1 |
Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment EpitheliumTanaka, Elly M., Zhu, Yu, Carido, Madalena, Meinhardt, Andrea, Kurth, Thomas, Karl, Mike O., Ader, Marius 18 January 2016 (has links) (PDF)
A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.
|
2 |
Utvärdering och implementering av antikroppen anti-NKX3.1 för diagnostik av metastaserande prostata-adenocarcinom / Evaluation and implementation of anti-NKX3.1 antibody for diagnosis of metastatic prostate adenocarcinomaBorglund, Kajsa January 2021 (has links)
Prostata-adenocarcinom är den vanligaste cancerformen hos män. Anti-PSA och anti-P501S är frekvent använda antikroppar för diagnostisering av prostata-adenocarcinom i vävnadsbiopsier, men kan ha svag eller negativ infärgning av antigenen. Antikroppen anti-NKX3.1 har visat sig ha en högre sensitivitet och starkare infärgning än anti-PSA och anti-P501S vid prostata-adenocarcinom. Det rekommenderas att använda anti-NKX3.1 tillsammans med anti-PSA och/eller anti-P501S för att diagnostisera metastaserande prostata-adenocarcinom. Studiens syfte var att utvärdera och implementera antikroppen anti-NKX3.1 för diagnostisering av metastaserande prostata-adenocarcinom. Positiva vävnadskontroller (prostatavävnad och testisvävnad) och negativ vävnadskontroll (appendix) färgades med anti-NKX3.1 i Roche Ventana Benchmark ULTRA med immunohistokemisk färgning. Förbehandlingarna CC1 mild, CC1 standard och Proteas 1 samt visualiseringskiten OptiView samt UltraView jämfördes. Spädningen optimerades från 1:50-1:200. Vävnadssnitten graderades sedan från 1 (ingen infärgning)-6 (starkast infärgning). Visualiseringskitet OptiView samt förbehandlingen CC1 standard gav den starkaste infärgningen med anti-NKX3.1 och valdes som det optimala visualiseringskitet respektive förbehandlingen. Med tanke på materialkostnad och smidighet valdes spädningen 1:100 som den optimala spädningen. / Prostate adenocarcinoma is the most common form of cancer in men. Anti-PSA and anti-P501S are frequently used antibodies for diagnosing of prostate adenocarcinoma in tissue biopsies but may show weak or negative staining of the antigens. Antibody anti-NKX3.1 has been shown to have a higher sensitivity and stronger staining then anti-PSA and anti-P501S in prostate adenocarcinoma. It is recommended to use anti-NKX3.1 along with anti-PSA and/or anti-P501S to diagnose metastases of prostate adenocarcinoma. The purpose of the study was to evaluate and implement the antibody anti-NKX3.1 for diagnosis of metastatic prostate adenocarcinoma. Positive tissue controls (prostate and testis) and negative tissue control (appendix) were stained with anti-NKX3.1 immunohistochemical staining in Roche Ventana Benchmark ULTRA machine. The pre-treatments CC1 mild, CC1 standard and Protease 1 and the visualization kits OptiView and UltraView were compared. The dilution was optimized from 1:50-1:200. Immunohistochemical staining of the tissue was graded from 1 (no staining) - 6 (strongest staining). Visualization kit OptiView and pre-treatment CC1 standard gave the strongest staining with anti-NKX3.1 and were chosen as the optimal visualization kit and pre-treatment. Considering the material cost and flexibility, the 1:100 dilution was chosen as the optimal dilution.
|
3 |
Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment EpitheliumTanaka, Elly M., Zhu, Yu, Carido, Madalena, Meinhardt, Andrea, Kurth, Thomas, Karl, Mike O., Ader, Marius 18 January 2016 (has links)
A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.
|
4 |
Localisation of kallikreins in the prostate and association with prostate cancer progressionBui, Loan Thuy January 2006 (has links)
At present, prostate cancer is a significant public health issue throughout the world and is the second leading cause of cancer deaths in older men. The prostate specific antigen or PSA (which is encoded by the kallikrein 3/KLK3 gene) test is the current most valuable tool for the diagnosis and management of prostate cancer. However, it is insufficiently sensitive and specific for early diagnosis, for staging of prostate cancer or for discriminating between benign prostatic hyperplasia (BPH) and prostate cancer. Recent research has revealed another potential tumour marker, glandular kallikrein 2 (KLK2 gene/hK2 protein), which may be used alone or in conjunction with PSA to overcome some of the limitations of the PSA test. Twelve new kallikrein gene family members have been recently identified and, like hK2 and PSA, many of these genes have been suggested to be involved in carcinogenesis. In this study, the cellular localisation and level of expression of several of these newer kallikreins (KLK4, KLK5, KLK7, KLK8 and KLK11) was examined in prostate tissue, to provide an understanding of the association of their expression with prostatic diseases and their potential as additional biomarkers. Like PSA and hK2, the present observation indicated that each of these proteins, hK4, hK5, hK7, hK8 and hK11, was detected within the cytoplasm of the secretory cells of the prostate glands. For the first time, all of these newly-identified proteins were shown to be expressed in prostatic intraepithelial neoplasia (PIN) lesions, in comparison to normal glands and cancer lesions. In addition to cytoplasmic secretory cell expression, the localisation of hK4 to the basal cells and nuclei in prostatic lesions was intriguing. The intensity of hK4 staining in prostate tissue was strongest in comparison to the other newly-identified kallikrein proteins (hK5, hK7, hK8 and hK11). Therefore, KLK4/hK4 expression was characterised further to define this cellular localisation and examined in non-prostatic tissue and also in a larger number of prostate tissues in an attempt to determine its potential value as a biomarker for prostate disease. Three hK4 antipeptide polyclonal antibodies, derived against N-terminal, mid-region and C-terminal hK4 amino acid sequences, were used. The hK4 N-terminal antipeptide antibody was used to demonstrate the cellular localisation of hK4 in kidney, salivary glands, liver, testis, colon carcinoma, heart, endometrium and ovarian cancer, for the first time. The presence of hK4 in these non-prostate tissues was consistent with the previous reports using RT-PCR. The dual cytoplasmic and nuclear localisation of hK4 observed in the prostate above was also seen in these tissues. Although hK4 was found widely expressed in many human tissue types, indicating that it is not prostate specific in its expression, the highest expression level of hK4 was seen in the prostate. Therefore, detailed expression patterns and levels of KLK4 mRNA and hK4 protein in the normal prostate and prostatic diseases and histopathological lesions were investigated and reported for the first time in this study. Twelve benign prostatic hyperplasia (BPH), 19 adenocarcinoma (Gleason grade 2-5) and 34 bone metastases from prostate cancer were analysed. Using in situ hybridisation, the expression of KLK4 mRNA was detected in the cytoplasm of the secretory cells of both normal and diseased prostate tissue. KLK4 mRNA was also noted in both secretory and basal cells of PIN lesions, but the basal cells of normal glands were negative. Using the hK4 N-terminal and mid-region antipeptide antibodies, hK4 was predominantly localised in the cytoplasm of the secretory cells. The intensity of hK4 staining appeared lowest in normal and BPH, and increased in PIN lesions, high Gleason grade prostate cancer and bone metastases indicating the potential of hK4 as a histopathological marker for prostatic neoplasias. Further studies are required with a larger cohort to determine its utility as a clinical biomarker. Small foci of atypical cells, which were found within normal glands, were also intensely stained. Surprisingly, hK4 protein was found in the nucleus of the secretory cells (but not the basal cells) of high grade PIN and Gleason grade 3 prostate cancer. The detection of KLK4 mRNA and hK4 protein in PIN lesions and small foci of atypical cells suggests that up-regulation of KLK4 expression occurs early in the pathology of prostate carcinogenesis. The finding of basal cell expression is not typical for the kallikreins and it is not clear what role hK4 would play in this cell type. With the use of the hK4 C-terminal antipeptide antibody, the staining was mainly localised in the nuclei of the secretory cells of the prostate glands. Although the nuclear localisation was readily noted in more than 90% of epithelial cells of the prostate gland with the C-terminal antibody, no difference in staining intensity was observed among the histopathological lesions of the prostate. The prominent nuclear localisation with the C-terminal antipeptide antibody was also shown to be distributed throughout the nucleus by using confocal microscopy. Further, by using gold-labelled particles for electron microscopy, the intracellular localisation of these hK4 antipeptide antibodies was reported here for the first time. Similar to the immunohistochemical results, the cytoplasm was the major site of localisation with the N-terminal and mid-region antipeptide antibodies. To further characterise the involvement of KLK4/hK4 in human prostate cancer progression, the transgenic adenocarcinoma mouse prostate (TRAMP) model was used in this study. In this study, mouse KLK4 (also known as enamel matrix serine protease -1, EMSP-1) was shown to be expressed in the TRAMP prostate for the first time. Previous studies had only shown the developing tooth as a site of expression for EMSP-1. The level of EMSP-1 mRNA expression was increased in PIN and prostate cancer lesions of the TRAMP model, while negative or low levels of EMSP-1 mRNA were seen in normal glands or in control mouse prostate tissue. The normal mouse prostate did not stain with any the three hK4 antipeptide antibodies. hK4 N-terminal and mid-region antipeptide antibodies showed positive staining in the cytoplasm of the epithelial cells of PIN and cancer lesions of the mouse prostate. The C-terminal antipeptide antibody showed distinctively nuclear staining and was predominantly localised in the nuclei of the glandular cells of PIN and cancer lesions of the mouse prostate. The expression patterns of both the mRNA and protein level for mouse KLK4 strongly supported the observations of KLK4/hK4 expression in the human prostate and further support the utility of the TRAMP model. Overall, the findings in this thesis indicate a clear association of KLK4/hK4 expression with prostate cancer progression. In addition, several intriguing findings were made in terms of cellular localisation (basal as well as secretory cells; nuclear and cytoplasmic) and high expression in atypical glandular cells and PIN, perhaps indicating an early involvement in prostate disease progression and, additionally, utility as basal cell and PIN histological markers. These findings provide the basis for future studies to confirm the utility of hK4 as a biomarker for prostate cancer progression and identify functional roles in the different cellular compartments.
|
Page generated in 0.0752 seconds