 2% (vol/vol) porcine serum, 100 U penicillan/mL, 100 µg of strepmycin/mL, and 20 µg of gentamicin/mL (HS), low serum which consisted of HS without 10% FBS (LS), and LS supplemented with 10% (wt/vol) porcine plasma (PP). Treatments for the differentiation and protein synthesis assays consisted of either HS or LS media that either contained porcine plasma at 10% (wt/vol; PPP) or 0% (wt/vol; PPN). The HS-PFM had a greater proliferation rate compared to the LS and PP-PFM, and PP-PFM had a greater proliferation rate compared to LS-PFM. The LG fetuses’ PFM had a reduced proliferation rate compared to SM and ME fetuses’ PFM, which were similar. The PPP-PFM had a decreased myotube diameter compared to PPN-PFM. Small fetuses’ PFM had a greater myotube diameter compared to ME and LG fetuses’ PFM, and ME fetuses’ PFM had a greater myotube diameter compared to LG fetuses’ PFM. The proliferation rate of PP-PSC was decreased compared to the HS- and LS-PSC, and HS-PSC had a greater proliferation rate compared to LS-PSC. The PPP-PSC had greater differentiation capacity and myotube diameter than PPN-PSC. In conjunction these results indicate divergent myogenic development among different fetal sizes within a litter and suggest that porcine plasma supplementation stimulates myogenic progenitor cell activity in an age specific manner.