5_Oct_17_SJ_BME236_2024

Page 1: Introduction

• Presenter: Sebastian Jessberger

• Affiliation: Brain Research Institute, University of Zurich

• Contact: essberger@hifo.uzh.ch

Page 2: Dialogue/Context

• A humorous exchange indicating a casual conversation.

Slide 3: Introduction to Stem Cell Research - Part 1

Key Points:

• Fundamental Questions in Stem Cell Research:

  • Definition: What qualifies a cell as a stem cell?

  • Source: How can embryonic stem (ES) cells be obtained?

  • Pluripotency: What characteristics make an ES cell pluripotent?

  • Challenges: What are the ethical and technical issues associated with ES cells?

  • Necessity: Is research involving ES cells still essential in modern science?

  • Applications: What potential do human pluripotent cells hold for therapy and research?

Explanation of Visuals:

• This slide lists critical questions framing the exploration of stem cell biology.

• These questions guide understanding and highlight the focus areas in the field, including definition, derivation, properties, challenges, and applications.

Glossary:

• Embryonic Stem (ES) Cells: Pluripotent stem cells derived from the early embryo.

• Pluripotency: The ability of a cell to differentiate into almost all types of cells in the body.

Key Takeaway: This slide sets the stage for discussions about stem cell biology, emphasizing the need to address fundamental biological and ethical questions to unlock the full potential of pluripotent stem cells in science and medicine.

Slide 4: Potential Stem Cells with Neural Capability – Totipotent Cells

Key Points:

• Totipotent Cells:

  • Have the capability to form all types of cells, including both embryonic and extra-embryonic tissues (e.g., placenta).

  • Source: Zygote immediately after fertilization.

  • Non-self-renewing; this capability is limited to the early stage after fertilization.

Explanation of Visuals:

• Diagram:

  • Highlights the totipotent zygote stage and its ability to produce any cell type.

  • Represents the zygote as a single blue sphere, indicating its unrestricted potential.

Glossary:

• Totipotent: Cells that can form the entire organism and supporting tissues like the placenta.

• Zygote: The first developmental stage after the union of egg and sperm.

Key Takeaway: Totipotent cells represent the earliest stage of development, with the potential to differentiate into any cell type necessary for forming an entire organism.

Slide 5: Potential Stem Cells with Neural Capability – Pluripotent Cells

Key Points:

• Pluripotent Cells:

  • Formed after the totipotent stage.

  • Can self-renew and differentiate into nearly all cell types in the body but not extra-embryonic tissues.

  • Source: Embryonic stem cells derived from the blastocyst.

Explanation of Visuals:

• Diagram:

  • Illustrates the transition from totipotent to pluripotent stem cells.

  • Adds a new layer to the hierarchy, showing pluripotent cells capable of extensive differentiation.

Glossary:

• Blastocyst: An early stage of embryo development containing pluripotent stem cells.

• Pluripotent: Cells with the ability to become any type of tissue within the body.

Key Takeaway: Pluripotent stem cells have broad differentiation potential, making them crucial for regenerative medicine and research.

Slide 6: Potential Stem Cells with Neural Capability – Multipotent Cells

Key Points:

• Multipotent Cells:

  • More restricted compared to pluripotent cells.

  • Can self-renew and differentiate into a limited range of cell types within a specific tissue or organ.

    • Stem cells of brain can only make brain, stem cells of skin can only make skin … etc.

    • They can be on multiple positions or organs, but are as said restricted to a tissue type.

    • Multipotent, they can not only make one cell type in e.g. brain tissue, but multiple cell types in brain tissue

  • Source: Found in embryos or adult tissues such as the brain or blood.

Explanation of Visuals:

• Diagram:

  • Shows the stepwise restriction of differentiation potential from pluripotent to multipotent cells.

  • Emphasizes the specialization of these cells to certain tissue lineages.

Glossary:

• Multipotent: Cells that can develop into multiple, but limited, cell types within a specific tissue or organ system.

Key Takeaway: Multipotent stem cells are specialized for tissue repair and maintenance within specific organ systems.

Slide 7: Potential Stem Cells with Neural Capability – Neural Progenitors

Key Points:

• Neural Progenitor Cells:

  • Have the narrowest differentiation potential, limited to neural lineages (e.g., brain or spinal cord cells).

  • Do not self-renew indefinitely.

  • Source: Neural tissue, including the brain and spinal cord.

Explanation of Visuals:

• Diagram:

  • Adds neural progenitor cells as the most restricted type in the hierarchy.

  • Differentiation potential narrows further as shown by the progression in the chart.

Glossary:

• Neural Progenitor Cells: Specialized cells that give rise to specific neural cell types but have limited self-renewal. Can only make one type of cells

• Lineage Restriction: The progressive narrowing of differentiation capabilities as cells specialize.

Key Takeaway: Neural progenitor cells represent the final step in stem cell specialization, critical for generating specific neural tissues.

Slide 8: Comprehensive Overview of Neural Stem Cell Potential

Key Points:

• Stem cells can be categorized based on their differentiation potential:

  • Totipotent: Zygote stage, forms all cells (embryonic and extra-embryonic).

  • Pluripotent: Embryonic stem cells, derived from the blastocyst, can form nearly all body tissues.

  • Multipotent: Tissue-specific stem cells, restricted to a lineage (e.g., neural or blood cells).

  • Progenitor Cells: Limited potential, e.g., neural progenitors forming specific neurons or glial cells.

  • Differentiated Cells: Final stage, fully specialized for a specific function, e.g., brain neurons.

Explanation of Visuals:

• A hierarchical diagram illustrates the stepwise restriction of differentiation potential, progressing from totipotent to differentiated cells.

• Each stage is represented by a unique symbol (e.g., zygote, blastocyst, and tissue-specific stem cells).

Glossary:

• Differentiation: Process where stem cells specialize into specific cell types.

• Glial Cells: Supportive cells in the nervous system.

• Neuron: A cell specialized for transmitting signals in the nervous system.

Key Takeaway: Stem cell potential narrows progressively from totipotency to fully specialized cells, crucial for understanding tissue repair and development.

TO REMEMBER:

The zygote is not technically considered a stem cell, but it has the highest developmental potential, known as totipotency, which allows it to develop into an entire organism, including both embryonic and extra-embryonic tissues (such as the placenta).

Key Differences Between Zygote and Stem Cells:

1. Totipotency vs. Stem Cell Properties:

   • The zygote is totipotent, meaning it can give rise to all cell types of the body and extra-embryonic structures.

   • Stem cells, such as pluripotent stem cells, can only differentiate into cells of the three germ layers (ectoderm, mesoderm, endoderm) but not extra-embryonic structures.

2. Self-Renewal:

   • The zygote does not self-renew; it divides and differentiates into other cell types.

   • Stem cells (such as pluripotent and multipotent stem cells) have the ability to self-renew, maintaining their population over time.

3. Function:

   • The zygote's primary function is to initiate development and form a multicellular organism.

   • Stem cells primarily serve to replenish specific tissues or differentiate into specialized cells.

Conclusion:

While the zygote has the ability to generate all cell types (making it totipotent), it is not classified as a stem cell because it lacks self-renewal capabilities, which is a defining characteristic of stem cells.

3 Main Hierarchies:

• Multipotent → Embryonic Stem cells

• Pluripotent → Somatic cells that are in tissues

• Limited stem cells (Progenitor) → Can only make one cell type in a given tissue.

Slide 9: Visual Representation of ES Cells

Key Points:

• Embryonic stem (ES) cells:

  • Pluripotent cells capable of differentiating into nearly any tissue.

  • Exhibit unique morphological characteristics under the microscope (e.g., dense colonies with distinct borders).

Explanation of Visuals:

• A microscopic image of an embryonic stem cell colony is shown:

  • Highlights the tightly packed, round appearance of ES cells.

  • Demonstrates the organized structure and clear boundaries typical of healthy ES cell colonies.

Glossary:

• Morphology: The physical appearance and structure of cells.

• Colony: A cluster of cells derived from a single progenitor cell.

Key Takeaway: Microscopic visualization of ES cells provides insight into their growth and morphology, crucial for identifying and maintaining these cells.

Slide 10: Isolation of Embryonic Stem (ES) Cells

Key Points:

1. Cleavage Stage Embryo:

   • The process starts with a developing embryo at the cleavage stage.

2. Cultured Blastocyst:

   • The embryo develops into a blastocyst, which contains an inner cell mass (ICM) that will be used to derive embryonic stem cells.

3. Isolation of Inner Cell Mass (ICM):

   • The ICM is carefully isolated from the blastocyst, as it contains pluripotent cells that can differentiate into various cell types.

4. Culturing on Feeder Cells:

   • The isolated ICM is plated on a layer of irradiated mouse fibroblast feeder cells, which provide support and essential factors for the growth of ES cells.

5. Dissociation and Replating:

   • The cells are dissociated and replated on fresh feeder cells to allow for further expansion and maintenance.

6. Established ES Cell Culture:

   • After several rounds of replating, a stable embryonic stem cell culture is established, which can be used for further research and applications.

Explanation of Visuals:

• The diagram illustrates the step-by-step progression from the early embryo stage to the establishment of embryonic stem cell cultures.

• Key stages include the isolation of the inner cell mass and its growth on feeder cells.

Glossary:

• Blastocyst: A hollow structure in early embryonic development that contains the inner cell mass, which gives rise to the embryo.

• Inner Cell Mass (ICM): A cluster of cells within the blastocyst that are pluripotent and can develop into any cell type in the body.

• Feeder Cells: Supportive cells (often fibroblasts) that provide nutrients and signaling factors to maintain the undifferentiated state of stem cells.

• Pluripotency: The ability of a stem cell to differentiate into any cell type of the three germ layers (ectoderm, mesoderm, and endoderm).

Key Takeaway: The process of isolating embryonic stem cells from blastocysts involves the careful extraction of the inner cell mass and culturing on feeder cells, leading to the establishment of pluripotent cell lines for research and therapeutic applications.

Once this is done, you can basically keep the cells forever. Since the selfrenew

Slide 11: Maintaining Pluripotency in ES Cells

Key Points:

• Environmental Stimuli: External factors influence the state of embryonic stem (ES) cells by triggering intracellular signaling pathways.

• Signal Transduction Pathways: These pathways transmit signals from the environment to the nucleus to regulate gene expression.

• Chromatin Regulators: Proteins that modify DNA packaging to control access to genes required for maintaining pluripotency.

• Transcription Factors: Key proteins that bind DNA and regulate the expression of genes necessary to sustain the pluripotent state.

• miRNAs (microRNAs): Small non-coding RNAs that fine-tune gene expression by targeting mRNA for degradation or translation repression.

• Target Genes: Pluripotency-related genes that are activated or repressed to maintain the self-renewal capacity of ES cells.

Explanation of Visuals:

• The diagram shows how environmental stimuli (e.g., growth factors, signaling molecules) influence stem cell behavior.

• The top part represents the incoming signals from the environment, which are processed via signal transduction pathways, leading to activation of transcription factors and chromatin regulators.

• Transcription factors (illustrated as blue circles) regulate key genes needed for pluripotency.

• Chromatin regulators (green circles) modify the structure of DNA to keep pluripotency genes active.

• Red blocks represent target genes that maintain the stem cell state.

• miRNAs (red arrows) play a regulatory role by interacting with transcription factors to fine-tune gene expression.

Glossary:

• Pluripotency: The ability of a cell to develop into all cell types of the three germ layers (ectoderm, mesoderm, and endoderm).

• Signal Transduction: The process by which a cell converts an external signal into a functional response.

• Transcription Factors: Proteins that control the rate of gene transcription by binding to specific DNA sequences.

• Chromatin Regulators: Molecules that alter chromatin structure to influence gene accessibility and expression.

• miRNA (MicroRNA): Small RNA molecules that regulate gene expression by targeting messenger RNA (mRNA).

Slide 12: Keeping an ES Cell Pluripotent: The Major Players

Key Points:

• Core Transcription Factors:

  • Oct4 and Sox2: Essential regulators that prevent differentiation and maintain self-renewal.

  • Nanog: Supports stem cell identity by preventing differentiation signals.

• Signaling Pathways Regulating Pluripotency:

  • Positive Signals:

    • FGF (Fibroblast Growth Factor): Promotes differentiation through the ERK signaling pathway.

    • Notch, BMP, Activin, Wnt: Signaling pathways involved in balancing self-renewal and differentiation.

    • LIF (Leukemia Inhibitory Factor): Maintains pluripotency by preventing differentiation in mouse ES cells.

  • Inhibition Strategies to Maintain Pluripotency:

    • FGFR/MEK Inhibitors: Block differentiation signals to retain stemness.

    • BMP4: Supports self-renewal under controlled conditions.

Explanation of Visuals:

• Panel A (Top):

  • Shows the role of FGF4 signaling in differentiation, leading to lineage commitment.

  • Oct4 and Sox2 maintain the pluripotent state but are influenced by external factors like FGF, Notch, and BMP.

  • The signaling cascade through ERK promotes differentiation.

• Panel B (Bottom):

  • Highlights the role of inhibitors (e.g., FGFR/MEK inhibitors) in blocking differentiation pathways.

  • LIF signaling is shown to support self-renewal and prevent differentiation into specific lineages.

Glossary:

• Oct4 and Sox2: Master transcription factors essential for maintaining stem cell pluripotency.

• Nanog: A transcription factor that helps sustain the pluripotent state by blocking differentiation signals.

• FGF (Fibroblast Growth Factor): A growth factor that promotes cell proliferation and differentiation.

• BMP (Bone Morphogenetic Protein): A signaling molecule involved in embryonic development and cell fate decisions.

• LIF (Leukemia Inhibitory Factor): A cytokine that supports pluripotency by preventing differentiation in mouse embryonic stem cells.

• FGFR/MEK Inhibitors: Chemicals used to block pathways that promote differentiation, helping to maintain an undifferentiated state.

Slide 13: Key Regulators of Pluripotency

Key Points:

• Pluripotency in embryonic stem (ES) cells is regulated by a core network of transcription factors that maintain their undifferentiated state and prevent differentiation.

• OCT4, SOX2, and NANOG are the central transcription factors essential for maintaining pluripotency by:

  • Activating genes involved in self-renewal and repressing differentiation signals.

  • Forming a positive feedback loop where they bind to their own promoters and reinforce their continuous expression, ensuring a stable pluripotent state.

  • Cooperating to regulate additional genes critical for stem cell identity and function.

• Chromatin regulators (e.g., histone acetyltransferases) modify chromatin structure to promote active gene transcription by making DNA more accessible to transcription factors.

• MicroRNAs (miRNAs) fine-tune pluripotency by post-transcriptionally regulating the expression of pluripotency-associated genes and suppressing differentiation-related pathways.

• The positive feedback loop ensures that once the pluripotency state is established, it remains stable by continuously reinforcing the expression of OCT4, SOX2, and NANOG, creating a self-sustaining circuit.

• This self-reinforcing mechanism allows ES cells to retain their pluripotent potential while remaining responsive to external differentiation signals when required.

Explanation of Visuals:

• The diagram illustrates the transcriptional regulatory network for pluripotency:

  • Top section: Shows how OCT4 and SOX2 activate key downstream pluripotency genes, ensuring the cell remains undifferentiated.

  • Middle section: Highlights chromatin remodeling factors that facilitate open chromatin structure, allowing efficient gene expression.

  • Bottom section: Depicts the suppression of differentiation-associated genes by repressive chromatin modifications, ensuring the pluripotent state is maintained.

Glossary:

• OCT4 and SOX2: Master transcription factors that activate genes promoting self-renewal and pluripotency in embryonic stem cells.

• NANOG: A transcription factor that works alongside OCT4 and SOX2 to reinforce the pluripotency network and inhibit differentiation.

• Chromatin remodeling: The dynamic modification of chromatin structure to regulate gene expression by controlling DNA accessibility.

• Transcription Factor: A protein that binds to specific DNA sequences to regulate the transcription (activation or repression) of target genes.

• MicroRNAs (miRNAs): Small RNA molecules that post-transcriptionally regulate gene expression to fine-tune cellular processes.

• Positive feedback loop: A regulatory mechanism where transcription factors reinforce their own expression by activating their own promoters, creating a self-sustaining system to maintain the pluripotent state.

Key Takeaway:

• The pluripotency of ES cells is maintained through a tightly controlled transcriptional network, where OCT4, SOX2, and NANOG reinforce their own expression via a positive feedback loop. Chromatin modifications and microRNAs further fine-tune this regulation, ensuring long-term maintenance of pluripotency while preventing premature differentiation.

Slide 15: Testing Pluripotency of ES Cells

Key Points:

• Pluripotency is validated through specific tests:

  • In vitro differentiation: ES cells are cultured under conditions promoting differentiation into three germ layers.

  • In vivo teratoma formation: ES cells form teratomas containing tissues from all germ layers when injected into mice.

  • Chimera formation: ES cells contribute to various tissues when introduced into embryos.

Explanation of Visuals:

• Top section shows ES cell colonies isolated from blastocysts.

• Bottom section explains how ES cells are tested for their ability to form:

  • Teratomas in mice.

  • Contributions to embryonic and adult tissues in chimeric organisms.

Glossary:

• Teratoma: A benign tumor containing different tissue types derived from all three germ layers.

• Chimera: An organism containing cells from two different zygotes.

• Germ Layers: The three layers of cells (ectoderm, mesoderm, and endoderm) that give rise to all tissues.

Key Takeaway: Pluripotency of ES cells is rigorously tested using in vitro and in vivo methods to confirm their ability to generate all tissue types.

Key Points:

• The pluripotency of embryonic stem (ES) cells is assessed by their ability to contribute to all cell types in a developing embryo.

• The process involves several key steps:

  • Cell isolation and culture: ES cells are isolated from the inner cell mass of a mouse blastocyst and cultured in vitro.

  • Genetic modification: A specific gene is introduced into cultured ES cells via genetic engineering.

  • Selection: Cells that have successfully incorporated the gene are selected.

  • Blastocyst injection: Selected ES cells are injected into a blastocyst, which is then implanted into a surrogate mother.

  • Chimeric offspring: If the ES cells contribute to the embryo, the resulting offspring will contain both host and donor cell-derived tissues.

  • Breeding: The chimeric mouse can pass the introduced genetic material to its offspring, confirming pluripotency.

Explanation of Visuals:

• The first panel illustrates the isolation of ES cells from a mouse blastocyst and their culture in a dish.

• The second panel provides a step-by-step depiction of the genetic modification process, selection of transformed cells, injection into the blastocyst, and implantation into a surrogate mother.

• The outcome shows a transgenic mouse that carries the modified genes.

Glossary:

• Chimera: An organism containing cells from two different genetic lineages.

• Blastocyst: An early-stage embryo consisting of an outer layer (trophoblast) and an inner cell mass, which gives rise to the embryo proper.

• Surrogate mother: A female used to carry an embryo to term after implantation.

• Gene integration: The process of inserting a gene into the genome of a cell to assess its function.

Key Takeaway:

• Pluripotency of ES cells is confirmed by their ability to integrate into the developing embryo and give rise to multiple cell types, contributing to functional tissues in offspring.

Slide 16: Metabolic Regulation and Neural Stem Cells

Key Points:

• Pluripotency can also be tested by differentiating ES cells in vitro into specialized cell types representative of all three germ layers:

  • Ectoderm: Forms neurons and skin cells.

  • Mesoderm: Forms heart muscle cells and blood cells.

  • Endoderm: Forms liver and intestinal cells.

• The process involves the following steps:

  • Fertilization of an oocyte by sperm, leading to the development of a blastocyst.

  • Isolation of the inner cell mass from the blastocyst and propagation in culture.

  • Controlled differentiation of ES cells into various specialized cell types.

• This assay allows researchers to observe whether ES cells retain their pluripotent capacity under laboratory conditions.

Explanation of Visuals:

• The first part of the diagram shows the fertilization process, leading to the formation of a blastocyst.

• The next part illustrates how the inner cell mass is isolated and cultured in vitro to establish pluripotent stem cells.

• The final section depicts the differentiation of ES cells into various specialized cell types, including heart muscle cells, neurons, and liver cells.

Glossary:

• Ectoderm: The outer germ layer that gives rise to the nervous system and skin.

• Mesoderm: The middle germ layer that forms muscles, bones, and blood.

• Endoderm: The inner germ layer that develops into the digestive tract and associated organs.

• In vitro differentiation: The process of directing stem cells to develop into specific cell types in a laboratory setting.

Key Takeaway:

• In vitro differentiation of ES cells into specialized cell types confirms their pluripotent potential and provides insights into their developmental capabilities.

Slide 18: Linking Neurogenesis to Cognition

Key Points:

• Neurogenesis-Cognition Link:

  • Studies explore the impact of genetic mutations on neurogenesis and cognitive functions.

  • Mouse models are used to investigate behavioral and cognitive changes.

• Approach: Combining genetic studies, molecular analyses, and animal models to map how brain development affects cognition.

Explanation of Visuals:

• The slide depicts:

  • Family tree analysis showing inheritance of mutations.

  • Imaging data linking mutations to altered neurogenesis and behavioral effects in mice.

Glossary:

• Cognition: Mental processes involved in learning, memory, and reasoning.

• Mouse Model: Laboratory mice used to study human disease processes.

Key Takeaway: Mutations in neurogenesis-related genes directly impact cognitive functions, as demonstrated in model organisms.

Wild type human embryonic stem cells (hESCs) refer to embryonic stem cells that possess the naturally occurring, unmodified genetic sequence found in humans. These cells have not been altered through genetic manipulation or mutations and serve as a reference or control in scientific studies.

Key Characteristics of Wild Type hESCs:

• Pluripotency: They can differentiate into any of the three germ layers (ectoderm, mesoderm, and endoderm), giving rise to all cell types in the body.

• Normal Genetic Makeup: Their DNA sequence is unaltered, making them useful for studying baseline cellular behaviors.

• Self-Renewal: They can divide indefinitely under appropriate culture conditions while maintaining their pluripotency.

• Research Control: Used as a standard for comparison against genetically modified or disease-specific stem cells.

Applications:

• Disease modeling: Studying normal vs. disease-affected cells.

• Drug screening: Evaluating drug effects on healthy human cells.

• Regenerative medicine: Investigating potential therapies for replacing damaged tissues with healthy cells.

Example: If scientists introduce a mutation into hESCs to study a disease, they would compare the results to wild type hESCs to understand how the mutation affects cell function.

In summary, wild type hESCs are unmodified stem cells that serve as an essential baseline for understanding normal human development and disease mechanisms.

Slide 19: FASN Mutation and NSC Phenotypes

Key Points:

• Genetic Modification:

  • The slide illustrates the introduction of the R1819W mutation in the FASN gene in human embryonic stem cells (hESCs).

  • The mutation was introduced using ssODN (single-stranded oligodeoxynucleotides) and a guide RNA (gRNA) targeting the wild-type (WT) sequence.

  • Restriction enzyme analysis (PfoI and RsaI) and gel electrophoresis confirmed the successful mutation.

• Cell Phenotype Analysis:

  • Expression of neural stem cell (NSC) markers, such as Pax6 and PLZF, were analyzed using immunofluorescence microscopy.

  • Cells carrying the R1819W mutation showed differences in marker expression compared to WT, suggesting a shift towards an NSC phenotype.

• Proliferation Analysis:

  • EdU incorporation assay was performed to assess cell proliferation.

  • The results (shown in the lower panel) indicate a significant decrease in proliferation in the R1819W mutant cells compared to WT.

  • A quantification graph on the right shows a statistical decrease in EdU-positive cells in the mutant group.

Explanation of Visuals:

1. Top-left section:

   • Sequence alignment of the WT and mutated FASN gene.

   • Schematic of the gene locus showing exon structure and restriction enzyme sites.

   • Gel electrophoresis confirming successful genetic modification.

2. Top-right section:

   • Pax6 and PLZF staining images show marker expression in WT vs. mutant cells.

   • Brightfield images show morphological differences between WT and mutant cells.

3. Bottom section:

   • EdU (red) and DAPI (blue) staining to assess proliferation.

   • Bar graph quantifying the percentage of proliferating cells, indicating a decrease in proliferation in the mutant.

Glossary:

• FASN (Fatty Acid Synthase): An enzyme involved in lipid biosynthesis, critical for cell membrane formation and energy storage.

• NSC (Neural Stem Cells): Multipotent stem cells that can differentiate into neurons, astrocytes, and oligodendrocytes.

• Pax6: A transcription factor crucial for neural development and NSC identity.

• PLZF (Promyelocytic Leukemia Zinc Finger): A protein involved in stem cell maintenance and self-renewal.

• EdU Assay: A method to measure cell proliferation by incorporating thymidine analogs into newly synthesized DNA.

• DAPI (4',6-diamidino-2-phenylindole): A fluorescent stain that binds to DNA, commonly used for nuclear staining.

Key Takeaway:

The FASN^R1819W mutation in hESCs induces characteristics of neural stem cells in 2D cultures, as shown by increased expression of NSC markers and reduced proliferation rates.

Slide 20: Regionalized Forebrain Organoids

Key Points:

• Organoid Technology:

  • Forebrain organoids are lab-grown structures mimicking the human brain's forebrain region.

  • Derived from human pluripotent stem cells to study brain development and disorders.

• Applications:

  • Understanding brain structure organization.

  • Modeling neurological diseases and developmental disorders.

Explanation of Visuals:

• Images showcase 3D forebrain organoids stained for specific neural markers.

  • Key markers identify regional specialization within the organoids.

  • Brightly colored staining highlights distinct regions of brain-like structures.

Glossary:

• Organoids: Miniaturized and simplified versions of organs grown in vitro for research.

• Pluripotent Stem Cells: Cells capable of differentiating into any cell type in the body.

Key Takeaway: Forebrain organoids provide a powerful tool to study human brain development and disorders in a controlled laboratory environment.

Slide 21: Modeling FASN Mutations in Mice and Human Cells

Key Points:

• Mutation Modeling:

  • Human FASN mutations are studied using genetically engineered mice and human cells.

  • These models replicate the neurodevelopmental effects of FASN dysfunction.

• Findings:

  • Mutations lead to altered brain structures and impaired neural differentiation.

  • Imaging highlights changes in neural tissue morphology.

Explanation of Visuals:

• Top Panel: Microscopic images comparing normal (WT) and mutated cells.

  • Differences in neural stem cell differentiation and tissue structure are evident.

• Bottom Panel: Stained brain sections show disrupted organization in mutant models.

Glossary:

• FASN: An enzyme critical for fatty acid synthesis.

• Neural Differentiation: The process where stem cells develop into specialized neural cells.

Key Takeaway: FASN mutations disrupt neural development, providing insight into metabolic contributions to brain disorders.

Slide 22: Modeling Human FASN Mutations in Mice and Human Cells

Key Points:

• Study of Impaired Neurogenesis:

  • Human and mouse models link FASN mutations to disrupted neurogenesis and cognitive deficits.

  • Models exhibit altered neural circuitry and decreased cognitive performance.

• Approach:

  • Multi-level analysis combining genetic, molecular, and behavioral studies.

Explanation of Visuals:

• Family tree diagram traces inheritance of FASN mutations.

• Imaging highlights impaired neural differentiation and function in models.

Glossary:

• Neurogenesis: Formation of new neurons in the brain.

• Cognition: Mental processes like learning and memory.

Key Takeaway: FASN mutations impair neurogenesis and cognitive functions, linking metabolic pathways to brain development.

Slide 23: Development of Specialized Cells

Key Points:

• Process Overview:

  • A donor oocyte undergoes enucleation, removing its nucleus.

  • A somatic cell from the patient (e.g., skin or bone marrow cell) is used to provide a nucleus, which is transferred into the enucleated egg via Somatic Cell Nuclear Transfer (SCNT).

  • After nuclear transfer and activation, the egg develops through the morula stage into a blastocyst, which contains the inner cell mass (ICM), a source of pluripotent embryonic stem cells.

  • The stem cells are propagated in culture and differentiated into specialized cells such as:

    • Heart muscle cells

    • Neurons

    • Blood cells

    • Liver cells

    • Pancreatic islet cells

    • Intestinal cells

• Therapeutic Applications:

  • Differentiated stem cells can be transplanted back into patients to treat conditions like:

    • Diabetes

    • Alzheimer's disease

    • Parkinson's disease

    • Spinal cord injuries

    • Cancer

    • Cardiovascular diseases

    • Rheumatoid arthritis

Explanation of Visuals:

1. Upper section:

   • Depicts the process of SCNT, showing enucleation of the donor egg, nuclear transfer, and development into a blastocyst.

2. Middle section:

   • Highlights the inner cell mass as the source of pluripotent embryonic stem cells.

3. Lower section:

   • Shows different cell types derived from stem cells and their potential therapeutic applications.

Challenges and Risks of Stem Cell Therapy:

• Tumor Formation (Teratomas): Pluripotent stem cells have the potential to form tumors if their growth is not properly controlled.

• Immune Rejection: Even though cells are derived from the patient, immune responses may still occur, especially in allogeneic transplants.

• Ethical Concerns: The use of embryonic stem cells raises ethical and moral considerations.

• Technical Challenges: Efficient differentiation and integration into target tissues remain complex and require further research.

• Regulatory Issues: Safety and long-term effects need rigorous clinical trials and approval processes.

Glossary:

• Somatic Cell Nuclear Transfer (SCNT): A technique where a nucleus from a somatic cell is transferred to an enucleated egg cell to create stem cells.

• Pluripotent Stem Cells: Cells that can differentiate into any cell type in the body but cannot form an entire organism.

• Blastocyst: A structure formed in early embryonic development that contains the inner cell mass, the source of embryonic stem cells.

• Differentiation: The process by which stem cells develop into specialized cell types.

• Enucleation: The removal of the nucleus from a cell to prepare it for nuclear transfer.

Key Takeaway:

SCNT allows the creation of patient-specific pluripotent stem cells, offering promising therapeutic potential for treating various diseases. However, ethical concerns, tumor risks, and technical challenges must be carefully managed.

Slide 24: Parkinson's Disease

Key Points:

• Definition: Parkinson's disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, a critical brain region for motor control.

• Symptoms:

  • Tremors.

  • Rigidity and slowed movement.

  • Postural instability.

• Neuronal Degeneration:

  • Dopaminergic neuron loss leads to impaired dopamine signaling, essential for motor coordination.

Explanation of Visuals:

• Top Image: Diagram of a healthy vs. Parkinson's-affected brain shows shrinkage and discoloration in the substantia nigra.

• Middle Image: Microscopic views highlighting degeneration in dopaminergic neurons.

Glossary:

• Substantia Nigra: A brain region involved in movement control.

• Dopaminergic Neurons: Nerve cells that produce dopamine, a neurotransmitter critical for motor and emotional functions.

Key Takeaway: Parkinson's disease results from the progressive loss of dopamine-producing neurons, leading to motor and non-motor symptoms.

Slide 25: Dopamine Neurons Derived from Human ES Cells

Key Points:

• Stem Cell Therapy Potential:

  • Human embryonic stem (ES) cells can be used to generate dopaminergic neurons.

  • These neurons are transplanted into animal models of Parkinson's disease to restore motor functions.

• Clinical Findings:

  • Successful transplantation of ES-derived neurons leads to a reduction in motor symptoms.

Explanation of Visuals:

• Graph: Displays improvement in rotational behavior in animal models after transplantation.

  • Blue line: Rotational behavior decreases (improvement).

  • Red line: Dopamine secretion increases over time, indicating neuronal integration.

Glossary:

• Embryonic Stem Cells (ES Cells): Pluripotent cells capable of developing into any cell type, including dopaminergic neurons.

• Rotational Behavior Test: A method to assess motor function in Parkinson's disease animal models.

Key Takeaway: Dopaminergic neurons derived from human ES cells show promise in restoring motor functions in Parkinson's models.

Slide 26: Discussion on ES Cell Research

Key Points:

• Ethical Debate: The use of embryonic stem cells for research and therapy has sparked ethical, religious, and political discussions.

• Milestone Event: In September 2001, U.S. President George W. Bush met Pope John Paul II to discuss the ethical implications of human ES cell research.

• Balance of Promise and Ethics:

  • Promising medical applications versus ethical concerns over the use of human embryos.

Explanation of Visuals:

• Photograph: Historical meeting between President Bush and Pope John Paul II to deliberate on ES cell research.

Glossary:

• Ethical Debate: Discussions on moral implications of using human embryos for research.

• ES Cell Research: Investigation of pluripotent stem cells derived from early embryos.

Key Takeaway: The use of ES cells raises significant ethical questions despite their vast potential in treating diseases like Parkinson's.

Slide 27: Do We Really Need ES Cell Research?

Key Points:

• Importance of ES Cells:

  • ES cells can differentiate into any cell type, making them invaluable for studying disease mechanisms and developing therapies.

  • They are currently unmatched by adult stem cells in pluripotency and therapeutic potential.

• Challenges:

  • Ethical concerns limit research opportunities.

  • Alternatives like induced pluripotent stem cells (iPSCs) are being explored but still lack the maturity of ES cell applications.

Explanation of Visuals:

• Microscopic Image: Depicts a human ES cell colony, showing undifferentiated cells capable of dividing and forming various tissues.

Glossary:

• Induced Pluripotent Stem Cells (iPSCs): Adult cells reprogrammed to a pluripotent state to bypass ethical concerns.

• Pluripotency: The ability of a cell to differentiate into any type of tissue.

Key Takeaway: While promising alternatives like iPSCs are emerging, ES cell research remains critical for understanding and treating complex diseases.

Slide 28: Do We Really Need Embryonic Stem Cells?

Key Points:

• Induced Pluripotent Stem Cells (iPSCs): Scientists have demonstrated that fibroblasts (skin cells) can be reprogrammed into pluripotent stem cells without the need for embryos.

• Yamanaka Factors: The reprogramming process involves introducing four key transcription factors, they just OVEREXPRESSED those 4 factors:

  • OCT4: Maintains self-renewal and pluripotency.

  • SOX2: Essential for maintaining embryonic stem cell identity.

  • KLF4: Involved in cell proliferation and survival.

  • c-MYC: Enhances cell proliferation and reprogramming efficiency.

• Mouse Model Validation:

  • Reprogrammed fibroblasts were integrated into embryos, resulting in chimeric mice containing iPS-derived cells.

  • These mice developed normally, demonstrating the pluripotency of iPSCs.

• Ethical and Practical Advantages:

  • iPSCs eliminate the need to use embryos, addressing ethical concerns.

  • They offer personalized therapeutic applications without immune rejection.

Explanation of Visuals:

• Top-left diagram: Depicts the process of reprogramming fibroblasts to iPSCs and their contribution to mouse embryos.

• Middle image: Shows a chimeric mouse with fluorescently labeled cells derived from iPSCs.

• Bottom images: Various embryonic developmental stages containing iPS-derived cells.

Glossary:

• iPSCs (Induced Pluripotent Stem Cells): Somatic cells reprogrammed to an embryonic-like state.

• Chimeric Mouse: A mouse containing both host and donor-derived cells, confirming pluripotency.

• Fibroblasts: Cells found in connective tissue that produce collagen and structural framework.

Key Takeaway: iPSCs created using the Yamanaka factors offer a promising alternative to embryonic stem cells, enabling advancements in regenerative medicine without ethical concerns.

Slide 29: Human iPS Cells

Key Points:

• Human Fibroblasts Reprogrammed into iPSCs: Human skin cells have been successfully converted into pluripotent stem cells using the Yamanaka factors.

• Validation of Pluripotency:

  • iPSCs express key pluripotency markers such as OCT4, SOX2, and NANOG.

  • They can differentiate into all three germ layers—ectoderm, mesoderm, and endoderm.

• Potential Applications:

  • iPSCs provide a valuable tool for regenerative medicine, disease modeling, and personalized therapies.

• Pluripotency Testing:

  • In vivo teratoma assays confirm the differentiation potential of iPSCs, producing tissues from all germ layers.

Explanation of Visuals:

• Top images: Comparison of fibroblasts and derived iPSCs under a microscope.

• Middle section: Immunofluorescence staining highlights the expression of pluripotency markers.

• Bottom histology images: Teratoma formation assay showing differentiation into various tissues (e.g., neurons, muscle, gut).

Glossary:

• Yamanaka Factors: A set of transcription factors (OCT4, SOX2, KLF4, c-MYC) used to induce pluripotency in somatic cells.

• Pluripotency Markers: Proteins (e.g., NANOG, OCT4, SOX2) that indicate the potential of cells to develop into any cell type.

• Teratoma Formation: A test in which injected iPSCs form tumors containing tissues from all germ layers.

Key Takeaway: Human iPSCs generated with Yamanaka factors can differentiate into diverse cell types, providing a powerful tool for medical research and therapy without relying on embryonic stem cells.

Slide 30: Reprogramming Somatic Cells to a Pluripotent State

Key Points:

• Human Fibroblasts Reprogrammed into iPSCs: Human skin cells have been successfully converted into pluripotent stem cells using the Yamanaka factors.

• Validation of Pluripotency:

  • iPSCs express key pluripotency markers such as OCT4, SOX2, and NANOG.

  • They can differentiate into all three germ layers—ectoderm, mesoderm, and endoderm.

• Potential Applications:

  • iPSCs provide a valuable tool for regenerative medicine, disease modeling, and personalized therapies.

• Pluripotency Testing:

  • In vivo teratoma assays confirm the differentiation potential of iPSCs, producing tissues from all germ layers.

Explanation of Visuals:

• Top images: Comparison of fibroblasts and derived iPSCs under a microscope.

• Middle section: Immunofluorescence staining highlights the expression of pluripotency markers.

• Bottom histology images: Teratoma formation assay showing differentiation into various tissues (e.g., neurons, muscle, gut).

Glossary:

• Yamanaka Factors: A set of transcription factors (OCT4, SOX2, KLF4, c-MYC) used to induce pluripotency in somatic cells.

• Pluripotency Markers: Proteins (e.g., NANOG, OCT4, SOX2) that indicate the potential of cells to develop into any cell type.

• Teratoma Formation: A test in which injected iPSCs form tumors containing tissues from all germ layers.

Key Takeaway: Human iPSCs generated with Yamanaka factors can differentiate into diverse cell types, providing a powerful tool for medical research and therapy without relying on embryonic stem cells.

Slide 31: Reprogramming: Sequential Activation of Pluripotency Markers

Key Points:

• Sequential Activation of Pluripotency Markers:

  • Fibroblasts are reprogrammed into induced pluripotent stem cells (iPSCs) through the introduction of transcription factors (e.g., Yamanaka factors).

  • Activation of pluripotency markers occurs in a stepwise fashion over approximately 30 days.

  • Early markers: Alkaline phosphatase (AP) and SSEA1 are expressed first.

  • Later markers: Key transcription factors Oct4 and Nanog appear later in the reprogramming process, indicating successful induction of pluripotency.

  • Transgene expression is required throughout the reprogramming process to achieve pluripotency.

• Experimental Approach:

  • Fibroblasts carrying fluorescent reporters for pluripotency genes (e.g., Oct4-GFP, Nanog-GFP) are infected with viruses carrying reprogramming factors.

  • The reprogramming process is tracked over time using fluorescence to monitor the activation of pluripotency markers.

  • GFP expression becomes detectable at different time points, reflecting the stochastic nature of reprogramming.

  • Some cells acquire pluripotency markers earlier, while others require more time.

Explanation of Visuals:

• Panel A:

  • A timeline of marker activation from day 0 to day 30, showing progressive expression of different pluripotency markers (AP, SSEA1, Oct4, Nanog).

  • The black bar indicates the period of transgene expression required for reprogramming.

• Panel B:

  • A schematic representation of the experimental workflow:

    1. Fibroblasts with Oct4-GFP/Nanog-GFP are infected with viral factors.

    2. Reprogramming is tracked at various time points (day 6, 12, 20, 25, and 31).

    3. Cells that successfully reprogram express GFP at different times, reflecting variability in reprogramming efficiency.

Glossary:

• Pluripotency Markers: Proteins expressed in stem cells that indicate their ability to differentiate into various cell types.

• Oct4 and Nanog: Key transcription factors necessary for maintaining stem cell pluripotency.

• SSEA1 (Stage-Specific Embryonic Antigen-1): A surface marker commonly used to identify early pluripotent cells.

• GFP (Green Fluorescent Protein): A marker used to visualize gene expression in living cells.

• Transgene Expression: The artificial introduction of genes to induce cellular changes.

Key Takeaway: The reprogramming of fibroblasts into iPSCs is a gradual and stochastic process, involving the sequential activation of pluripotency markers, with some cells responding faster than others.

Slide 32: Four Strategies to Induce Reprogramming of Somatic Cells

Key Points:

• Methods to Induce Reprogramming:

  1. Nuclear Transfer: Transplanting somatic cell nuclei into enucleated oocytes.

  2. Cell Fusion: Fusing somatic cells with embryonic stem cells to induce pluripotency.

  3. Explant Culture: Culturing somatic cells under specific conditions to promote reprogramming.

  4. Direct Reprogramming: Introducing specific transcription factors (Oct4, Sox2, etc.) to somatic cells.

• Each method varies in complexity and efficiency, with direct reprogramming being the most commonly used.

Explanation of Visuals:

• The diagram illustrates the four reprogramming techniques, highlighting the pathways and outcomes for each method (e.g., pluripotent state).

Glossary:

• Nuclear Transfer: Technique of transferring a cell nucleus into another cell without a nucleus.

• Explant Culture: Growth of tissue outside the body to study cell behavior.

Key Takeaway: Multiple strategies can induce reprogramming of somatic cells, with direct reprogramming being a preferred, efficient method using transcription factors.

Slide 33: Patient-Specific Disease Modeling

Key Points:

• Purpose of Patient-Specific iPSCs:

  • iPSCs are used to create specialized cells for modeling diseases like Diabetes, Crohn’s Disease, and Parkinson’s Disease.

  • Applications include drug testing, biochemical analysis, and gene expression profiling.

• Personalized Approach:

  • iPSCs allow the study of diseases in a patient-specific context, offering tailored therapeutic insights.

Explanation of Visuals:

• The diagram shows differentiated cells derived from iPSCs, including muscle, nerve, and pancreatic beta cells, emphasizing their role in disease modeling.

Glossary:

• Beta Cells: Cells in the pancreas responsible for insulin production.

• Expression Analysis: Technique for studying gene expression patterns in cells.

Key Takeaway: Patient-specific iPSCs enable the modeling of complex diseases, facilitating personalized drug testing and understanding disease mechanisms.

Slide 34: A Model for Neural Development and Treatment of Rett Syndrome

Key Points:

• Rett Syndrome:

  • A neurological disorder caused by mutations in the MECP2 gene.

  • iPSCs are used to model neural development in Rett Syndrome patients.

• Study Outcomes:

  • Research demonstrates altered neuronal growth and differentiation in iPSCs from Rett Syndrome patients.

  • Provides insights into potential therapeutic approaches.

Explanation of Visuals:

• The image displays the title and methodology of the study.

• The process of deriving neurons from patient-specific iPSCs is highlighted.

Glossary:

• MECP2 (Methyl-CpG-Binding Protein 2): A gene implicated in neurological development.

• Neural Development: The process of growth and differentiation of neurons.

Key Takeaway: iPSC models provide valuable tools for understanding Rett Syndrome and testing potential treatments for neuronal dysfunction.

Slide 35: Control iPSCs vs. Rett Syndrome iPSCs

Key Points:

• Comparison of Control vs. Disease iPSCs:

  • Rett Syndrome iPSCs show reduced neuronal differentiation, smaller soma size, and altered synapse numbers compared to control iPSCs.

• Implications:

  • The findings highlight the cellular-level deficits in Rett Syndrome, offering targets for therapeutic intervention.

Explanation of Visuals:

• The comparison chart and images illustrate differences in differentiation, soma size, and synapse density between control and Rett Syndrome iPSCs.

Glossary:

• Soma Size: The size of the cell body of a neuron.

• Synapse: The junction where neurons communicate.

Key Takeaway: Control iPSCs and Rett Syndrome iPSCs exhibit distinct differences in neuronal development, emphasizing the utility of iPSC models in studying and treating neurological disorders.

Slide 36: Modeling Schizophrenia Using Human Induced Pluripotent Stem Cells

Key Points:

One of the challenges of embryonic stem (ES) cell therapy is the uncertainty of the genetic factors underlying the disease. In conditions like schizophrenia, the genetic basis is complex and not fully understood, making it difficult to predict how transplanted stem cells might behave or whether they could be affected by the same underlying genetic predispositions.

• Schizophrenia Modeling:

  • iPSCs derived from schizophrenia patients are used to investigate neuronal abnormalities associated with the disorder.

• Neuronal Differences:

  • Schizophrenia iPSCs show differences in neuronal growth, connectivity, and activity compared to normal iPSCs.

• Applications:

  • iPSCs provide a platform to study schizophrenia-specific neuronal changes and test treatments.

Explanation of Visuals:

• Microscopic images compare neurons derived from schizophrenia and normal iPSCs, showing differences in structure and markers (e.g., MAP2 and SOX2 staining).

• Graphs depict reduced synaptic activity and altered electrophysiological properties in schizophrenia iPSCs.

Glossary:

• MAP2 (Microtubule-Associated Protein 2): A protein marker for mature neurons.

• SOX2: A marker for stemness and neuronal progenitors.

• Electrophysiology: The study of electrical activity in neurons.

Key Takeaway: Human iPSCs reveal schizophrenia-related neuronal deficits, offering insights into disease mechanisms and potential therapeutic interventions.

Slide 37: Neuronal Function in Schizophrenia iPSCs

Key Points:

• Functional Deficits:

  • Schizophrenia iPSCs show significant impairments in:

    • Synaptic activity.

    • Neuronal connectivity.

    • Action potential firing.

• Therapeutic Testing:

  • These models allow researchers to test drugs aimed at restoring normal neuronal function.

Explanation of Visuals:

• Multiple bar graphs and traces highlight:

  • Reduced synaptic signals.

  • Decreased action potential frequencies.

  • Altered neuronal excitability in schizophrenia iPSCs.

Glossary:

• Action Potential: A brief electrical impulse that represents neuron firing.

• Synaptic Activity: Communication between neurons at synapses.

Key Takeaway: Functional deficits in schizophrenia iPSCs provide a model to understand disease mechanisms and explore potential therapeutic drugs.

Slide 38: Development of Specialized Cells

Key Points:

• Process Overview:

  • Patient-derived cells can be reprogrammed into iPSCs and differentiated into specialized cell types for therapeutic use.

  • Examples include neurons, beta cells (for diabetes), and cardiac cells.

• Personalized Medicine:

  • This approach enables tailored treatments for individual patients.

Explanation of Visuals:

• A diagram illustrates the process of:

  1. Obtaining a patient biopsy.

  2. Reprogramming cells into iPSCs.

  3. Differentiating them into specific cell types for therapy or research.

Glossary:

• Beta Cells: Insulin-producing cells of the pancreas.

• Cardiac Cells: Cells that make up heart tissue.

Key Takeaway: iPSCs can be used to generate specialized cells, advancing personalized medicine and regenerative therapies.

Slide 39: Human iPSC-Derived Dopaminergic Neurons in Parkinson’s Model

Key Points:

• Parkinson’s Treatment:

  • Dopaminergic neurons derived from human iPSCs were transplanted into a primate model of Parkinson’s disease.

  • Results showed improved motor functions and dopamine production.

• Therapeutic Promise:

  • iPSC-derived neurons offer potential for treating neurodegenerative diseases.

Explanation of Visuals:

• A graph compares motor performance between treated and untreated Parkinson’s primates, showing significant improvement in the treated group.

• The methodology illustrates iPSC differentiation into dopaminergic neurons and subsequent transplantation.

Glossary:

• Dopaminergic Neurons: Neurons that produce dopamine, a key neurotransmitter.

• Motor Functions: Movements controlled by the central nervous system.

Key Takeaway: iPSC-derived dopaminergic neurons show therapeutic efficacy in improving motor functions in Parkinson’s models, highlighting their clinical potential.

Slide 40: Introduction to Somatic Stem Cells

Key Points:

• Somatic Stem Cells Overview:

  • These are stem cells found in various tissues of the adult body.

  • Responsible for tissue maintenance, repair, and regeneration.

• Heart as an Example:

  • Somatic stem cells contribute to the repair of heart tissue, although their regenerative capacity may be limited compared to embryonic stem cells.

• Therapeutic Potential:

  • They hold promise for treating degenerative diseases and injuries.

Explanation of Visuals:

• The image shows the human heart and vasculature, emphasizing the role of somatic stem cells in cardiac tissue repair.

Glossary:

• Somatic Stem Cells: Stem cells that reside in adult tissues and can differentiate into specific cell types.

• Regeneration: The process of tissue repair and renewal.

Key Takeaway: Somatic stem cells are crucial for maintaining and repairing tissues in the adult body, with potential applications in regenerative medicine.

Slide 41: Part 2: Somatic Stem Cells

Key Points:

• Somatic Stem Cell Distribution:

  • Somatic stem cells are present in various organs such as the brain, liver, intestine, skin, and bone marrow, where they support tissue maintenance and repair.

  • Each organ contains specialized stem cells that are adapted to meet the organ’s unique regenerative requirements.

  • These stem cells have a more restricted differentiation potential compared to embryonic stem cells, primarily giving rise to specific cell types within their tissue of origin.

• Importance of Studying Somatic Stem Cells:

  • Research into somatic stem cells holds promise for developing regenerative medicine therapies to treat degenerative diseases and injuries.

  • Understanding their mechanisms can help enhance wound healing, organ regeneration, and cell-based therapies for conditions such as liver cirrhosis, neurodegeneration, and blood disorders.

Explanation of Visuals:

• The diagram visually represents different organs where somatic stem cells are located, such as the intestine, lungs, skin, and bones, showing their diverse presence and potential roles in tissue homeostasis.

• Arrows indicate the direction of cell differentiation within each organ system.

Glossary:

• Somatic Stem Cells: Adult stem cells found in specific tissues responsible for repair and maintenance of that tissue.

• Differentiation Potential: The ability of stem cells to transform into specific cell types within their respective tissues.

• Regenerative Medicine: A field of medicine focused on replacing or regenerating human cells, tissues, or organs to restore normal function.

Key Takeaway:
Somatic stem cells are integral to maintaining and repairing tissues in various organs, offering potential therapeutic applications in regenerative medicine.

Slide 42: Santiago Ramón y Cajal’s Perspective on Regeneration

Key Points:

• Historical Belief:

  • Ramón y Cajal, a pioneering neuroscientist, believed that the adult nervous system was incapable of regeneration.

  • He famously stated that nerve paths in adults are fixed and unchangeable.

• Modern Understanding:

  • Research has since disproven this belief, showing that adult neural stem cells exist and contribute to brain plasticity.

Explanation of Visuals:

• The image shows Santiago Ramón y Cajal, highlighting his contributions to neuroscience.

• His quote reflects early misconceptions about neural regeneration.

Glossary:

• Neurogenesis: The formation of new neurons in the brain.

• Plasticity: The brain's ability to adapt and reorganize.

Key Takeaway: While early scientists believed the adult nervous system could not regenerate, modern findings have revealed the presence of neural stem cells and their regenerative capabilities.

Slide 43: Early Evidence of Cell Division in the Adult Brain

Key Points:

• Key Discovery:

  • In 1964, researchers provided the first evidence of cell division in the adult brain.

  • This overturned the belief that neurogenesis is restricted to development.

• Implications:

  • Opened new avenues for studying brain plasticity and regenerative medicine.

Explanation of Visuals:

• The figure from a 1964 Nature publication shows dividing cells in the adult brain, marking a groundbreaking discovery in neuroscience.

Glossary:

• Neural Stem Cells: Stem cells in the brain capable of generating neurons and glial cells.

• Glial Cells: Support cells in the nervous system that protect and nourish neurons.

Key Takeaway: The discovery of adult brain cell division revolutionized our understanding of neurogenesis and brain repair.

Slide 44: Analysis of Adult Neurogenesis

Key Points:

• BrdU Labeling Technique:

  • Bromodeoxyuridine (BrdU) is incorporated into the DNA of dividing cells during the S-phase of the cell cycle.

  • Antibodies specific to BrdU allow for visualization of newly formed neurons.

  • This method is widely used to track neurogenesis in the adult brain.

• Fluorescence Imaging:

  • Cells labeled with BrdU can be co-stained with neuronal markers such as NeuN to confirm neuronal identity.

  • The overlap of BrdU (red) and NeuN (green) in the provided image indicates successful neurogenesis.

Explanation of Visuals:

• The left side of the slide illustrates the mechanism of BrdU incorporation into DNA and subsequent detection via antibodies.

• The right side presents a microscopic image showing BrdU-labeled cells in red and neuronal marker NeuN in green, with colocalization appearing as yellow.

Glossary:

• BrdU (Bromodeoxyuridine): A synthetic thymidine analog that integrates into newly synthesized DNA strands.

• NeuN: A neuronal nuclear protein used as a marker for mature neurons.

• Neurogenesis: The process of forming new neurons in the brain.

Key Takeaway:
BrdU labeling allows for tracking of new neuron formation in the adult brain, helping to study neurogenesis and its implications for brain function and repair.

Slide 45: Plasticity and Repair of the Mammalian Brain

Key Points:

• Neuroplasticity and Brain Adaptation:

  • The brain can reorganize and adapt in response to various stimuli such as experience, learning, and injury.

  • Structural

    and functional changes in neural networks enable adaptation to environmental demands.

• Hippocampus as a Key Brain Region:

  • The hippocampus is crucial for memory formation and spatial navigation.

  • The analogy to "Asterix & Obelix's village" suggests its role as a central hub for cognitive functions and protection against neurological challenges.

Explanation of Visuals:

• The left side of the slide demonstrates how experience, learning, and injury influence brain plasticity.

• The right side humorously compares the hippocampus to a fortified village (Asterix & Obelix), emphasizing its protective and adaptive functions.

Glossary:

• Neuroplasticity: The brain's ability to reorganize itself by forming new neural connections throughout life.

• Hippocampus: A brain region associated with memory, learning, and spatial navigation.

• Cognitive Function: The mental process that includes thinking, learning, and memory.

Key Takeaway:
The mammalian brain exhibits remarkable plasticity, allowing it to adapt to experience and injury through structural and functional changes, with the hippocampus playing a central role in memory and learning.

Slide 46: Life-Long Neurogenesis in Humans

Key Points:

• Neurogenesis in the Adult Brain:

  • Life-long neurogenesis occurs in the hippocampus of mammals, including humans, though the extent is debated.

  • Studies have reported the presence of immature neurons (DCX+ cells) in the dentate gyrus of older individuals.

• Function of Adult Neurogenesis:

  • It plays a role in learning and memory, contributing to cognitive flexibility and adaptation to new experiences.

• Neurogenesis and Neuropsychiatric Disorders:

  • Altered or reduced neurogenesis is linked to disorders such as depression and aging-related cognitive decline.

Explanation of Visuals:

• The left side of the slide shows anatomical illustrations of the rodent and human brain, highlighting the hippocampal dentate gyrus.

• The right side features an image of immature neurons (DCX+ labeled) in the hippocampus, indicating ongoing neurogenesis in an aging brain.

Glossary:

• Hippocampus: A brain region critical for memory and learning.

• DCX (Doublecortin): A marker protein used to identify newly formed neurons.

• Neurogenesis: The process of generating new neurons from neural stem cells.

Key Takeaway:
Adult neurogenesis persists in the hippocampus and is crucial for cognitive functions, but it may decline with age and neuropsychiatric conditions.

Slide 47: Dynamic Regulation of Adult Neurogenesis

Key Points:

• Influence of Environment and Lifestyle:

  • Factors such as physical exercise, enriched environments, and cognitive stimulation can enhance neurogenesis.

  • Conversely, stress and aging can negatively impact the generation of new neurons.

• Behavioral Paradigms Used in Research:

  • Running wheels, enriched housing, and spatial learning tasks (e.g., maze tests) are used to study neurogenesis regulation.

• Neuronal Adaptation:

  • Newly generated neurons can integrate into existing circuits, aiding in functional recovery and adaptation.

Explanation of Visuals:

• The images show experimental setups used to study neurogenesis, such as running wheels for exercise and maze tests for cognitive stimulation.

• The upward arrow towards neurons suggests enhanced neurogenesis in response to positive environmental stimuli.

Glossary:

• Enriched Environment: A setting that provides sensory, cognitive, and physical stimulation.

• Plasticity: The brain's ability to reorganize and adapt by forming new neural connections.

• Cognitive Stimulation: Activities that challenge the brain to enhance mental function.

Key Takeaway:
Adult neurogenesis is dynamically regulated by environmental and lifestyle factors, which can either enhance or impair neural regeneration.

Slide 48: Dynamic Regulation of Adult Neurogenesis

Key Points:

• Adaptability of Neurogenesis:

  • Adult neurogenesis dynamically responds to external and internal influences, including:

    • Enriched environments, physical activity, and dietary factors (promote neurogenesis).

    • Aging, chronic stress, and disease conditions (suppress neurogenesis).

• Neuronal Morphology:

  • Newly formed neurons develop complex structures, such as dendrites, contributing to connectivity and function in neural circuits.

Explanation of Visuals:

• Left panel: Illustrations and images show external factors influencing neurogenesis (e.g., exercise, lifestyle changes).

• Right panel: A schematic representation of newly developed neurons with branching dendrites, emphasizing their integration into neural circuits.

Glossary:

• Neurogenesis: The process of generating new neurons in the brain.

• Dendrites: Tree-like extensions of neurons that receive signals from other cells.

Key Takeaway: Adult neurogenesis is a flexible process influenced by lifestyle and environmental factors, with potential implications for brain health and function.

Slide 49: Hippocampal Neurogenesis in 3D Visualization

Key Points:

• 3D Imaging of Neurons:

  • Advanced imaging techniques reveal detailed 3D structures of neurons in the hippocampus.

  • Highlights neuronal connections in specific subregions, such as CA3 and dentate gyrus.

• Role in Learning and Memory:

  • These regions are critical for spatial navigation, memory formation, and pattern separation.

Explanation of Visuals:

• 3D rendered image of hippocampal neurons shows their intricate structures and spatial relationships in the CA3 region.

• Neurons are labeled with green fluorescent proteins for visualization.

Glossary:

• CA3: A subregion of the hippocampus involved in forming and recalling memories.

• Fluorescent Protein (e.g., GFP): A protein that emits light, used to visualize cells under a microscope.

Key Takeaway: 3D imaging of hippocampal neurons provides insight into their complex structures and roles in learning and memory.

Slide 50: Hippocampal Neurogenesis: Far Away

Key Points:

• Visualizing Neurogenesis:

  • Advanced imaging methods, such as Nestin-GFP labeling, allow visualization of neural stem cells in the hippocampus.

  • Focuses on regions like the dentate gyrus where neurogenesis is prominent.

• Connectivity Across Brain Regions:

  • Demonstrates connections between the hippocampus, cortex, and other regions, indicating the integration of new neurons into broader circuits.

Explanation of Visuals:

• Left image: Highlights specific brain regions (e.g., cortex, corpus callosum, dentate gyrus).

• Right image: Shows green fluorescent-labeled neurons (Nestin-GFP) in the hippocampus.

Glossary:

• Nestin: A protein expressed in neural stem cells, used as a marker for neurogenesis.

• Corpus Callosum: A bundle of nerve fibers connecting the two hemispheres of the brain.

Key Takeaway: Advanced imaging methods enable precise visualization of neural stem cells and their integration into brain circuits.

Slide 51: Using Intravital Imaging to Follow Neural Stem Cells

Key Points:

• Intravital Imaging Approach:

  • A specialized imaging technique is used to track neural stem cells within living organisms over time.

  • The technique involves the use of a 1040-nm laser beam and a high-magnification objective to visualize cells deep within the brain.

  • A cranial window is implanted in the skull, allowing for repeated imaging sessions without additional surgical procedures.

• Experimental Model:

  • The genetic model used for tracking cells is Ascl1CreERT² / tdTOMATO, which labels neural stem cells with a fluorescent marker (tdTOMATO) to facilitate their visualization.

  • The imaging timeline consists of a 2-week waiting period post-tamoxifen (Tam) induction, followed by regular imaging sessions over 60 days.

• Observations:

  • Two types of cells are visualized during the study:

    • R cells (Radial cells): Show characteristic morphology and positioning within the dentate gyrus (DG).

    • NR cells (Non-radial cells): Appear in different locations, such as the granular cell layer (GCL), with distinct properties.

  • Time-lapse imaging helps to monitor cell movement, proliferation, and differentiation dynamics.

Explanation of Visuals:

• Left side: Diagram of the intravital imaging setup, illustrating the positioning of the microscope objective over a surgically implanted cranial window in the hippocampus, targeting areas such as CA1 and DG (dentate gyrus).

• Right side: Microscopic images of R and NR cells, highlighting their morphology and position in the brain. A timeline at the bottom indicates the imaging schedule over the course of 60 days.

Glossary:

• Intravital Imaging: A technique that allows for real-time visualization of cellular processes in living organisms.

• Cranial Window: A transparent implant placed in the skull to facilitate long-term imaging of the brain.

• Tamoxifen (Tam): A drug used to activate CreERT² recombinase, leading to gene expression in specific cells.

• tdTOMATO: A fluorescent protein used as a marker to visualize cells in live imaging experiments.

• Dentate Gyrus (DG): A part of the hippocampus involved in neurogenesis and memory formation.

• Radial Cells: Neural stem cells in the dentate gyrus that have a radial glial-like morphology, characterized by an elongated process extending from the cell body to the brain surface, playing a role in guiding newborn neurons.

• Non-Radial Cells: Neural progenitor cells that lack the elongated radial process and exhibit different migration and differentiation properties compared to radial cells.

Key Takeaway:
Intravital imaging provides a powerful method for tracking the behavior of neural stem cells over time, allowing researchers to study their role in brain plasticity and regeneration.

Slide 53: Constructing Lineage Trees of Adult Neural Stem Cells

Key Points:

• Lineage Mapping of Stem Cells:

  • Traces the division history and differentiation paths of adult neural stem cells (NSCs).

  • Lineage trees illustrate how one NSC can give rise to multiple cell types.

• Insights from Imaging:

  • Use of advanced imaging techniques (e.g., live-cell tracking) to observe NSC divisions and lineage transitions.

Explanation of Visuals:

• Left panel: Sequential images show dividing NSCs and their progeny.

• Right panel: Lineage trees represent cell divisions over time, highlighting the diversity of progeny from a single NSC.

Glossary:

• NSCs (Neural Stem Cells): Cells in the brain capable of self-renewal and differentiation into neurons, astrocytes, and oligodendrocytes.

• Lineage Tree: A graphical representation of cell divisions and differentiation pathways.

Key Takeaway: Lineage trees of adult NSCs reveal the complex dynamics of stem cell divisions and the diversity of cell types they produce.

Slide 54: One Mother and Her Daughters

Key Points:

• Aspects of Neural Stem Cell (NSC) Research:

  • Stem cell dynamics: Understanding how NSCs behave over time.

  • Mode of cell division: Investigating whether NSCs divide symmetrically or asymmetrically.

  • Migration and integration: How newly formed neurons migrate and incorporate into brain circuits.

  • Functional properties: Evaluated using genetically encoded calcium indicators (GECIs).

  • Self-renewal potential: The ability of NSCs to sustain neurogenesis over time.

• Evidence from Studies:

  • Some studies suggest long-term self-renewal, indicating NSCs can persist and continuously produce new cells.

  • Other studies report quick depletion, implying that the NSC pool may diminish rapidly under certain conditions.

Explanation of Visuals:

• A fluorescent image of an NSC (mother) and its progeny (daughters), highlighting the extensive branching of newly generated neurons.

• A textual summary of research findings that support either sustained NSC activity or rapid depletion.

Glossary:

• Self-Renewal: The process by which NSCs produce identical stem cells to maintain their population.

• Migration: The movement of newly formed neurons to different regions of the brain.

• GECIs (Genetically Encoded Calcium Indicators): Tools used to monitor cellular activity in live cells.

Key Takeaway:
Understanding NSC behavior and their ability to self-renew is crucial for developing therapies aimed at neurodegenerative diseases.

Slide 55: Distinct Behavior of NSCs in the Hippocampus

Key Points:

• Comparing NSC Behaviors Using Different Genetic Lineages:

  • Ascl1CreERT² (Neurogenic Burst):

    • NSCs show a rapid burst of activity, leading to a depletion of the stem cell pool.

    • Associated with transient, high neurogenesis.

  • GliCreERT² (Niche Maintenance):

    • NSCs are maintained over time, contributing to a steady rate of neurogenesis.

    • Helps preserve the neural stem cell niche for long-term function.

• Lineage Tracing of NSCs:

  • Tracking NSC fate in the hippocampus reveals differences in their long-term contributions to neurogenesis.

  • Clonal analysis shows variation in the retention and depletion of NSCs.

Explanation of Visuals:

• Graphs Comparing NSC Dynamics:

  • The left graph (Ascl1CreERT²) illustrates a steep increase in neuron generation followed by depletion.

  • The right graph (GliCreERT²) shows a slower, sustained production of new neurons over time.

• Scatter Plot:

  • Demonstrates the proportion of NSCs retained over time between the two genetic models, highlighting the longevity of GliCreERT² cells compared to Ascl1CreERT².

Glossary:

• Neurogenic Burst: A phase of rapid NSC division and differentiation.

• Niche Maintenance: The ability of NSCs to persist and contribute steadily to neurogenesis over time.

• Lineage Tracing: A method used to track the fate of cells over time in a living organism.

Key Takeaway:
NSCs exhibit different behaviors depending on their genetic lineage; some rapidly differentiate and deplete, while others maintain a stable stem cell pool to ensure long-term neurogenesis..

Slide 56: Molecular Signature of Diverse Hippocampal Stem Cells

Key Points:

• Single-Cell Analysis:

  • Reveals distinct molecular profiles of hippocampal stem cells.

  • Identifies unique gene expression patterns associated with specific NSC behaviors.

• Functional Specialization:

  • NSCs exhibit molecular diversity, influencing their role in neurogenesis or niche support.

Explanation of Visuals:

• Left panel: Diagrams show experimental setups for single-cell RNA sequencing.

• Right panel: Scatter plots illustrate clusters of NSCs based on gene expression profiles, highlighting functional diversity.

Glossary:

• Single-Cell RNA Sequencing: A technique to analyze gene expression in individual cells.

• Molecular Signature: A unique set of genes expressed by a cell, reflecting its function.

Key Takeaway: Molecular profiling of hippocampal NSCs highlights their diversity and specialized roles in maintaining brain health.

Slide 57: Diversity of Hippocampal Stem Cells

Key Points:

• Functional Diversity:

  • Hippocampal neural stem cells (NSCs) exhibit molecular and functional diversity.

• Long-Term Self-Renewal:

  • Some hippocampal NSCs can self-renew for over 100 days.

• Research Questions:

  • How do these NSCs contribute to life-long neurogenesis?

  • How do their dynamics evolve with aging?

Explanation of Visuals:

• Diagram: Shows the differentiation pathway of hippocampal stem cells into astrocytes, proliferative progenitors, and neurons, emphasizing their diverse roles.

Glossary:

• Hippocampus: A brain region involved in memory and learning.

• Neurogenesis: The process of generating new neurons from NSCs.

Key Takeaway: Hippocampal NSCs exhibit diverse functional roles, including sustaining life-long neurogenesis, but questions remain about their behavior over time.

Slide 58: Prior History of a Given Cell

Key Points:

• Understanding a cell’s lineage history provides insight into its developmental and functional roles.

• Challenges in Lineage Tracing:

  • Mapping the division and differentiation history of individual cells in complex tissues remains a critical research challenge.

Explanation of Visuals:

• Lineage tree: Represents potential pathways of division and differentiation for a single stem cell.

Glossary:

• Lineage Tracing: A method to determine the ancestry of a cell by tracking its divisions.

Key Takeaway: Tracing the prior history of individual cells is essential to understanding their contributions to development and function.

Slide 59: iCOUNT: Investigating Division History of a Cell

Key Points:

• Purpose of iCOUNT:

  • iCOUNT is a genetic lineage tracing technique used to monitor cell division history by utilizing fluorescent markers.

  • The method involves tracking changes in the expression of fluorescent proteins in histone-tagged cells, providing insights into cell proliferation over time.

• Mechanism of iCOUNT:

  • A genetic construct is introduced into the cell’s genome, incorporating histone H3.1 (H3f3) fused to fluorescent proteins.

  • The construct contains loxP sites flanking the mCherry (red) marker, followed by GFP (green).

  • Activation of Cre recombinase results in the removal of the mCherry sequence, switching the cell's fluorescence from red to green.

  • As cells divide, the proportion of cells expressing GFP increases, providing a measure of the number of divisions.

• Interpretation of Fluorescence:

  • Initially, all cells exhibit red fluorescence (mCherry-tagged histones).

  • After Cre recombinase activation, the switch to GFP occurs progressively across divisions.

  • The distribution of red and green cells indicates cell proliferation and division rates.

Explanation of Visuals:

• Left Diagram:

  • Illustrates how stem cells and their progeny transition from mCherry to GFP, showing lineage tracking within a neural environment.

• Top Right Diagram:

  • Depicts the genetic construct with the histone H3f3 gene before and after Cre recombinase activity, showing the transition from mCherry to GFP labeling.

• Bottom Right Diagram:

  • Represents the progressive dilution of mCherry-labeled histones and the increase of GFP in subsequent cell generations, with percentages indicating division dynamics.

Glossary:

• Histone H3.1 (H3f3): A protein that helps package DNA into chromatin and is used here to monitor cellular replication.

• Cre recombinase: An enzyme that facilitates site-specific recombination at loxP sequences, enabling genetic modifications.

• loxP site: A specific DNA sequence recognized by Cre recombinase to facilitate genetic excision.

• mCherry: A red fluorescent protein used to tag histones for cell tracking.

• GFP (Green Fluorescent Protein): A green marker used to track gene expression and cell lineage.

• Cell Lineage Tracing: A technique to study the progeny of a single cell over time.

Key Takeaway:
iCOUNT is a powerful genetic tool that utilizes histone-tagged fluorescent markers to track cell division history, providing crucial insights into cell proliferation and differentiation in developmental biology.

Slide 60: iCOUNT Results and Analysis

Key Points:

• Fluorescent Analysis:

  • GFP intensity reflects the division history of cells.

  • Increased fluorescence correlates with more divisions.

• Results:

  • Data reveal patterns of division and self-renewal in different cell types.

Explanation of Visuals:

• Graphs: Show fluorescence levels in various cells, indicating their division history.

• Bar charts: Compare division rates and GFP expression across different populations.

Glossary:

• Fluorescence Intensity: The brightness of a fluorescent marker, indicating protein expression or activity.

• Self-Renewal: The ability of a stem cell to divide and maintain its population.

Key Takeaway: The iCOUNT technique quantifies division history, highlighting the self-renewal potential and behavior of different cell types.

Slide 61: iCOUNT in Vivo

Key Points:

• iCOUNT Technology Application:

  • Tracks the division of cells in living organisms using genetic marking and fluorescence.

• Targeted Regions:

  • iCOUNT identifies dividing cells in various brain regions, including the habenula, medial ganglionic eminence (MGE), tegmentum, liver, gut, and somites.

• Markers:

  • Combines fluorescent markers (e.g., mCherry and GFP) to distinguish dividing cell populations.

Explanation of Visuals:

• Left Panel:

  • Cross-sectional image of a mouse brain showing regions labeled with iCOUNT markers.

  • Key areas such as the habenula, MGE, and tegmentum are highlighted.

• Right Panels:

  • Individual panels display fluorescence for mCherry (red) and GFP (green) markers across different tissues:

    • Habenula and MGE: High GFP activity indicating active cell division.

    • Liver and Gut: Dual fluorescence indicating cell proliferation.

    • Somites: Distinct GFP-positive cells reflect specific division activity.

Glossary:

• Habenula: A brain region involved in processing aversive stimuli.

• MGE (Medial Ganglionic Eminence): A structure in the developing brain contributing to interneuron formation.

• Somites: Blocks of mesoderm in vertebrate embryos that develop into skeletal muscles, vertebrae, and dermis.

• mCherry/GFP: Fluorescent proteins used as markers for visualizing cell activity.

Key Takeaway: iCOUNT in vivo highlights dividing cells in multiple tissues, revealing dynamic patterns of cell proliferation and differentiation within living organisms.

Slide 62: iCOUNT in the Adult Brain

Key Points:

• Adult Brain Application:

  • iCOUNT technology is used to track cell division in adult brain cells.

• Genetic Marking:

  • H2B-GFP marker identifies dividing cells, revealing active stem cell populations in specific brain regions.

• Findings:

  • Division dynamics are visualized in the hippocampus using fluorescence imaging.

Explanation of Visuals:

• Fluorescent Images:

  • Upper panels: Highlighted areas show GFP-labeled cells in the hippocampal region.

  • Lower panels: Red and green fluorescence indicate cells in different stages of division.

Glossary:

• H2B-GFP: A fusion protein combining histone H2B and GFP to mark dividing cells.

• TAM (Tamoxifen): A chemical used to activate CreERT2 for lineage tracing.

Key Takeaway: iCOUNT enables visualization and tracking of dividing cells in the adult brain, focusing on regions like the hippocampus where neurogenesis occurs.

Slide 63: Quantitative Analysis of iCOUNT

Key Points:

• Division Frequency:

  • Graphs indicate varying division frequencies across cell populations.

• Cell Type Comparison:

  • Different types of neural stem cells (NSCs) show distinct division patterns.

• Significant Observations:

  • Quantitative data link division activity to specific NSC subpopulations.

Explanation of Visuals:

• Graphs and Heatmaps:

  • Show statistical analysis of division activity across populations.

  • Distribution and intensity of GFP fluorescence reflect division rates.

Glossary:

• Heatmap: A graphical representation of data where individual values are represented by color.

Key Takeaway: Quantitative data from iCOUNT reveal distinct division patterns, advancing our understanding of NSC behavior.

Slide 64: iCOUNT in Human ESC-Derived Organoids

Key Points:

• Human Application:

  • iCOUNT is applied to human embryonic stem cell (ESC)-derived brain organoids.

• Organoid Development:

  • Organoids mimic the development of human brain structures in vitro.

• Observations:

  • GFP labeling highlights cell proliferation and differentiation processes.

Explanation of Visuals:

• Left panel: Graph shows division activity over time.

• Middle and right panels: Fluorescent images illustrate distinct zones of dividing cells in organoids.

Glossary:

• Organoids: Miniature, simplified versions of an organ grown in vitro from stem cells.

• ESC (Embryonic Stem Cells): Pluripotent stem cells derived from early-stage embryos.

Key Takeaway: iCOUNT provides insights into cell division and differentiation within human brain organoids, offering a model for neurodevelopmental research.

Slide 65: Molecular Signature of Previous Cell Divisions

Key Points:

• Objective:

  • Investigating how previous cell divisions impact molecular signatures in neural stem cells (NSCs).

  • The iCOUNT technique is used to track cell division history via fluorescent markers (mCherry and tdTomato).

• Flow Cytometry Analysis:

  • Fluorescence-based sorting distinguishes cells based on their division history.

  • Cells expressing high levels of GFP indicate multiple divisions, while mCherry-positive cells indicate fewer divisions.

• Cell Type Identification via t-SNE Analysis:

  • Different cell populations such as neurons, immature neurons, basal progenitors, and NSCs are identified based on gene expression.

  • Distinct clusters suggest transcriptional differences correlated with division history.

• Key Findings:

  • Dividing and non-dividing NSCs exhibit different transcriptional profiles.

  • Cell division history may influence differentiation potential and cellular identity.

Explanation of Visuals:

• Left Panel: Flow cytometry plots showing the distribution of cells with different division histories (mCherry vs. GFP labeling).

• Right Panel: t-SNE clustering visualizes distinct cell populations based on their gene expression signatures, with neurons and NSCs forming separate clusters.

Glossary:

• t-SNE (t-distributed Stochastic Neighbor Embedding): A dimensionality reduction technique used to visualize high-dimensional data.

• Flow Cytometry: A method to analyze cell populations based on fluorescence and size.

• Basal Progenitors: Neural progenitor cells contributing to neurogenesis.

• Apical Progenitors: Stem cells lining the brain ventricles that generate neurons and glial cells.

Key Takeaway:
Previous cell division events leave distinct molecular signatures that can influence the differentiation and function of neural stem cells.

Slide 66: Functional Significance of Identified Genes/Pathways

Key Points:

• Objective:

  • Exploring how gene expression changes based on division history impact cell function.

  • Focus on the role of specific genes identified through the iCOUNT approach in cellular responses.

• Forebrain Organoids Experiment:

  • Overexpression and CRISPR/Cas9 deletion experiments performed on forebrain organoids to study gene function.

  • Changes in cell proliferation observed at different time points (3, 5, and 7 days).

• Functional Analysis Across Tissues:

  • iCOUNT analysis applied to different tissues, including the hippocampus, hematopoietic stem cells (HSCs), and spleen, showing division-dependent diversity in gene expression.

• Key Findings:

  • Specific genes related to cell division influence response to tissue damage or stress.

  • Functional diversity observed depending on prior division history.

Explanation of Visuals:

• Left Panel: Images and graphs showing cell proliferation in forebrain organoids with gene manipulation.

• Right Panel: Flow cytometry plots comparing gene expression profiles across different tissues (hippocampus, HSCs, and spleen).

Glossary:

• Organoids: 3D cellular models mimicking organ structures and functions.

• CRISPR/Cas9: A gene-editing tool used to delete or modify genes.

• Hematopoietic Stem Cells (HSCs): Stem cells that give rise to blood and immune cells.

Key Takeaway:
Gene expression patterns influenced by cell division history have significant functional implications in various tissues, including responses to stress and injury..

Slide 67: Things to Know

Answers to Key Points:

1. Potency of different types of stem cells:

   • Stem cell potency refers to their ability to differentiate into various cell types. The main types are:

     • Totipotent: Can generate all cell types, including embryonic and extraembryonic tissues (e.g., zygote).

     • Pluripotent: Can differentiate into all three germ layers (ectoderm, mesoderm, endoderm), such as embryonic stem cells (ESCs) and iPSCs.

     • Multipotent: Differentiate into a limited range of cell types within a specific lineage (e.g., neural stem cells in the hippocampus).

     • Unipotent: Can produce only one cell type, such as muscle stem cells.

   • Measuring potency involves assessing the differentiation capacity and division history of cells to predict their future behavior. Techniques such as the iCOUNT method help track the division history and potency of stem cells.

2. What is an embryonic stem cell? What is an induced pluripotent stem cell (iPSC)?

   • Embryonic Stem Cells (ESCs): Derived from the inner cell mass of blastocysts, ESCs are pluripotent and can differentiate into any cell type of the body. They are conventionally used to study diseases and serve as a potential source for regenerative therapies.

   • Induced Pluripotent Stem Cells (iPSCs): Generated by reprogramming somatic cells (e.g., fibroblasts) using the Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC). Overexpression of these factors restores pluripotency, allowing iPSCs to behave similarly to ESCs. Both ESCs and iPSCs are valuable for disease modeling, drug testing, and potential cell replacement therapies.

3. What can you “do” with iPSCs?

   • iPSCs have multiple applications, including:

     • Disease modeling: Studying diseases such as neurodegenerative disorders by creating patient-specific cells.

     • Drug screening: Testing the effects of drugs on patient-derived cells.

     • Regenerative medicine: Providing a source for cell transplantation in diseases like Parkinson's, Alzheimer's, or diabetes.

     • Gene therapy: Introducing genetic corrections in patient-specific cells before re-implantation.

   • iPSCs can help understand diseases by mimicking developmental processes in vitro and predicting treatment responses.

4. Which are the neurogenic niches of the adult brain?

   • The adult brain retains the ability to generate new neurons primarily in two neurogenic niches:

     • Hippocampus (dentate gyrus): Essential for learning and memory, it continuously adds new neurons throughout life.

     • Subventricular zone (SVZ) of the lateral ventricles: Produces new neurons that migrate to the olfactory bulb.

   • Neurogenesis in these niches plays a role in cognitive function, mood regulation, and recovery from injury.

5. How can you visualize newborn cells in the adult brain?

   • Various techniques are used to visualize newborn cells in the adult brain:

     • Intravital imaging: Using techniques like multiphoton microscopy with fluorescent reporters (e.g., tdTOMATO) to track neural stem cells over time.

     • BrdU (Bromodeoxyuridine) labeling: Incorporates into dividing cells' DNA and is detected using immunohistochemistry.

     • Fluorescent reporter systems: Genetic labeling of newborn cells using Cre-lox recombination systems to track cell lineage and behavior.

     • Histological analysis: Examining brain sections with markers such as DCX (Doublecortin) to identify newly formed neurons.

   • These techniques allow researchers to track the history of cell division, predict cell behavior, and study the integration of new neurons into brain circuits.

Key Takeaway:
Understanding the potency of stem cells, their sources, and their applications in neurogenesis provides valuable insights for developing regenerative therapies and studying brain function. Advanced imaging techniques and cell tracking methods are crucial for visualizing and assessing the impact of newly generated neurons in the adult brain.

Slide 68: Stem Cells and Their Progeny Are Pretty Exciting

Key Points:

• Humorous comic strip to close the presentation.

• Stresses the importance of stem cells in science and their vast potential for future discoveries.

Explanation of Visuals:

• A comic strip adds a light-hearted conclusion to the technical presentation, emphasizing the enthusiasm and curiosity driving stem cell research.

Glossary:

• Progeny: The offspring or descendants of cells, referring to the differentiated cells that arise from stem cells.

Key Takeaway: Stem cells remain a fascinating area of research with enormous potential for breakthroughs in understanding biology and treating diseases.