Chu Lectures

Podcast for these Lectures: https://notebooklm.google.com/notebook/354c2e61-1476-4362-a85a-0e2e1a8ddb85/audio

Lecture 10 Summary: Stem Cells

Stem Cells: Characteristics and Types

1. Definition: Stem cells are cells capable of:

   • Self-renewal: Maintaining their pool over time.

   • Differentiation: Generating specialized cells for tissue growth and repair.

2. Types of Stem Cells:

   • Pluripotent Stem Cells (PSCs): Can differentiate into any cell type within the adult body (e.g., embryonic stem cells, iPSCs).

   • Tissue-Specific (Somatic) Stem Cells: Found in organs like the bone marrow or intestines, limited to forming specialized cells within that tissue

     1. Found in bone marrow (haematopoietic stem cell), restricted to the blood lineage → RBC’s, WBC’s, platelets 

3. Potency Levels:

   • Totipotent: Capable of forming all embryonic and extraembryonic tissues.

   • Pluripotent: Capable of forming all cell types derived from the three germ layers.

   • Multipotent: Capable of making multiple types of specialized cells 

Defining Pluripotency in Cancer Cells

Many different types of tissues were observed in a mouse teratocarcinoma tumor. Normally tissue types would be more homogenous, so it was hypothesized that at the time of tumor development there was some kind of stem cell playing a role. 

• How can this be determined? 

  • Chimeric assay/germline transmission: Cells derived from the tumor can be inserted into developmental context (ie. a blastocyst), and if they contribute to development, this tells us the tumor cells have stem cell properties and confirms contributions to embryonic development.

Why is this research important?

• 3D culture (organoids): miniature, simplified versions of organs developed in vitro. These structures mimic the functionality and architecture of actual organs more accurately than traditional 2D cell cultures.

• Scale up (bioreactor): Provides sufficient cell quantities for clinical applications, such as regenerative therapies and transplantation. Facilitates research and development in tissue engineering and drug testing on a large scale.

• Assembly (Bio-3D-printing): using stem cells and biomaterials to construct tissues and organs layer by layer, replicating the precise architecture of human organs. Opens the possibility of creating transplantable organs, addressing organ donor shortages.

• Disease modeling: Stem cells, particularly induced pluripotent stem cells (iPSCs), can be used to create models of specific diseases by differentiating into affected cell types (e.g., neurons for neurological diseases). Facilitates personalized medicine by using patient-derived iPSCs to study how their disease progresses and which treatments might be effective.

Gene Targeting in Embryonic Stem (ES) Cells in Mice

1. Gene Targeting in ES Cell Culture:

   • Mouse ES cells are cultured under specific conditions to maintain pluripotency.

2. Construction of Targeting Vector:

   • A vector is engineered to include a gene of interest flanked by homologous sequences to the target locus. It also contains a selectable marker (e.g., antibiotic resistance gene) to identify successful integrations.

3. ES Cell Transfection:

   • The targeting vector is introduced into ES cells via electroporation or other methods. Homologous recombination allows the vector to integrate into the target genomic location.

4. Proliferation of Targeted ES Cells:

   • Cells are cultured with selective agents (e.g., antibiotics) to isolate those that incorporate the targeting vector. Successfully targeted cells are expanded and later used to create chimeric mice for further study.

Regulation of pluripotency

Extrinsic Factors: Pluripotency is influenced by signals from the microenvironment (niche) around the embryonic stem cells. In ex vivo models this is supplied from the growth media/feeder. Studies showed that you could impact pluripotency by replacing a factor in the conditioned media. 

• Example: continual addition of Leukemia inhibitory factor (LIF) activates the STAT3 pathway to maintain pluripotency in mouse ES cells.

Intrinsic Factors: Pluripotency is maintained by the intrinsic expression of core transcription factors that regulate the gene network specific to stem cell identity. 

Somatic Cell Nuclear Transfer (SCNT): context for reprogramming 

• SCNT is a groundbreaking technique that demonstrated the potential for reprogramming differentiated somatic cells into an embryonic-like state. This process involves transferring the nucleus of a somatic cell into an enucleated egg, which reprograms the nucleus to a totipotent or pluripotent state, capable of giving rise to a new organism

Gurdon’s work with tadpoles: transferred nuclei from differentiated intestinal cells of tadpoles into enucleated frog eggs. The egg reprogrammed the transferred nucleus, and some of these embryos developed into normal tadpoles. 

• This work proved that cellular differentiation is reversible and that the cytoplasm of an egg contains factors capable of reprogramming a somatic nuclei.

Deriving iPSCs from somatic cells 

The Process:

• Yamanaka identified four key transcription factors—Oct4, Sox2, Klf4, and c-Myc—that could reprogram somatic cells (e.g., fibroblasts) into a pluripotent state.

• These factors were introduced into somatic cells using retroviral vectors.

• Reprogrammed cells exhibited characteristics of embryonic stem cells, such as self-renewal and the ability to differentiate into any cell type.

Significance:

• iPSCs bypass the ethical concerns of using embryonic stem cells.

• They provide a personalized source of stem cells, reducing the risk of immune rejection.

Applications include disease modeling, regenerative medicine, and conservation efforts

Lecture 11 Summary: Reprogramming

Novel Reprogramming Strategies

In Vivo Reprogramming by Ectopic Expression of OSKM

In vivo reprogramming involves directly reprogramming cells within a living organism using transcription factors like Oct4, Sox2, Klf4, and c-Myc (OSKM). This method bypasses the need for cell extraction and transplantation, offering a novel way to treat age-associated and degenerative conditions.

1. Ameliorating Age-Associated Hallmarks:

   • Studies show that transient expression of OSKM in aged mice reduces cellular markers of aging, such as DNA damage and epigenetic drift.

   • Improved organ function and tissue regeneration have been observed, suggesting reprogramming can rejuvenate aged cells.

     1. Restoring Vision: A notable application is the restoration of optic nerve injury and vision in animal models through in vivo reprogramming. OSKM expression reprograms retinal ganglion cells to a more youthful state, promoting regeneration.

Lecture 11 Summary: Reprogramming

Novel Reprogramming Strategies

In Vivo Reprogramming by Ectopic Expression of OSKM

In vivo reprogramming involves directly reprogramming cells within a living organism using transcription factors like Oct4, Sox2, Klf4, and c-Myc (OSKM). This method bypasses the need for cell extraction and transplantation, offering a novel way to treat age-associated and degenerative conditions.

1. Ameliorating Age-Associated Hallmarks:

   • Studies show that transient expression of OSKM in aged mice reduces cellular markers of aging, such as DNA damage and epigenetic drift.

   • Improved organ function and tissue regeneration have been observed, suggesting reprogramming can rejuvenate aged cells.

     1. Restoring Vision: A notable application is the restoration of optic nerve injury and vision in animal models through in vivo reprogramming. OSKM expression reprograms retinal ganglion cells to a more youthful state, promoting regeneration.

Challenges in In Vivo Reprogramming Research

• Tumorigenesis: Continuous OSKM expression can lead to uncontrolled proliferation and tumor formation.

• Partial Reprogramming Risks: Incomplete reprogramming may result in cellular instability and unintended differentiation.

• Target Specificity: Delivering reprogramming factors to the correct cells without affecting surrounding tissues remains challenging.

• Immune Response: Host immune systems may react to the viral vectors used to deliver transcription factors.

Applications of PSCs in Cell Replacement Therapy

Pluripotent stem cells (PSCs) offer a renewable source of cells that can replace damaged or diseased tissues, providing a foundation for regenerative medicine.

1. Retinal Pigmented Epithelium (RPE) Cell Therapy for Macular Degeneration (AMD):

   • AMD Overview: A leading cause of blindness, characterized by the degeneration of RPE cells.

   • Therapy:

     • PSCs are differentiated into RPE cells and transplanted into the retina.

     • These cells integrate into the retinal structure, replacing damaged RPE and supporting photoreceptor survival.

   • Progress: Clinical trials have shown promising results, with patients experiencing improved or stabilized vision.

2. Mitochondrial Replacement Therapy (MRT):

   • Context: Defective mitochondria cause severe genetic disorders.

   • Therapy:

     • PSCs are used to generate healthy mitochondria or replace defective ones in affected cells.

     • This technique helps restore cellular energy production and function.

   • Applications: MRT is being explored for mitochondrial diseases and in regenerative therapies requiring metabolic stability.

Applications of Pluripotent Stem Cells

1. Regenerative Medicine:

   • PSCs are used to generate specific cell types for repairing or replacing damaged tissues, such as neurons for Parkinson’s disease or cardiomyocytes for heart failure.

2. Species Conservation:

   • iPSCs are being explored to create gametes for endangered species, like the northern white rhino, to preserve genetic diversity and combat extinction.

3. Cell Therapy:

   • Patient-specific iPSCs minimize immune rejection for therapies like restoring vision in macular degeneration.

4. Drug Screening:

   • PSCs differentiated into disease-relevant cell types enable high-throughput drug testing, reducing reliance on animal models.

5. Cellular Agriculture:

   • PSCs can produce lab-grown meat and dairy products, offering sustainable and ethical alternatives to traditional agriculture.

6. Personalized Medicine:

   • Patient-derived iPSCs provide a platform to study individual responses to drugs or develop tailored treatments.

Challenges in Generating iPSCs and ESCs from Non-Rodent/Primate Mammals

Generating iPSCs and ESCs from species beyond rodents and primates, such as livestock and wildlife, has been challenging due to:

• Species-Specific Differences: Variations in epigenetics and signaling pathways complicate reprogramming.

• Limited Knowledge: Lack of comprehensive genomic and molecular tools for many species.

Relevance:

• In veterinary medicine, PSCs could improve livestock health, treat animal diseases, and enhance breeding programs.

• For species conservation, creating iPSCs from endangered species can aid in cloning, creating gametes, and studying genetic resilience.

Ethical Concerns and Societal Debate

Advancing PSC technology raises several ethical concerns that require societal dialogue, including:

• Embryo Use: ESC research involves destroying embryos, raising moral questions about the beginning of life.

• Genetic Modification: The potential to misuse PSCs for genetic enhancement or cloning raises concerns about “designer organisms.”

• Animal Welfare: Using animals to generate iPSCs for conservation or agriculture poses questions about exploitation.