DEV3032 L5 Lecture 5 PDF

🥽 Lecture 5: The Stem Cell Niche Lecture 5: The Stem Cell Niche 1  Define the stem cell niche Stem cell niche refers to the microenvironment where the stem cells reside and receive stimuli to determine their fate...

🥽 Lecture 5: The Stem Cell Niche Lecture 5: The Stem Cell Niche 1  Define the stem cell niche Stem cell niche refers to the microenvironment where the stem cells reside and receive stimuli to determine their fate It’s important because the niche can really have a very dramatic effect on how stem cells behave Understanding stem cell niche assist us with controling how stem cells grow and understanding how to transplant stem cells The niche can determine of a stem cell produces normal tissue or a tumour Embryonic stem cell differentiation is highly context dependent → different Outcomes Based on Environment: Injecting mouse embryonic stem cells into a blastocyst results in normal mouse development with patches of injected cell-origin tissues. Injecting the same cells under the skin of a host mouse leads to teratocarcinoma formation, a complex tumor composed of various abnormal cell types. Contextual Influence: The environment plays a crucial role: In a developmental context (blastocyst), injected cells integrate normally and function normally throughout the animal's life. In a non-developmental context (under the skin), the same cells form tumors, highlighting the importance of environment in determining cell behavior. Lecture 5: The Stem Cell Niche 2  Schofield - Niche Hypothesis Stem cells reside in fixed compartments or niches that are conductive to the maintenance of stem cell properties The niche is a compartment that provides signals to stem cells such as secreted and cell surface molecules to control the proliferation and fate of stem cells. Describe 2 key examples of stem cell niches Hematopoietic Stem Cell (HSC) Niche in the Bone Marrow: Lecture 5: The Stem Cell Niche 3  Location: This niche is located in the bone marrow. Key Components: Hematopoietic Stem Cells (HSCs) (shown in green): These are the stem cells responsible for generating all the different types of blood cells. Endothelial Cells: These cells line the blood vessels in the bone marrow and are located close to HSCs. They play a crucial role in maintaining the niche environment by providing signals and a structural framework. Mesenchymal Stem Cells (MSCs): These multipotent stromal cells contribute to the HSC niche by secreting factors that support HSC maintenance and differentiation. Neurons: Nerve cells are also present in the niche, contributing to the regulation of HSCs through various signals. Extracellular Matrix (ECM): The ECM, including components like fibronectin, provides a scaffold for the cells in the niche and influences cell behavior through mechanical and biochemical signals. Lecture 5: The Stem Cell Niche 4  Secreted Factors: These are various signaling molecules that are essential for the regulation of HSC activity within the niche. Function: This niche supports the maintenance, self-renewal, and differentiation of HSCs, ensuring a continuous supply of blood cells throughout life. Skin Stem Cell Niche in the Epidermis: Location: This niche is located in the basal layer of the skin’s epithelium, near the basement membrane. Key Components: Epidermal Stem Cells (shown in green): These stem cells reside in the basal layer and are responsible for the continuous renewal of the skin. Lecture 5: The Stem Cell Niche 5  Basement Membrane: A specialized structure that separates the epidermis from the underlying dermis, providing a physical and biochemical barrier. Differentiation Gradient: As stem cells divide and differentiate, they move upward through the epidermal layers, becoming more specialized before eventually shedding from the skin’s surface. Key Growth Factors: Wnt: A critical signaling molecule involved in maintaining the undifferentiated state of epidermal stem cells. EGF (Epidermal Growth Factor): Another important growth factor that promotes cell proliferation and survival, playing a key role in skin regeneration and repair. Function: This niche is essential for the continuous renewal of the skin and for repair processes, such as healing wounds or regenerating skin after damage. Describe experimental systems where the properties of stem cell niches can be examined Assay to Determine Stem Cell Properties: The Stem Cell Microenvironment can be perturbed to assay function → An assay that involves depleting the niche of existing stem cells (e.g., through irradiation) and then introducing a candidate cell population into the niche. Lecture 5: The Stem Cell Niche 6  The behavior of the transplanted cells—whether they integrate into the niche, survive, and produce differentiated progeny—provides evidence of whether the candidate cells are stem cells and whether the location is a functional stem cell niche. Example of Bone Marrow Irradiation: Irradiating bone marrow to deplete hematopoietic stem cells and then transplanting donor stem cells. This method is used to confirm both the identity of the stem cells and the functionality of the bone marrow niche. This example also highlights how different microenvironments can influence stem cell behavior, including survival, differentiation, and potential tumor formation. — A variety of components contribute to stem cell niches Cells/cellular components, secreted factors, immune cells, extracellular matrix, physical forces, hypoxia. metabolism Lecture 5: The Stem Cell Niche 7  Niche components can drive pathological processes such as cancer! A major example of a pathological niche is the tumor microenvironment, where the surrounding tissue environment supports and sustains cancer cells In tumors, there is crosstalk between cancer cells (including cancer stem cells) and the surrounding cells in the microenvironment → drives tumour growth by not only supporting the survival and proliferation of cancer cells but also aids in the spread of cancer (metastasis). Intrinsic properties of the cancer cell together with the organ microenvironment can determine the extent of metastatic spread. Therapeutic implication → rather than targeting the tumour themselves - disrupting the supportive signals within the niche, you could weaken the tumor or prevent it from spreading Lecture 5: The Stem Cell Niche 8  Explain the key features of the Drosophila testis and how this system has been used to identify key niche signals. Stem cells reside at the tip of the testes where they are surrounded by niche cells Scale & anatomy of the testis Drosophila (fruit fly) testis is remarkably large relative to the fly's body, occupying much of the abdomen Sperm tails are particularly long, extending along the length of the testis Stem cell location Stem cells reside at the tip of the testis → as cells move along the testis, they differentiate, ultimately forming sperm. Research significance The testis is non-essential for the fly - genetic studies can manipulate stem cells or niche cells without affecting fly viability Lecture 5: The Stem Cell Niche 9  Germline stem cells (S) → somatic hub cells (*) and somatic stem cell progenitors (P) provide niche signals to the germline stem cells Diagram Stem cells (purple) are actively dividing, as shown by the dividing cell highlighted in the image Somatic hub cells (green) - stem cells are in contact these cells → hub cells maintain stem cell identity and regulating their division through signaling interactions Niche Cells (yellow) - Surrounding the stem cells are yellow cells known as niche or supporting cells → provide the necessary environment for stem cell maintenance and differentiation TGFβ family member (Dpp) is expressed in hub cells in the niche DPP is secreted from the hub and is received by receptors on the nearby stem cells Role of DPP: DPP is crucial for regulating stem cell numbers. It maintains the stem cell population by signaling to the stem cells, ensuring they remain undifferentiated and capable of self-renewal. Lecture 5: The Stem Cell Niche 10  Experiment with DPP Overexpression: In a normal fly testis (shown in photomicrograph B), DPP is expressed only in the hub, resulting in a controlled population of stem cells localized to the tip of the testis. When DPP expression is artificially increased outside of the hub (i.e., in other areas of the testis), it leads to a dramatic expansion of the stem cell population. This overexpression creates a "stem cell tumor," where an abnormally large number of stem cells occupy a much larger area of the testis than normal. Lecture 5: The Stem Cell Niche 11  Name a key cell type that supplies niche signals in the mammalian testis. Lecture 5: The Stem Cell Niche 12  Sertoli Cells (Niche Cells): Sertoli cells are large, yellow cells within the mammalian testis that serve as niche cells. They have numerous extensions or "arms" that wrap around developing germ cells, providing structural support and essential signals. The Sertoli cell is a critical niche cell in the mammalian testis, essential for supporting the development and differentiation of spermatogonia into mature sperm. In the testis, there's a progression of germ cells: Undifferentiated Spermatogonia: These are the stem cells that give rise to sperm. They are located near the Sertoli cells and progress through differentiation as they move through the testis. Differentiating Germ Cells: As spermatogonia differentiate, they develop into more mature forms, such as spermatids, which are closer to becoming sperm. Germ cells are lost when the niche is disrupted Mutant - disruption in Wnt signalling Wnt Signaling Pathway: Wnt is a secreted growth factor that interacts with the Frizzled receptor on the surface of a target cell. This interaction triggers a cascade of intracellular events, which are Lecture 5: The Stem Cell Niche 13  crucial for various cellular processes, including maintaining stem cells. Secreted by Sertoli cells in the testes Experimental Setup: The experiment involved disrupting the Wnt signaling pathway in mice to observe its effects on the testis, particularly on the spermatogonial stem cells. Wild Type (Normal): Testis shows normal differentiation of sperm cells. The tubules are filled with differentiating sperm, and their tails are visible as little lines in the center of the tubules. Mutant Mouse (Wnt Disrupted): When Wnt signaling is disrupted in the mutant mouse, a significant gap appears in the tubules, indicating a loss of germ cells. The remaining cells around the edges of the tubules are primarily Sertoli cells (referred to as "cittoli cells" in the text). Outline the key cell types and signals in the intestinal stem cell niche. Lecture 5: The Stem Cell Niche 14  Small intestinal epithelium is composed of crypts and villi Colonic epithelium consists of crypts but lacks villi Stem cells reside near the base of crypts and are responsible for continuously renewing the entire epithelial cell layer Four differentiated cell types are found in the intestinal epithelium → absorptive enterocytes, Goblet cells, Paneth cells and enteroendocrine cells. Enterocytes Location: Found along the villi of the intestine. Function: Absorption of nutrients and maintenance of intestinal barrier function. Goblet Cells Lecture 5: The Stem Cell Niche 15  Location: Distributed throughout the intestinal epithelium, more abundant in the villi. Function: Secrete mucus to protect and lubricate the intestinal lining. Paneth Cells Location: Positioned at the base of the crypts near ISCs. Function: Provide niche support by secreting antimicrobial peptides and growth factors. Enteroendocrine Cells Location: Scattered throughout the intestinal epithelium. Function: Produce hormones that regulate digestion and gut motility. The intestinal crypt is a stem cell niche Intestinal stem cells are located at the base of the crypts, nestled between specialised cells called Paneth cells. ^ These stem cells are responsible for the continuous renewal of the intestinal epithelium. All cell types within the intestinal lining are derived from these stem cells. Lecture 5: The Stem Cell Niche 16  The first type of cell that intestinal stem cells produce are called transient amplifying cells. Niche cells & signals The stem cells in the crypts are supported by a surrounding niche that consists of various cell types and signaling molecules. Niche cells → Telocytes, stromal cells & macrophages Key signal → Wnt, Notch, EGF, Neuregulin 1 (NRG1) Wnt Signaling Source: Secreted by Paneth cells and mesenchymal cells. Function: Activates β-catenin signaling pathway, crucial for ISC self-renewal and proliferation. Notch Signaling Source: Interaction between ISCs and neighboring cells, including Paneth cells. Function: Regulates cell fate decisions and differentiation, balancing the stem cell pool and progenitor differentiation. Egf (Epidermal Growth Factor) Source: Produced by various cells including Paneth cells and enterocytes. Function: Stimulates cell proliferation and survival in the intestinal epithelium. Describe the function of Wnt and Notch signalling in the intestine. Wnt Signalling Pathway Lecture 5: The Stem Cell Niche 17  Function: The Wnt signaling pathway is crucial for regulating cell proliferation in the intestine. Activity: In the Crypts: Wnt signaling is highly active in the intestinal crypts, where it drives the proliferation of intestinal stem cells (ISCs). This activity ensures a continuous supply of new cells to replace those that are lost during normal turnover. In the Villi: Wnt signaling is inactive in the villi, where mature cells are positioned. In this region, cell proliferation is minimal, as the primary function is to support nutrient absorption rather than generating new cells. Notch Signalling Pathway Lecture 5: The Stem Cell Niche 18  Function: The Notch signaling pathway plays a critical role in determining cell fate within the intestinal epithelium, influencing whether cells become absorptive or secretory. Activity: Absorptive Cells: When Notch signaling is active, cells in the crypts are more likely to differentiate into absorptive enterocytes. These Lecture 5: The Stem Cell Niche 19  are the cells that line the villi and are responsible for nutrient absorption. Secretory Cells: When Notch signaling is inactive, cells are more likely to differentiate into secretory cells, such as goblet cells (which secrete mucus) and enteroendocrine cells (which produce hormones). Describe biological assays that define intestinal stem cells. Stem cells produce all the differentiated cell types present in the intestinal epithelium Paneth cells, enterocytes, goblet cells, enteroendocrine cells But how can intestinal stem cells be identified? Genetic strategy → EG) Linear tracing Candidate stem cells are genetically marked. Introduced marker allows stem cells and differentiated progeny to be observed. Isolation and culture strategy → EG) organoid culture Candidate cells are isolated and cultured Lecture 5: The Stem Cell Niche 20  Cultured cells should have the capacity to form all differentiated cell types of the tissue in vitro. Upon transplantation cells should be able to replace the tissue Provide an example of an intestinal stem cell marker. Lineage tracing from Lgr5 stem cells reveals the fate of individual stem cell clones Key components needed for lineage tracing: LGR5 Gene Locus with GFP: LGR5: A marker for intestinal stem cells. GFP (Green Fluorescent Protein): Attached to the LGR5 promoter, so cells expressing LGR5 will also express GFP and fluoresce green. This allows visualization of stem cells under a fluorescence microscope. CRE Recombinase Gene: CRE: An enzyme that can induce recombination at specific DNA sequences (LOXP sites). CRE-ER2: A modified form of CRE that remains in the cytoplasm until activated by tamoxifen. Reporter Construct with LACZ: LACZ: A gene encoding β-galactosidase, an enzyme that turns cells blue when a substrate is added. LOXP Sites: Sequences flanking a stop codon in the LACZ gene. The stop codon prevents LACZ expression until the CRE enzyme recombines between LOXP sites and removes the stop codon. Procedure for lineage tracing: 1. Initial Setup: Intestinal tissue is genetically engineered so that LGR5-positive stem cells express both GFP and CRE-ER2. The tissue also contains a reporter construct with LACZ and LOXP sites flanking a stop codon. 2. Induction with Tamoxifen: Lecture 5: The Stem Cell Niche 21  Tamoxifen activates CRE-ER2, allowing CRE to enter the nucleus and induce recombination. CRE recombines between the LOXP sites, removing the stop codon, and activates LACZ expression. 3. Observation: Day 1: Only the stem cells are labeled blue due to the activation of LACZ by CRE. 24 Hours Later: Progeny of the stem cells, which have divided and begun to differentiate, also retain the blue color. Several Days Later: Progeny move up the crypts, differentiate into various cell types, and eventually become part of the villi. The blue labeling spreads through the crypts as more cells derived from the original stem cell differentiate into various cell types (e.g., goblet cells, enteroendocrine cells). Cell Sloughing: Eventually, differentiated cells at the top of the villi are shed. Describe organoid cultures and transplantation assays Lecture 5: The Stem Cell Niche 22  Isolation and Culture Strategy 1. Identifying and Culturing Stem Cells: Goal: To isolate and culture intestinal stem cells, then assess their ability to differentiate into all cell types of the intestinal epithelium. Technique: Use a genetically engineered mouse (e.g., LGR5 GFP mouse) where LGR5-positive stem cells are marked with green fluorescence. This allows for easy identification and isolation of these cells using fluorescence-activated cell sorting (FACS). 2. Organoid Cultures: Development: Cultured LGR5-positive stem cells can form organoids, which are 3D structures resembling miniature intestines, or “mini-guts.” Findings: These organoids exhibit crypt-like structures and contain all differentiated cell types typical of the intestinal epithelium, indicating that the stem cells are functional and capable of producing the full spectrum of intestinal cells. 3. Key Niche Signals: Lecture 5: The Stem Cell Niche 23  Signals for Growth: Key signals from the intestinal niche that support stem cell growth and organoid formation include: Paneth Cells: Produce Wnt3a, EGF, and Uregulin. Stromal Cells: Produce R-spondin. Telocytes: Secrete BMP molecules. Role of Signals: These signals are crucial for maintaining stem cell proliferation and differentiation in vitro. Transplantation Experiment Objective: To demonstrate the functional capability of stem cells by transplanting them into an injured organ. Study: Organoids derived from EGFP-transgenic mice were transplanted into a mouse model of colitis (ulcerative colitis) in Japan. Results: The transplanted organoids incorporated into the damaged intestinal tissue, repaired ulcers, and restored normal epithelial structure, showing their potential for therapeutic applications. Lecture 5: The Stem Cell Niche 24 Lecture 5: The Stem Cell Niche 25