W2 prac: Characterisation and differentiation of pluripotent and adult stem cells
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Questions and Answers

What is the primary function of the Oct3/4 protein in stem cells?

  • Inducing apoptosis
  • Maintaining pluripotency (correct)
  • Facilitating cellular respiration
  • Regulating cell division rates
  • In what cellular location is Oct3/4 primarily found?

  • Nucleus (correct)
  • Cytoplasm
  • Mitochondria
  • Cell membrane
  • Which advantage does DAPI provide in cell analysis?

  • Determines cell metabolism
  • Identifies apoptotic cells (correct)
  • Directly measures RNA levels
  • Visualizes protein structures
  • Which of the following statements about Oct3/4 is accurate?

    <p>It is essential for inner cell mass formation.</p> Signup and view all the answers

    What kind of staining method is DAPI considered to be?

    <p>Fluorescent</p> Signup and view all the answers

    What type of fluorescence would pluripotent cells exhibit due to the Oct4-GFP reporter?

    <p>Green fluorescence</p> Signup and view all the answers

    What role does Oct3/4 play in reprogramming somatic cells?

    <p>It serves as a reprogramming factor.</p> Signup and view all the answers

    Which fluorescent markers can be combined with DAPI staining?

    <p>GFP and mCherry</p> Signup and view all the answers

    What is the primary role of Oct3/4 in cells?

    <p>Regulating gene expression</p> Signup and view all the answers

    Which fluorescent tag is used to indicate pluripotency in the cell?

    <p>GFP</p> Signup and view all the answers

    What can be inferred if DAPI-stained nuclei co-localize with Oct3/4 stained cells?

    <p>Oct3/4 is localized within the nuclei</p> Signup and view all the answers

    If 80 out of 100 DAPI-stained nuclei show green fluorescence, what is the estimated percentage of pluripotent cells?

    <p>80%</p> Signup and view all the answers

    What is a key advantage of using GFP, mCherry, and DAPI together in imaging?

    <p>They allow multiplexing and simultaneous visualization</p> Signup and view all the answers

    Under well-maintained iPSC cultures, what is the expected percentage of pluripotent cells?

    <p>70-80%</p> Signup and view all the answers

    Why is the overlapping fluorescence of Oct3/4 and DAPI considered significant?

    <p>It validates GFP reporter functionality</p> Signup and view all the answers

    What effect do differentiation signals have on pluripotency percentages?

    <p>They significantly decrease pluripotency</p> Signup and view all the answers

    What is the main cause of the constant red fluorescence observed in pluripotent cells with the CAG-mCherry reporter?

    <p>The CAG promoter drives constant expression of mCherry.</p> Signup and view all the answers

    What fluorescence pattern is expected in fully differentiated cells?

    <p>High blue and no green fluorescence.</p> Signup and view all the answers

    Which statement best describes the observation of Oct3/4 signal in a mixed population of cells?

    <p>Pluripotent stem cells show green fluorescence while differentiated cells do not.</p> Signup and view all the answers

    Why is the Oct3/4 reporter signal expected to localize to the nucleus?

    <p>Oct4 is a transcription factor that operates within the nucleus.</p> Signup and view all the answers

    What change occurs in green fluorescence (GFP) as cells begin to differentiate?

    <p>It decreases or becomes absent in differentiating cells.</p> Signup and view all the answers

    What type of fluorescence is detected in all pluripotent cells, regardless of Oct4 expression?

    <p>Constant red fluorescence from mCherry.</p> Signup and view all the answers

    Which of the following best describes the fluorescence characteristics of differentiating cells?

    <p>Consistent red fluorescence and reduced or absent green fluorescence.</p> Signup and view all the answers

    How does the presence of blue fluorescence (DAPI) differ between pluripotent and differentiated cells?

    <p>It is consistently present in both cell types.</p> Signup and view all the answers

    Which morphological characteristic is NOT indicative of a pluripotent stem cell colony?

    <p>Individual cells easily distinguishable</p> Signup and view all the answers

    What change in colony morphology suggests cells are undergoing differentiation?

    <p>Fuzzy edges and loss of compactness</p> Signup and view all the answers

    Which combination of reporter genes confirms pluripotency in iPSCs?

    <p>CAG-mCherry and Oct4-GFP together</p> Signup and view all the answers

    What is one of the main advantages of using DAPI for staining?

    <p>It allows visualization of nuclei in all cell types irrespective of their state</p> Signup and view all the answers

    In which cell compartment does DAPI primarily localize during visualization?

    <p>Nucleus</p> Signup and view all the answers

    What indicates a cell has started to differentiate in terms of its physical appearance?

    <p>Spreading out of cells with increased cytoplasm</p> Signup and view all the answers

    Which observation might signify differences in pluripotency among cell lines under the microscope?

    <p>Variations in colony morphology</p> Signup and view all the answers

    What effect does differentiation have on the nucleus-to-cytoplasm ratio?

    <p>It decreases during differentiation</p> Signup and view all the answers

    What is the purpose of mCherry in the dual-reporter line?

    <p>To consistently mark all cells regardless of their pluripotent state</p> Signup and view all the answers

    Which method directly assesses the ability of embryonic stem cells to generate diverse cell types?

    <p>Directed Differentiation Assays</p> Signup and view all the answers

    What risk is associated with undifferentiated cells in a clinical context?

    <p>Uncontrolled growth and tumorigenesis</p> Signup and view all the answers

    Which of the following is not a method for assessing pluripotency?

    <p>Direct Fluorescence Microscopy</p> Signup and view all the answers

    What is a key factor that researchers must ensure before using ESC or iPSC-derived cells in therapies?

    <p>Complete differentiation and removal of undifferentiated cells</p> Signup and view all the answers

    Which markers are commonly analyzed to confirm pluripotency?

    <p>Oct4, Sox2, and Nanog</p> Signup and view all the answers

    Why is long-term monitoring important in clinical applications of stem cells?

    <p>To monitor for potential tumorigenesis</p> Signup and view all the answers

    How do organoids relate to normal tissue architecture?

    <p>Organoids can partially replicate normal tissue architecture</p> Signup and view all the answers

    Which of the following describes a key advantage of using organoids in research?

    <p>They provide a physiologically relevant 3D environment.</p> Signup and view all the answers

    What is a limitation of using organoids compared to whole organ systems?

    <p>Organoids often lack blood vessels, limiting their growth.</p> Signup and view all the answers

    Which application of organoids does not involve drug testing?

    <p>Studying immune responses.</p> Signup and view all the answers

    In terms of cellular diversity, how do organoids compare to traditional cell cultures?

    <p>Organoids provide a more complex mix of multiple cell types.</p> Signup and view all the answers

    What is not considered a major advantage of organoids over 2D cell cultures?

    <p>Lower costs of production.</p> Signup and view all the answers

    What challenges about organoids can affect reproducibility in studies?

    <p>Variability in organoid formation and function.</p> Signup and view all the answers

    Which statement accurately reflects a potential ethical challenge associated with organoid research?

    <p>Patient-derived organoids may raise questions about consent and ownership.</p> Signup and view all the answers

    Which functional aspect is not fully replicated by organoids?

    <p>Complete immune cell interactions.</p> Signup and view all the answers

    Study Notes

    Pluripotent Stem Cell Colonies

    • Compact, well-defined colonies with smooth edges are indicative of pluripotent stem cells
    • Cells have a high nucleus-to-cytoplasm ratio with prominent nucleoli and minimal cytoplasm
    • Colonies are tightly packed, individual cells are not easily distinguishable

    Differentiating Colonies

    • Changes in colony morphology, such as irregular or fuzzy edges, loss of compactness, or cells migrating out of the colony, may indicate differentiation
    • Increased cytoplasm and a decreased nucleus-to-cytoplasm ratio might also be observed

    Cell Line Differences

    • iPSCs with the Oct4-GFP reporter exhibit green fluorescence, indicating pluripotency. Control iPSCs without the reporter will not fluoresce.
    • iPS cell lines with both CAG-mCherry and Oct4-GFP reporters will show both red and green fluorescence if pluripotent and actively expressing Oct4
    • Differences in colony morphology can also be observed based on whether the cells are maintaining pluripotency or differentiating

    DAPI Staining

    • DAPI stains all cell nuclei in a culture
    • Binds strongly to DNA allowing visualization of cell nuclei regardless of cell type or state
    • Specifically stains the nucleus, providing clear visualisation of nuclear material

    Advantages of DAPI Staining

    • Universal nuclear marker for identifying and counting all cells in a sample
    • Can be combined with other fluorescent markers (such as GFP and mCherry) for correlated nuclear and protein expression analysis
    • Useful for assessing cell health, identifying apoptotic or dead cells by detecting nuclear morphology changes

    Oct3/4 Protein Function and Localization

    • Oct3/4 (also known as POU5F1) is a transcription factor responsible for maintaining pluripotency and regulating self-renewal
    • Essential for embryonic development, particularly for forming the inner cell mass of the blastocyst and all three germ layers
    • A key factor for reprogramming somatic cells into induced pluripotent stem cells (iPSCs)
    • Localized to the nucleus

    Pluripotent Cell Profiles

    • Green Fluorescence (GFP): Indicates pluripotency through the expression of Oct4
    • Red Fluorescence (mCherry): A constitutive marker for all cells in the specified cell line regardless of pluripotency status in the cell
    • Blue Fluorescence (DAPI): All pluripotent cells have blue-stained nuclei

    Differentiating Cell Profiles

    • Reduced or No Green Fluorescence (GFP): Indicates decreasing Oct4 expression during differentiation.
    • Red Fluorescence (mCherry): Differentiating cells will retain red fluorescence, as mCherry expression is constitutive
    • Blue Fluorescence (DAPI): Differentiating (and differentiated) cells will have blue-stained nuclei

    Co-localization of DAPI and Oct3/4

    • Antibody staining for Oct3/4 should show fluorescent overlap with DAPI-stained nuclei, confirming nuclear presence of Oct3/4
    • Validates the accuracy of GFP reporter in reflecting Oct3/4 localization

    Percentage of Pluripotent Cells

    • By analyzing the proportion of green fluorescent cells relative to total DAPI-stained nuclei, the percentage of pluripotent cells can be estimated
    • Typically, over 70-80% of well-maintained iPSC cultures should be pluripotent, but exact percentages can vary based on culture conditions and differentiation signals

    Organoid Advantages over 2D Cell Cultures

    • 3D structure that better mimics cell localization in tissues
    • More cell diversity, enabling more accurate studies
    • Functional modelling to better replicate tissue functions

    Organoid Limitations

    • Lack of vascularization, potentially impeding the modelling of full organ complexity
    • Variability in reproducibility, making standardization challenging
    • Scale/size limitations, unable to completely replicate organism-level interactions or environments

    Ethical Challenges in Organoid Use

    • Consent for use of human cells
    • Potential chimerism through combining human and animal cells
    • Misuse concerns about creating more complex or functional human tissues
    • Issues regarding intellectual property

    Intestinal Organoid Structures

    • Intestinal organoids can have crypt-like structures and villi-like protrusions which resemble architecture of the small intestine
    • Cell types, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, will be present

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    Explore the crucial roles and functions of the Oct3/4 protein in stem cells through this quiz. Answer questions about its location, advantages of DAPI in analysis, and its implications in pluripotency and reprogramming. Test your knowledge on fluorescent markers and their application in stem cell research.

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