ipscs and adult scs

• Adult SCs - from specific organs like hematopoietic and brain
• Pluripotent SCs - from embryo or reprogrammed adult SCs as iPSCs
• Adult SCs - multipotent or unipotent, limited to their region
• Pluripotent SCs - pluripotent or totipotent, and can differentiate into all three germ layers
• Adult SCs - self renewal is limited to the ability to repair and maintain tissues
• Pluripotent SCs - self-renewal is extensive for long-term propagation for growing organisms or organoids for disease modelling
• Adult SCs - relatively genetically stable, won't differentiate aberrantly and aren't tumorigenic
• Pluripotent - genetically unstable and quite tumorigenic
• DMD Mice - CRISPR/Cas9 used to introduce the DMD mutation into mouse embryonic stemcells
• DMD Mice - used for pathophysiology studies and to test therapeutic interventions
• DMD Mice - used for pre-clinical trials, drug screens and testing gene therapy efficiacy
• CF humans - CRISPR/Cas9 to introduce or correct mutations in CFTR gene
• CF humans - iPSCs can be used for isogenic controls
• CF humans - used for disease modelling, drug screens, identifying functional compounds
• Hematopoietic stem cells are isolated from beta-thalassemia patients, where a viral vector can be used to introduce corrections and reinfused
• CRISPR/Cas9 technology has also been used to edit genes in hematopoietic stem cells (HSC) taken from beta-thalassemia patients.
• Beta thalassemia mice have been developed using CRISPR/Cas9 technology to create a mouse model with the same genetic defect as human patients.
• In sickle cell anemia, CRISPR/Cas9 technology has been used to target and repair the mutation responsible for producing abnormal hemoglobin molecules.
• The use of CRISPR/Cas9 technology in HSCs allows for the correction of the genetic defect responsible for beta-thalassemia, potentially providing a cure for this inherited blood disorder.
• This approach involves introducing a guide RNA into bone marrow stem cells, which directs Cas9 to cut out the faulty DNA sequence and replace it with a healthy one.
• CRISPR/Cas9 technology is being explored as a potential treatment option for other inherited blood disorders such as severe combined immunodeficiency (SCID), also known as "bubble boy" disease.
• Viruses like adeno-associated virus (AAV), lentivirus, and retrovirus have been utilized for gene delivery due to their ability to infect specific types of cells and integrate into the genome.
• Electroporation uses electric pulses to temporarily open pores on the surface of cells, allowing nucleic acids to enter.
• iPSCs can be differentiated into various cell types affected by the disease, such as neurons, cardiomyocytes, hepatocytes, or pancreatic cells
• Differentiation protocols involve using growth factors, cytokines, and small molecules to induce iPSCs to develop into desired cell types
• Recapitulating disease-relevant cell types allows the study of disease mechanisms in a context closely resembling the affected tissues
• Direct reprogramming has shown success in generating functional neuronal progenitors from fibroblasts
• The use of patient-specific iPSCs enables personalized medicine approaches that consider individual genetic backgrounds and environmental influences
• Directed differentiation of iPSCs provides an alternative source of cells for drug testing and toxicity screening compared to animal models
• IPSCs have been used to model diseases like Alzheimer's, Parkinson's, Huntington's, and ALS
• Neuron-like cells generated from patient iPS cells exhibit similar electrophysiological properties compared to primary neurons
• iPSCs offer a platform to study complex, polygenic diseases by comparing isogenic iPSC lines with and without specific risk alleles or combinations of risk alleles
• iPSC-derived neurons can exhibit disease-specific phenotypes, including protein aggregation and cellular dysfunction.
• iPSC-based disease models can be used to identify biomarkers associated with disease onset, progression, or response to therapy.
• • Collect somatic cells like skin fibroblasts or blood cells
  • Reprogram these somatic cells into iPSCs using OCT4, SOX2, KLF4, and MYC.
• Quality Control and Characterization:

  • Validate the pluripotent nature of generated iPSCs using markers like OCT4, NANOG, and SSEA4.
  • Perform karyotyping and confirm the absence of transgenes used during reprogramming.
• Genetic Editing for Disease-Specific Mutations:

  • Use CRISPR/Cas9 or other gene-editing techniques to introduce disease-specific mutations or correct mutations in iPSCs.
  • Create isogenic control lines with corrected mutations for comparison.
• Disease Phenotype Validation:

  • Characterize differentiated cells to ensure they exhibit disease-specific phenotypes.
  • For neurodegenerative diseases, check for protein aggregation or neuronal dysfunction. In cardiac diseases, assess contractile function and electrophysiological properties.
• Functional Assays:

  • Perform functional assays to assess disease-related cellular functions.
  • For example, study neurotransmitter release and uptake in neurons or evaluate contractility and calcium handling in cardiomyocytes.
• Long-Term Culturing for Disease Progression Studies:

  • Establish long-term cultures to study disease progression over time.
  • Monitor changes in cellular morphology, function, and molecular markers associated with disease development.