Lecture 4: Manipulating the Genome

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Questions and Answers

What is the purpose of using Tamoxifen in the experimental setup?

To enhance the fluorescence of stem cells

To label differentiated cells in the villi

To induce differentiation of progenitor cells

To activate CRE-ER2 for recombination (correct)

What does the expression of LACZ indicate in the experimental model?

The stem cells have differentiated into intestinal cell types

The CRE enzyme has successfully recombined the LOXP sites (correct)

The stop codon is still intact and non-functional

The stem cells are not responding to Tamoxifen

Which cell types do the progeny of LGR5-positive stem cells differentiate into?

Only goblet cells and enterocytes

Exclusively stem cells without differentiation

Only enterocytes and stem cells

Goblet cells, enteroendocrine cells, and absorptive cells (correct)

What is the significance of gene expression in crypts within this model?

It reflects the ability of stem cells to continually regenerate Signup and view all the answers

What technique is primarily utilized to identify and isolate LGR5-positive stem cells?

Fluorescence-activated cell sorting (FACS) Signup and view all the answers

What role does the basement membrane play in the stem cell niche?

It separates the epidermis from the dermis and acts as a barrier. Signup and view all the answers

Which growth factor is specifically noted for maintaining the undifferentiated state of epidermal stem cells?

Wnt Signup and view all the answers

What is a method used to test whether a candidate cell population can function as stem cells?

Depleting the niche of existing stem cells and observing integration. Signup and view all the answers

What is the significance of the differentiation gradient in stem cell behavior?

It shows the progression of stem cells to more specialized forms. Signup and view all the answers

What aspect of stem cell niches does the assay involving bone marrow irradiation examine?

The integration and functionality of donor stem cells within the niche. Signup and view all the answers

Which of the following statements correctly describes the function of the stem cell niche?

It is crucial for continuous skin renewal and repair processes. Signup and view all the answers

What can be concluded if transplanted cells survive and produce differentiated progeny in a stem cell niche?

The transplanted cells may be stem cells and the niche is functional. Signup and view all the answers

What role do somatic hub cells play in the testis stem cell niche?

They maintain stem cell identity and regulate their division. Signup and view all the answers

Which of the following statements accurately describes the location of germline stem cells in Drosophila?

They reside at the tip of the testis surrounded by niche cells. Signup and view all the answers

What is the primary function of DPP in the stem cell niche?

To ensure stem cells maintain their undifferentiated state and self-renewal capability. Signup and view all the answers

How do niche cells contribute to the environment of germline stem cells?

By providing necessary signals and a suitable microenvironment. Signup and view all the answers

Which factor describes the relationship between germline stem cells and somatic stem cell progenitors?

Somatic progenitors provide niche signals to support germline stem cells. Signup and view all the answers

Why is the testis considered non-essential for the viability of Drosophila?

Genetic manipulation of stem or niche cells does not impact fly lifespan. Signup and view all the answers

What structural adaptation allows sperm tails to be particularly long in Drosophila?

The larger size of the testes accommodates longer sperm tails. Signup and view all the answers

How does the interaction between stem cells and somatic hub cells illustrate cell signaling?

Hub cells secrete signals that maintain stem cell identity and growth rate. Signup and view all the answers

What specific area does the testis occupy in Drosophila?

A significant part of the abdomen. Signup and view all the answers

What is the primary role of intestinal stem cells in the intestinal epithelium?

To generate differentiated cell types including Paneth cells and enterocytes Signup and view all the answers

Which genetic strategy can be used to identify intestinal stem cells?

Linear tracing with genetic markers Signup and view all the answers

What is the significance of the LGR5 gene in intestinal stem cell research?

It serves as a marker for intestinal stem cells Signup and view all the answers

What is the function of the CRE recombinase gene in lineage tracing?

To induce recombination at specific DNA sequences Signup and view all the answers

Which component is essential for viewing LGR5 marked cells in a fluorescence microscope?

Green Fluorescent Protein (GFP) Signup and view all the answers

What event is triggered by the interaction of Sertoli cells in the testes?

A cascade of intracellular events crucial for maintaining stem cells Signup and view all the answers

What does the presence of LACZ in the reporter construct indicate during lineage tracing?

Expression of β-galactosidase in response to CRE activity Signup and view all the answers

What notable change is observed in the mutant mouse with disrupted Wnt signaling?

Significant gaps indicating loss of germ cells Signup and view all the answers

When are intestinal stem cells typically isolated and cultured?

In steady-state conditions without injury Signup and view all the answers

Which cell types are found in the intestinal epithelium?

Absorptive enterocytes, Goblet cells, Paneth cells, and enteroendocrine cells Signup and view all the answers

Which factor is not required for lineage tracing using the LGR5 marker?

A distinct lineage marker for each differentiated cell type Signup and view all the answers

Where are intestinal stem cells located?

Near the base of the crypts Signup and view all the answers

Which cell type is produced by intestinal stem cells that aids in the immune response?

Paneth cells Signup and view all the answers

What is the primary function of enterocytes in the intestine?

Absorption of nutrients and maintenance of intestinal barrier Signup and view all the answers

What is a critical characteristic of cultured intestinal stem cells in organoid cultures?

The ability to form all differentiated cell types of the tissue Signup and view all the answers

What feature distinguishes colonic epithelium compared to small intestinal epithelium?

Lack of villi Signup and view all the answers

What do Sertoli cells predominantly surround in the testis?

Differentiating sperm and germ cells Signup and view all the answers

What happens to the tubules in the testis when Wnt signaling is functioning properly?

They show normal differentiation of sperm cells Signup and view all the answers

Which of these cell types are primarily found in the edges of tubules when Wnt signaling is disrupted?

Cittoli cells (Sertoli cells) Signup and view all the answers

What is one major role of Paneth cells in the intestinal niche?

Secretion of antimicrobial molecules Signup and view all the answers

Study Notes

The Stem Cell Niche

Refers to the microenvironment where stem cells reside and receive signals that determine their fate.
Crucial because the niche can dramatically affect stem cell behavior, including how they grow, differentiate, and if they form normal tissue or tumors.
Embryonic stem cell differentiation is context-dependent, with different outcomes depending on the environment (e.g., blastocyst vs. under the skin).
Niche defects can contribute to diseases.

Model Systems for Studying the Niche

Drosophila germline: A useful model system due to the large size of the fly's testis, which contains spermatogonia (stem cells) at the tip, visible along the length of the testis.
Mammalian stem cell niches: Examples include testis, blood, and skin.

Niche Hypothesis

Stem cells reside within fixed compartments/niches that promote their maintenance.
Niches provide signals to stem cells through secreted and cell surface molecules, regulating their proliferation and fate.

Key Components of the HSC Niche (Hematopoietic Stem Cell)

Location: Bone marrow.
Endothelial cells: Line blood vessels, provide signals, and structure for the HSC niche.
Mesenchymal stem cells (MSCs): Multipotent stromal cells that support HSC maintenance and differentiation by secreting factors.
Neurons: Contribute to HSC regulation through signaling.
Extracellular matrix (ECM): Provides a scaffold for cells in the niche and influences stem cell behaviour through mechanical and biochemical signals.

Key Components of the Skin Stem Cell Niche

Location: Basal layer of the skin's epithelium.
Epidermal Stem Cells: Responsible for continuous skin renewal.
Key Components: Physical factors (substrate elasticity), secreted factors, cell-cell contact, and ECM.

Key Components of the Intestinal Stem Cell Niche

Location: Base of the crypts in the intestinal epithelium.
Stem Cells: Responsible for renewing the entire epithelial layer.
Differentiated Cell Types: Absorptive enterocytes, goblet cells, Paneth cells, and enteroendocrine cells.
Key Signals: WNT, R-SPONDIN, NOOGIN, BMP, EGF, and NRG1.
These stem cells are nested between specialised cells called Paneth cells.

Defining Stem Cell Niches via Experiments

Replacement Assay: Depleting the niche of stem cells and then introducing candidate cells to assess whether they integrate, survive, and differentiate.
Drosophila Testis: Studying how signaling molecules like DPP influence the number and location of germline stem cells in the testis.

Niche Components and Cancer

Tumor microenvironment: Cancer cells interact with surrounding tissue, fostering their survival, proliferation, and spread to other organs (metastasis).
Microenvironment properties (e.g., signaling) can drive cancer progression and influence the spread of cancer cells through different organs, such as in colitis.

Experimental Systems for Studying Stem Cells

Organoid cultures: 3D structures that resemble, in miniature, the organ of interest. They allow researchers to evaluate the function of stem cells in producing different cell types characteristic of the organ.
Transplantation assays: Transplanting stem cells into a model of the organ disease (like colitis) to observe function and regeneration potential.

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Description

Test your knowledge on stem cell biology, focusing on key aspects such as LGR5-positive stem cells, gene expression in crypts, and the role of the basement membrane. This quiz covers experimental methods and the significance of various factors in maintaining stem cell niches and differentiation. Dive deep into the complexities of stem cell behavior in this informative quiz.