Prac 2 - Notes PDF

🔬 Prac 2 - Notes Activity 1: Assessing pluripotency TASK A 1) What morphological characteristics are indicative of a pluripotent stem cell colony? Compact, well-defined colonies with smooth edges....

🔬 Prac 2 - Notes Activity 1: Assessing pluripotency TASK A 1) What morphological characteristics are indicative of a pluripotent stem cell colony? Compact, well-defined colonies with smooth edges. The cells should have a high nucleus-to-cytoplasm ratio, with prominent nucleoli and minimal cytoplasm. The colonies should be tightly packed, and individual cells should not be easily distinguishable within the colony. 2) What observations may indicate colonies undergoing differentiation? Changes in colony morphology such as irregular or fuzzy edges, a loss of compactness, or cells beginning to migrate out of the colony. The cells may start to spread out, with increased cytoplasm, and the nucleus-to-cytoplasm ratio may decrease. Prac 2 - Notes 1  Differentiation is also indicated by the appearance of distinct cell types or structures within the colony. 3) Do you note any differences between the different cell lines on the slide? The iPSCs with the Oct4-GFP reporter should exhibit green fluorescence, indicating pluripotency, while the control iPSCs without the reporter would not fluoresce. The iPS cell line with both the CAG-mCherry and Oct4-GFP reporters should show both red and green fluorescence if the cells are pluripotent and actively expressing Oct4. Differences in colony morphology could also be observed, depending on whether or not the cells are maintaining pluripotency or starting to differentiate. TASK B Theory questions (1) What cells will be stained with the DAPI? (2) What compartment of the cell will be visualised with DAPI? (3) What is the advantage of staining cells with DAPI? (1) DAPI will stain all the nuclei of the cells in the culture. It binds strongly to DNA, allowing visualization of the cell nuclei regardless of the cell type or state. (2) DAPI specifically stains the nucleus of the cell. It binds to the DNA within the nucleus, providing a clear visualization of nuclear material. (3) Advantages: Universal Nuclear Marker: It allows for the identification and counting of all cells within a sample by highlighting their nuclei. Overlay with Fluorescent Reporters: DAPI staining can be easily combined with other fluorescent markers (like GFP and mCherry) to correlate nuclear information with specific protein expression. Assessing Cell Health: DAPI can help identify apoptotic or dead cells by detecting changes in nuclear morphology. Prac 2 - Notes 2  Ease of Use: It is a simple and rapid staining method compatible with live-cell imaging protocols. What is the function of the Oct3/4 protein? Where is it normally localized in a cell? Function of Oct3/4: Pluripotency Maintenance: Oct3/4 (also known as POU5F1) is a transcription factor critical for maintaining the pluripotent state of stem cells. It regulates the expression of genes involved in self- renewal and inhibits differentiation. Embryonic Development: Essential for the formation of the inner cell mass in the blastocyst, ensuring the development of all three germ layers. Reprogramming Factor: One of the key factors used to reprogram somatic cells into induced pluripotent stem cells (iPSCs). Localization of Oct3/4: Nuclear Localization: Oct3/4 is predominantly localized in the nucleus of the cell, where it binds to DNA and regulates gene expression. Its presence in the nucleus is indicative of its active role in transcriptional regulation. Given the fluorescent reporters and markers used in the experiment today, what profile would you expect to observe in the following cell types in the culture? PLURIPOTENT CELLS Green Fluorescence (GFP): Due to the Oct4-GFP reporter, pluripotent cells will exhibit green fluorescence, indicating active expression of the Oct4 gene. Red Fluorescence (mCherry): In the cell line harboring both CAG- mCherry and Oct4-GFP reporters, pluripotent cells will also show constitutive red fluorescence, as the CAG promoter drives constant expression of mCherry regardless of the cell state. Blue Fluorescence (DAPI): All pluripotent cells will have blue- stained nuclei. DIFFERENTIATING CELLS Prac 2 - Notes 3  Reduced or No Green Fluorescence (GFP): As Oct4 expression decreases during differentiation, differentiating cells will show diminished or absent green fluorescence. Red Fluorescence (mCherry): Cells with the CAG-mCherry reporter will continue to display red fluorescence, as mCherry expression is constitutive. Blue Fluorescence (DAPI): Differentiating cells will still have blue- stained nuclei. DIFFERENTIATED CELLS No Green Fluorescence (GFP): Fully differentiated cells will not express Oct4, resulting in no green fluorescence. Red Fluorescence (mCherry): In cell lines with the CAG-mCherry reporter, differentiated cells will retain red fluorescence. Blue Fluorescence (DAPI): All differentiated cells will have blue- stained nuclei. Experimental questions Are all cells exhibiting Oct3/4 signal (green fluorescence)? Expected Observation: Not all cells should exhibit Oct3/4 (green fluorescence). Only pluripotent stem cells actively expressing Oct4 will show green fluorescence. Differentiating and differentiated cells will either show reduced or no green fluorescence, depending on the stage of differentiation. Interpretation: A mixed population with both green-fluorescent (pluripotent) and non-fluorescent (differentiated) cells indicates ongoing differentiation within the culture. Is the Oct3/4 reporter signal localized to the nucleus? Why or why not? Expected Observation: Yes, the Oct3/4 reporter signal (green fluorescence from GFP) should be localized to the nucleus. Reasoning: Since Oct3/4 is a nuclear transcription factor, the GFP reporter linked to the Oct4 promoter will also localize to the nucleus where Oct3/4 exerts its function in regulating gene expression. Prac 2 - Notes 4  If you had done an antibody stain for the Oct3/4 protein, would you expect to see co-localisation of DAPI and Oct3/4 in the cells? Yes, Co-localization Expected: An antibody stain for Oct3/4 would show fluorescence overlapping with DAPI-stained nuclei. This is because Oct3/4 resides in the nucleus, and DAPI also stains the nuclear DNA. Interpretation: Co-localization confirms the nuclear presence of Oct3/4, validating that the GFP reporter accurately reflects the localization of the endogenous Oct3/4 protein. Based on your observations, what percentage of your cells do you think are in a pluripotent state? Assessment Approach: By analyzing the proportion of cells exhibiting green fluorescence (and red fluorescence in dual-reporter lines) relative to the total number of DAPI-stained nuclei, one can estimate the percentage of pluripotent cells. Expected Range: In well-maintained iPSC cultures, a high percentage (typically >70-80%) of cells should be pluripotent. However, the exact percentage can vary based on culture conditions and the presence of differentiation signals. Example Estimation: If, for instance, 80 out of 100 DAPI-stained nuclei show green fluorescence, approximately 80% of the cells are in a pluripotent state. We have imaged the cells using 3 different fluorescent tags today – GFP, mCherry, and DAPI. Why is this set of fluorescent tags an appropriate choice? Multiplexing Capability: Using GFP, mCherry, and DAPI allows simultaneous visualization of multiple cellular components and states without significant spectral overlap. GFP (Green Fluorescence): Indicates pluripotency through the Oct4-GFP reporter. mCherry (Red Fluorescence): Provides a constitutive marker to identify all cells in the dual-reporter line, regardless of their pluripotent state. Prac 2 - Notes 5  DAPI (Blue Fluorescence): Stains all nuclei, enabling cell counting and localization of fluorescent signals within the cell. Activity 2: Differentiation of embryonic stem cells List 3 other ways that a state of pluripotency can be assessed: a) Embryoid Body Formation: This in vitro method allows ESCs or iPSCs to form three-dimensional aggregates that can differentiate into cell types from all three germ layers. b) Directed Differentiation Assays: Specific protocols can induce ESCs or iPSCs to differentiate into particular lineages (e.g., neural, cardiac), confirming their ability to generate various cell types. c) Gene Expression Analysis: Quantitative PCR or RNA sequencing can be used to confirm the expression of key pluripotency markers (e.g., Oct4, Sox2, Nanog) and the suppression of differentiation markers. Given the ability of undifferentiated cells to form these highly complex tumours when introduced in vivo, discuss what implications this may have when researchers are considering using ESC or iPSC-derived cells in a clinical context The ability of undifferentiated cells to form complex teratomas indicates a high potential for uncontrolled growth if these cells are not fully differentiated before transplantation. This poses a risk of tumorigenesis in clinical applications. Therefore, stringent protocols must be established to ensure complete differentiation and removal of any remaining undifferentiated cells before these cells are used in therapies. Prac 2 - Notes 6  Additionally, long-term monitoring would be crucial to ensure safety in patients. Activity 3: Organoids THEORY QUESTIONS How well do organoids recapitulate normal tissue architecture? Organoids are designed to mimic the 3D structure and function of their tissue of origin. They can closely replicate the cellular diversity, organization, and some physiological functions of the original tissue, but they may not fully capture all aspects of tissue architecture, such as the full extent of vascularization or interactions with immune cells. List three applications of organoids: a) Disease Modeling: Organoids can be used to model diseases, providing insights into disease mechanisms and potential treatment strategies. b) Drug Testing: Organoids are used to screen and evaluate the efficacy and toxicity of new drugs in a more tissue-relevant context compared to 2D cultures. c) Personalized Medicine: Patient-derived organoids can be used to tailor treatments based on individual responses, allowing for more personalized therapeutic approaches. What are three advantages of using organoids over 2D cell culture to study and treat diseases? Prac 2 - Notes 7  a) 3D Structure: Organoids provide a more physiologically relevant 3D environment that better mimics the organization of cells in tissues. b) Cellular Diversity: Organoids contain multiple cell types found in the original tissue, allowing for more complex and accurate studies. c) Functional Modeling: Organoids can replicate some of the functions of the tissue, making them more suitable for studying disease mechanisms and treatment responses. What are three limitations of using organoids? a) Lack of Vascularization: Organoids often lack blood vessels, limiting their growth and the ability to model the full complexity of organs. b) Reproducibility: There can be variability in organoid formation and function, making standardization and reproducibility challenging. c) Scale and Size: Organoids are typically small and may not fully replicate the larger-scale interactions and environments of full organs. What are some potential ethical challenges in the use of organoids in biomedicine? a) Consent for Use of Human Cells: Ethical considerations around obtaining and using human cells to generate organoids. b) Chimerism Concerns: The creation of human-animal chimeras using organoids for research purposes. c) Potential for Misuse: Concerns about the misuse of organoid technology, particularly if they are used to create more complex or functional human tissues. d) Intellectual Property: Issues related to the ownership and patenting of organoid technologies. EXPERIMENTAL QUESTIONS As you observe these organoids, can you observe any intestinal-type structures in either the cultures or the sections? Yes, intestinal organoids often display crypt-like structures and villi-like protrusions that resemble the architecture of the small intestine. Prac 2 - Notes 8  What cell types (generally) might you expect to exist in these particular organoids? You might expect to see a variety of intestinal cell types, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, which are all involved in the function and maintenance of the intestinal epithelium. Prac 2 - Notes 9