Driever Stem cells

Stem cells can be classified based on several criteria, including their origin, differentiation potential, and developmental stage.

1. Origin: embryonic inner cell mass from blastocyst, somatic (found in various tissues in the body e.g. bone marrow, adipose tissue or skin), induced SC (by reprogramming)

2. differentiation potential / potency: Totipotent (everything [zygote), Multipotent, Oligopotent or Unipotent (only one cell type e.g. spermatogonia)

3. developemental stage: Zygote, embryonic stem cells from blastozyst, embryonic germ cells, adult stem cells

ES: embryonic stem cells —> derieved from inner cell mass of pre implantation embryo [blastocyst]

EG: embryonic germ cells —> derieved from primordial (initial) germ cells from fetus gonads

EC: embryonal carcinoma —> derieved from primordial germ cells but are usally detected as components of testicular cancer in adults (not naturally occuring)

Differences:

• origins, normal functions, and associations with health and disease

Commonalities:

• Pluripotency: All three types of cells are pluripotent, meaning they have the potential to differentiate into cells of all three germ layers (ectoderm, mesoderm, endoderm).

• Research Use: They are valuable tools in understanding development, differentiation, and disease.

• type of tumor that is typically found in reproductive organs (germ cell tumors) but can be found throughout whole body

• Embryonic stem cells (ES, EG, EC) inside vertebrates have unlimited proliferation potential and differentiate into teratomas (embryonic carcinomas)

• contain different types of tissues derived from the three germ layers

• potential to contain a variety of cell types, such as hair, teeth, bone, neural tissue, and even more complex structures. This ability to form tissues from different germ layers is a result of the pluripotent nature of the cells within the tumor

• mostly benign but can become cancerous

• pluripotent SC have telomerase —> no chromosomal aging (good for tumor)

  
  

  
  

• the introduction of cells or embryos from one individual into the developing embryo of another. This process results in an organism composed of cells with different genetic origins, methods:

  • Blastocyst Injection

  • Inner Cell Mass (ICM of blastocyst) Transfer

  • Induced Pluripotent Stem Cell (iPSC) Technology

  • Microinjection of Cells

• the formation of pluripotent stem cells depends on the POU (Pit-1, Oct4, Unc86) transcription factor Oct4

• pluripotency transcription factors regulate the expression of genes that are involved in maintaining the undifferentiated state and controlling the potential for differentiation

  • Oct4 —> maintaining the undifferentiated state of embryonic stem cells

  • Sox2 —> Co-expressed with Oct4, regulation of pluripotency and self-renewal

  • BMP and LIF —> balence differentiation versus maintenance of pluripotency

  • Nanog

  • Klf4 (krüppel like factor4) —> reprogramming of somatic cells into induced pluripotent stem cells (iPSCs)

  • c-Myc —> iPSC generation, along with Oct4, Sox2, and Klf4

The evolution of pluripotency involves the transition from single-celled to multicellular organisms, leading to the emergence of specialized cell types and the development of transcriptional regulatory networks. Pluripotency transcription factors, such as Oct4, Sox2, and Nanog, likely evolved as part of these networks, allowing cells to maintain a flexible, undifferentiated state capable of giving rise to various cell types. The conservation of pluripotency factors across species suggests their importance in the evolutionary development of complex organisms. The evolution of pluripotency is closely tied to the advantages it provides in adapting to changing environments and the establishment of precise developmental programs.

1. Core Pluripotency Transcription Factors:

   • Oct4 (Octamer-binding transcription factor 4): Central to pluripotency, Oct4 regulates genes involved in maintaining the undifferentiated state.

   • Sox2 (SRY (Sex Determining Region Y)-Box 2): Often cooperates with Oct4 to control pluripotency and self-renewal.

   • Nanog: Maintains pluripotency by suppressing genes associated with differentiation and promoting self-renewal.

2. Transcriptional Regulation:

   • Pluripotency transcription factors bind to specific DNA sequences within the promoters and enhancers of target genes, regulating their expression.

   • These factors often form complexes and interact with co-regulators to control gene expression.

3. Feedback Loops:

   • The core pluripotency factors often regulate each other's expression through positive feedback loops, reinforcing their collective role in maintaining pluripotency.

4. Epigenetic Regulation:

   • Pluripotency is associated with an open chromatin structure, allowing for the dynamic regulation of gene expression.

   • Epigenetic modifications, such as DNA methylation and histone modifications, play a crucial role in maintaining pluripotency by regulating gene accessibility.

Further interactions:

1. Signaling Pathways:

   • Pluripotency is influenced by various signaling pathways, including the Wnt, BMP, and FGF pathways.

   • These pathways interact with the core transcription factors to modulate gene expression and influence cell fate decisions.

2. MicroRNAs (miRNAs):

   • Small RNA molecules, such as miRNAs, contribute to the pluripotency network by post-transcriptionally regulating gene expression.

   • Some miRNAs target and modulate the levels of key pluripotency factors.

3. Cell Cycle Regulation:

   • Pluripotent stem cells exhibit a unique cell cycle profile, characterized by a short G1 phase and rapid cell division.

   • The pluripotency network is intertwined with cell cycle regulators to ensure efficient self-renewal.

4. Heterogeneity and Plasticity:

   • The pluripotency network exhibits dynamic states, and individual cells within a pluripotent population may have varying expression levels of key factors.

   • Cellular plasticity allows for the transition between different pluripotent states.

Somatic Cell Nuclear Transfer (SCNT):

• Donor Cell: A somatic cell (body cell) is taken from the animal to be cloned. This cell contains the full complement of DNA.

• Egg Cell Acquisition: An egg cell (oocyte) is retrieved from a female donor animal. The nucleus of the egg cell is removed (isolate meiotic spindle with glass needle) or inactivated, leaving an enucleated egg.

• Cell Fusion: The somatic cell and the enucleated egg are fused together using an electric current or a specialized chemical treatment. This creates a single cell with the complete set of DNA from the donor somatic cell.

• Stimulation of Development: The reconstructed cell is stimulated to divide and develop into an embryo.

• Implantation: The developing embryo is then implanted into the uterus of a surrogate mother for further development.

Further methods:

Embryo Splitting or Blastomere Separation (like SCNT but use developing embryo bot somatic cells)

Artificial Embryo Twinning (separating the early embryo to form two individuals)

Induced Pluripotent Stem Cells (iPSCs) —> controlled formation of various cell types why not whole organism in future

1. Low Efficiency and High Failure Rates:

2. Genetic and Epigenetic Abnormalities:

   • The process of nuclear transfer and reprogramming may not fully reset the epigenetic marks, leading to altered gene expression patterns and potential health issues.

3. Premature Aging and Telomere Shortening

4. Large Offspring Syndrome (LOS):

   • LOS can lead to birthing difficulties and neonatal health issues.

5. Mitochondrial DNA Mismatch:

   • SCNT involves using an enucleated egg, but the mitochondrial DNA comes from the egg donor. This can result in a mismatch between nuclear and mitochondrial DNA, potentially leading to mitochondrial dysfunction and health problems.

6. Ethical and Welfare Concerns

7. Environmental and Technical Challenges:

   • The success of somatic cloning can be influenced by environmental factors such as the conditions in which cells are cultured. Variability in these conditions can affect the efficiency and health of the resulting clones.

8. Species-Specific Variation:

   • Different species respond differently to somatic cloning, and success rates can vary. Some species may be more amenable to cloning, while others may face greater challenges.

9. Limited Reproductive Potential

10. Use of Surrogate Mothers:

    • The use of surrogate mothers in the cloning process raises ethical concerns, as these animals may experience health risks associated with carrying cloned embryos.

Telomeres function as protective caps that prevent the loss of genetic information during DNA replication. They become shorter with each cell division because the DNA polymerase is unable to copy the very ends of linear chromosomes, when the ends are to short the replicative senescence is erached and to avoid DNA errors the cell cannot divide anymore

Telomerase rebuilds these repetetive DNA sequences at the end of the chromosomes e.g. in stem cells

• Large offspring syndrome

• Growth / organ abnormalities

• premature aging (linked to telomere shortening)

• epicgenetic aberrations (imprints can cause trouble)

• sterility, behaviour aberrations, diseases (cardiovascular, lung, immunsystem)

• miRNA dysregulation

epigenetic marks

methylation imprints: male and female gamete specific imprints

Imprinting often causes expression of only one allele (methylation blocks or varies the reading frame for gene on mother or father chromosome)

• induced pluripotent stem cells (somatic cells reprogrammed to pluripotency —> ability to differentiate into various cell types representing all three germ layers)

• Specific transcription factors known as Yamanaka factors are introduced into the somatic cells —> they activate transcription of pluripotency genes. The original set of Yamanaka factors includes:

  • Oct4 (Octamer-binding transcription factor 4)

  • Sox2 (SRY (Sex Determining Region Y)-Box 2)

  • Klf4 (Kruppel-like factor 4)

  • c-Myc (Myc proto-oncogene)

• The reprogramming factors can be delivered into the cells using viral vectors (e.g., retroviruses or lentiviruses). The viruses integrate the reprogramming genes into the host cell's genome

• charcterisation, culturing and expansion (mayn weeks till the cells can be used for e.g. treatment tests with drugs)

1. Stochastic phase = early initiation phase

   • introduction of reprogramming factors (Oct4, Sox2 …) and dedifferentiation (deactivation of specializetion genes)

   • MET (mesenchymal to epithelial transition)

   • metabolic changes, DNA repair, RNA processing

2. Intermediate phase

   • activation of pluripotency genes, developemental regulators and glycolysis

3. late maturation and stabilization phase (deterministic phase)

   • activation of pluripotency circuits, cytoskeletal remodelling, cell adhesion and ECM proteins, vesicular transport

   • silencing of transgenes, epigenetic resetting

Yes, it is possible with methods that avoid genomic integration of reprogramming factors. These approaches aim to minimize the risk of introducing genetic mutations and increase the safety of iPSCs for potential therapeutic applications.

—> using non integrating viral vectors (e.g.of adenovirus or sendai virus or episomal vectors)

—> using modified RNAs or proteins to induce PSC (but inefficient)

—> using chemical reprogramming, or excisable transposons

• amputation

• wound closure by wound epidermis

• proliferating blastema

• pattern formation in the blastema

• morphogenesis, differentiation and longation

• complete regeneration

Bone make bone, muscle makes mucle, blood makes blood, skin makes skin (GFP marking experiment)

Regeneration from tissue specific stem cells (not pluripotent cells)

1. Dermal Cells:

   • Dermal cells, which are present in the skin, play a crucial role in limb regeneration. These cells contribute to the formation of the wound epidermis, a specialized structure that covers the wound site and serves as an essential signaling center for regeneration.

2. Epidermal Cells:

   • Epidermal cells at the wound site undergo dedifferentiation, a process where they revert to a more primitive state. These dedifferentiated epidermal cells form the apical epidermal cap, or wound epidermis, which is vital for coordinating the regeneration process.

3. Mesenchymal Cells:

   • Mesenchymal cells in the stump, including connective tissue cells, contribute to the formation of the blastema. The blastema is a mass of undifferentiated cells that serves as a source of progenitor cells for the regeneration of various limb structures.

4. Muscle Cells:

   • Muscle cells in the remaining part of the limb contribute to the regeneration of muscle tissue. Satellite cells associated with muscle fibers play a role in providing myoblasts for muscle regeneration.

5. Chondrocytes (Cartilage Cells):

   • Chondrocytes are cells that form cartilage. During limb regeneration, chondrocytes contribute to the regeneration of cartilaginous structures, such as the skeletal elements of the limb.

6. Osteoblasts (Bone-Forming Cells):

   • Osteoblasts are involved in the regeneration of bone structures in the amphibian limb. These cells contribute to the formation of new bone tissue to replace the lost or damaged skeletal elements.

7. Blood Vessel Cells:

   • Blood vessel cells play a role in providing the necessary vascular support for the regenerating tissues. Vascularization is crucial for the transport of nutrients and oxygen to the growing structures.

8. Nerve Cells:

   • Nerve cells are important for guiding the regenerating tissues and reestablishing connections with the nervous system. Proper innervation is essential for the functional recovery of the regenerated limb.

During limb regeneration in amphibians, the blastema is a key structure that acts as a reservoir of undifferentiated cells capable of giving rise to various cell types needed for tissue regrowth. The blastema is formed through the dedifferentiation of cells near the injury site and serves as a source of progenitor cells that differentiate into specific cell types required for the regeneration of the limb. The coordinated interactions among these cell types contribute to the successful and remarkable regeneration of the amphibian limb.

• specialized cells are directly converted into another cell type without passing through a pluripotent or multipotent intermediate state (like in iPSC)

• direct reprogramming factors (embryonic factors) necessary e.g. fibroblast into cardiomyocyte by GATA4 (MEF2C, TBX5) or Fibroblast to neuron conversion by Ascl1, Brn2 and Myt1l

stem cells in the umbilical cord:

– blood stem cells in umbilical cord blood

– mesenchymal “oligopotent” stem cells in “Wharton’s yelly” (Oct4!)

• good proliferation, low teratoma risk (lower then iPSC)

• postpartum umbilical cord tissue may be frozen in liquid nitrogen for whole life of person (easy to collect, harvest and store)

• Source of Hematopoietic Stem Cells (HSCs) —> can differentiate in various blood cells, and treat blood diseases

• aka. as somatic or adult stem cells

• tissue-specific stem cells are multipotent, meaning they can give rise to a limited range of cell types within a specific lineage

• located in tissue specific niches which provide microenvironment

• ablility for self renewal, tissue maintenance and repair

• Examples of Tissue-Specific Stem Cells:

  • Hematopoietic Stem Cells (HSCs): Found in the bone marrow, HSCs give rise to various blood cell types, including red blood cells, white blood cells, and platelets.

  • Neural Stem Cells: Located in the central nervous system, neural stem cells can give rise to neurons, astrocytes, and oligodendrocytes.

  • Epidermal Stem Cells: Found in the skin, epidermal stem cells contribute to the regeneration of the epidermis and its appendages.

• located in tissue specific niches which provide microenvironment (with niche cells, long range components and stromal cells that send specific signals) —> loss of attechment/niche signal results in loss of stem cell properties (same for the progeny of the stem cells that differentiate)

• Hematopoetic SC in the bone marrow

• Neural stem cells in the hippocampus and subventricular zone

• skin stem cells in basal layer of epidermis (on the basal membrane connect via adhesive molecules)

• intestine stem cells in the crypt (where WNT pathway activates proliferation and the produced cells differetiate controled by Notch (Delta) signaling and lose proliferation potential because WNT no longer active)

Lineage Tracing: Double label by BrdU + EdU for long term identification of stem cells (by marking DNA that is replicated in S-phase of mitosis) if a cell gets labeled 2 times in a few weeks its a stem cell

Functional Assays: analyse the colony formation of a in vitro cultre, stem cells have the ability to form colonies with multiple cell types and can differentiate

Screening for the stem cell specific genes/signal molecules: by CRISPR/Cas9 or scRNA sequencing

A stem cell niche is a specialized microenvironment within tissues that regulates the behavior of stem cells. It encompasses cellular and extracellular components, including neighboring cells, extracellular matrix, signaling molecules, and blood vessels. The niche influences stem cell activities such as self-renewal, differentiation, and quiescence through signaling pathways, cell-cell interactions, and physical characteristics. Stem cell niches are dynamic and respond to changes in the microenvironment, contributing to the regulation of tissue homeostasis and regeneration.

• mark and sort stem cells out of a dissociated tissue sample via Fluorescence-Activated or Magnetic-Activated Cell Sorting, stem cell specific antibodys with fluorescence or magnetic nanoparticals

• Density centrifugation or size exclusion filtration (if stem cell differs strongly in these parameters)

• functional assays and culture based methods

• in vivo isolation (extract the stem cell niche/colony)

• Stem cells themselves and their progeny

• mesenchymal or stromal cells

• ECM / the basal lamina

• long range components (endothelial or neurons that send long range signals)

Neurons subventricular zone

• Notch signaling (rezeptor vs ligand on two neighboring cells interact)

• WNT signaling (where stem cells are maintained and proliferate and where to differentiate)

• BMP as signal to differentiate (BMP inhibition in stem cells)

• Sonic Hedgehog signaling pathway to maintain stem cell pool in the niche (gradient from apical (high -> SC) to basal (low)

• ECM, stromal and blood vessel interaction in the niche

Bone marrow

• hypoxic regulation (SC where oxigen rich if not quiescent)

• Cytokine signaling

• sympathetic nervous system signaling (e.g. Noradrenaline)

• endosteal and vascular niches. HSCs are regulated by signals from these niches, with endosteal niches promoting quiescence and vascular niches supporting HSC proliferation and differentiation

Embryonic Stem (ES) Cells:

Advantages:

1. Pluripotency: ES cells are pluripotent, meaning they can differentiate into cells of all three germ layers. This broad differentiation potential makes them versatile for generating various cell types needed for tissue repair.

2. Proliferative Capacity: ES cells have a high proliferative capacity, allowing for the generation of large numbers of cells for therapeutic applications.

Disadvantages:

1. Ethical Concerns: The use of human embryonic stem cells raises ethical concerns related to the destruction of embryos during their extraction.

2. Immunorejection: Transplanted ES cells may face immune rejection if they are not a perfect match to the recipient, requiring immunosuppression.

3. Tumorigenicity: ES cells have the potential to form teratomas, tumors containing cells from all three germ layers. Rigorous purification and differentiation protocols are required to minimize this risk.

Induced Pluripotent Stem (iPS) Cells:

Advantages:

1. Patient-Specific: iPS cells can be generated from a patient's own cells, reducing the risk of immune rejection and ethical concerns associated with embryonic stem cells.

2. Pluripotency

3. Disease Modeling: iPS cells can be used to model diseases, providing insights into disease mechanisms and drug testing.

Disadvantages:

1. Reprogramming Efficiency: The reprogramming process to generate iPS cells can be inefficient, and variations in the quality of iPS cells may affect their utility.

2. Genetic Modifications: (when they are used cause of efficiancy)

3. Tumorigenicity

Tissue-Specific Stem Cells:

Advantages:

1. Tissue-Specific Commitment: Tissue-specific stem cells are committed to specific lineages, reducing the risk of forming teratomas and improving the safety of transplantation.

2. Natural Microenvironment: Tissue-specific stem cells reside in their natural microenvironment, or niche, facilitating better integration into the host tissue.

3. Lower Risk of Immune Rejection: When autologous (from the same individual) tissue-specific stem cells are used, the risk of immune rejection is reduced.

Disadvantages:

1. Limited Differentiation Potential: Tissue-specific stem cells are multipotent and can only differentiate into a restricted range of cell types within a specific lineage.

2. Limited Availability: The isolation and expansion of tissue-specific stem cells may be challenging, especially in cases where the cell population is small or difficult to obtain.

3. Aging Effects: Tissue-specific stem cells may be affected by aging and may exhibit reduced regenerative capacity over time.

Considerations for All Stem Cell Types:

1. Safety Concerns: All stem cell types carry safety concerns, particularly related to tumorigenicity and the potential for uncontrolled cell growth.

2. Regulatory Challenges: The use of stem cells in clinical applications is subject to regulatory challenges and ethical considerations that vary by region.