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Week 1: Intro, Lecture 4: Skin Cells, Lecture 3: Stem Cells, Lecture 2:…

- - - - Make customized patch implanted on defect cartilage
- - - - Extract cells
        
        Keratinocytes, Melanocytes, Fibroblasts, Endothelial cells
        
        All required for new skin to function
    - - Digest basement membrane
        
        Support/signalling layer between dermis/epidermis
    - - Isolate cells from step 1
    - - Dish culture for each cell type
        
        Keratinocytes, Melanocytes, Fibroblasts, Endothelial cells
  - - - Genes, proteins, etc.
    - - Dependent on thickness of skin being grafted
        
        Can possibly be remedied by using pre-vascularized skin in-vitro
- - - - Find donor pancreas from deceased
        
        Dissect & chop organs
      - Ricordi Chamber
        
        Chemically + mechanically digest islet cells to separate from pancreas
      - Separate Islets
        
        QC, test insulin production & # cells
      - Transplant to patient liver
        
        Pancreas is just below liver, share ducts
        
        Portal vein access
      - Cells begin to secrete insulin
    - - Inadequate supply of donor pancreas
      - Rejection
      - Long-term immuno-suppresant requirement
      - Lack of funding
- - - - Cells can divide ~40-60 times before apoptosis
        
        Telomeres
        
        Caps that protect genetic material/Ends on chromosomes
        
        Shorten a bit every division
        
        Cause unknown
- - - - Cells sourced from the donor, but back into the donor
        
        Skin, bone marrow, blood, etc
        
        i.e. Leukemia (WBCs), Cartilage
        
        Advantages:
        
        No rejection
        
        Challenges:
        
        Limited Supply
        
        Surgical/Medical intervention
        
        Graft vs. Cancer effect
        
        The tendency of the host's immune system to attack/reject the graft
    - - Cells transplanted from the donor to a recipient of the same species
        
        Organs, blood
        
        Advantages:
        
        Graft vs. Cancer effect reduced
        
        Healthier cells if host is affected w/ something
        
        Donor cells = donor immune system
        
        Fight cancer response of autologous cells
        
        Challenges:
        
        Risk of rejection
        
        Attack on healthy cells likely
        
        Infections b/c of immunosuppressants
    - - Cells transplanted from one identical twin to another
        
        Bone/blood marrow, cartilage/skin (TBD on testing)
        
        Advantages:
        
        Similar immune system, less chance of rejection
        
        Attack specific to unhealthy/cancer cells
        
        Minimal risk of new cancers appearing
        
        Challenges:
        
        New immune system much like recipients
        
        Therefore NO graft vs. cancer effect?
    - - Cells transplanted from one species to another
        
        Human-animal, pig preferred
        
        Advantages:
        
        Unlimited supply
        
        Genetically modifiable
        
        Challenges:
        
        Cross-species rejection
        
        Disease transmission
        
        Lifelong dependence on immunosuppressants
        
        Viable tissue life isn't the same as humas (15-20 years from pigs)
- - - - Avascular
        
        No blood for nourishment
        
        Receive nourishment from diffusion
      - Aneural
        
        No nerves
        
        Pain from surrounding bone/tissue
      - Alymphatic
        
        No lymph cells
    - - Chondral
        
        Just cartilage
      - Osteochondral
        
        Cartilage & underlying bone
    - - Sourcing
        
        Autograft (Autologous) or Allograft (Allogenic)
        
        In autograft, want to source from distal femur, as it is less load bearing
      - Growing
        
        Replicate cells in-vitro
      - Isolating
      - Transplant
        
        Repair damage
- - - - Preferably from hip for marrow, peripheral veins for blood
    - - Return blood
    - - Store/freeze stem cells
    - - Weaken immune system
- - - - Red blood cells
      - White blood cells
      - Platelets
- - - - Means they are capable of giving rise to several different cell types
  - - - Such as bone marrow or fat
- - - - Discovered a new way to produce embryonic-type stem cells from adult stem cells
        
        Narrowed 24 to 4 genes needed to wind back the developmental clock
        
        Boon for regenerative medacine
        
        Could reprogram other cells into iPSC, then use to grow organs for treatment
        
        Could reduce risk of immune system rejection
        
        Overcomes the ethical concerns of embryonic stem cells
      - In 2006, presented results in Toronto
        
        Won Nobel Prize in 2012 in Physiology or Medicine
- - - - Using adult DNA, clone Morag & Megan using embryonic cells
      - Used Nuclear transfer to clone Dolly
- - - - Totipotent Stem Cells
        
        Potential to divide to create a whole organism
        
        Pluripotent Stem Cells
        
        Can Divide into most/all cell types
        
        Can't develop into organisms on its own
    - - Nuclear transfer
        
        Remove nucleus from egg cell
        
        Put nucleus from cell w/ desired genes in to denucleated egg cell
        
        Clone
      - In-Vitro Fertilization (IVF) Technique
        
        Cultivate egg w/ sperm & develop embryo 5-7 days
        
        Remove blastocysts from embryo
        
        Grow in dish
  - - - Can differentiate into many cell types, but within their lineage
      - 3 Categories
        
        Hematopoietic (blood forming) Stem Cells
        
        Mesenchymal Stem Cells
        
        Can differentiate into bone, muscle/tendon, stroma, chondrocyte cells
        
        Neuronal Stem Cells
        
        Can generate neurons, oligodendrocytes (glial), and astroglia (glial)
- - - - Osteocytes respond to mechanical strain & send signals for bone formation
- - - - Covers
        
        Skin, passage linings
      - Absorbs, secretes, transports, protects, senses
      - Types:
        
        Squamous
        
        Cuboidal
        
        Columnar
        
        Stratified Squamous, Cuboidal, Columnar
    - - Supports & binds
        
        Bone, blood, lymph
      - 3 General Classifications
        
        Loose
        
        Loosely woven fibres that surround organs
        
        Consists of fibroblasts
        
        Make cells in all connective tissue types
        
        Dense
        
        Protective capsules around organs
        
        Attaches muscles to bones & links bones at joints
        
        Higher proportion of collagen fibre provides rigigity
        
        Specialized
        
        Blood, cartilage, bone, fat
    - - Contraction
      - Types
        
        Smooth
        
        Involuntary contraction & movement in internal organs
        
        Cardiac
        
        Pumping blood
        
        Skeletal
        
        Attached to bones, contraction makes movement possible
    - - Carries messages
      - 2 Types
        
        Neurons
        
        Specialized cells that transmit electrical pulses
        
        Glial Cells
        
        Cells that support, protect, & nourish neurons
  - - - Adhesive Proteins
        
        Sticks cells together & organize their structure
        
        Integrin, Fibronectans, Laminin
      - Structural Proteins
        
        Provide tensile & compressive strength
        
        Collagen, Elastin
      - Proteoglycans
        
        Attracts water
        
        Gel that cushions the cells
        
        Allows chemical signalling by binding various secreted signal molecules
- - - - Published book on animal & plant cells
        
        Considered one of the foundational theories on which modern cell biology is based
  - - - Discussed observations on plant & animal cells
      - Kinda got screwed
- - - - Major nutrients of cells
    - - Store energy, structural components, cell signalling
    - - Contain info
    - - Execute tasks directed by Nucleic acids
  - - - Cell shape
      - Cell movement (motility)
      - Intracellular movement
  - - - For coupling, transport, signalling
  - - - Whole membrane transforms to engulf large molecules
- - - - So why do they look and function differently?
  - - - RNA
        
        Proteins
  - - - The portion of DNA copied into RNA on their way to making a protein
- - - - Humans have ~22,000 genes which get activated depending on cell type, time & locations
  - - - Biochemical function
      - Structural features (e.g. cell shape)
    - - Differentiation continues until terminal differentiation is achieved (cell is mature)
        
        Example: Liver cells & neural cells both technically have enzyme to break down alcohol, but it is "turned off" in neural cells
- - - - Directly convert somatic cells into another type
        
        o -> o shape
      - Pros
        
        Autologous Transplantation
        
        ~6-16% efficiency (higher than iPSC)
        
        Low oncogenic (tumour) potential
        
        TF can be delivered directly to injury site
        
        Less time
      - Cons
        
        Lack of funding
        
        Costs a lot & takes a long time
        
        Reprogramming factors vary
        
        Requires guess & check for TFs
    - - Genetically reprogram somatic cells into embryonic stem cell state, then differentiate to any lineage
        
        /\ shape
      - Pros
        
        Same set of reprogramming factors in humans & animals
      - Cons
        
        Lack of funding
        
        Costs a lot and takes a long time
        
        Low efficiency: 0.02%
        
        Higher oncogenic potential
      - Both require reprogramming
        
        3 Approaches
        
        Exogeneous Approach
        
        Transgenes (genes from outside host's body) are introduced into the cell
        
        Kickstarts reprogramming process
        
        Lenti-viruses (HIV) are an example
        
        Can insert a DNA copy of its RNA genome into the DNA of the host
        
        Host DNA continuously recreates this new DNA
        
        We can use the understanding of how they work to manipulate & change cell function by controlling TFs
        
        5 Steps
        
        Plasmid packaged in a virus
        
        Clone coding DNA of select TFs & package into a virus
        
        Transfection
        
        Cells are encoded by the virus in the plasmid using the lenti-virus system
        
        Selection using antibiotics
        
        We can eliminate untransduced cells using antibiotic selection
        
        Induction
        
        Cells begin to transcribe the TF coding data as if it were its own
        
        Transdifferentiation
        
        TFs are substantially overexpressed
        
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        Challenges
        
        We don't know what TFs to overexpress
        
        Guess & check is required
        
        Low efficiency (~6-16%)
        
        Priming & preparing cells with specific TF & then inducing differentiation can improve efficiencies up to 34%
        
        Endogenous Approach
        
        Manipulate DNA to affect gene behaviour
        
        Add, remover, or alter particular locations in the genome
        
        Silence endogenous genes
        
        Can be performed w/ CRISPR/Cas9
        
        Made by Emmanuelle Charpentier & Jennifer Doudna
        
        They won the Nobel Prize in 2020
        
        Binds to a specifically coded guide RNA
        
        Can cut out viral (or any) DNA at a precisely chosen location
        
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        Pharmacological Approach
        
        Pharmacological agents that can induce an immunological response in cells
        
        Causes a cascade that triggers epigenetic remodelling
- - - - Replacement rates of cells
        
        Bone marrow, small intestine, epidermis (cuts) have fast turnover rate
        
        Liver is slow, replacing in ~1 year
      - Production rates of cells
    - - Fat cells (adipose)
        
        Limited #
        
        Grow in size & slight in # with weight gain
        
        Shrink w/ weight loss, but extra gained cells remain
      - Retinal Cells
      - Neurons in CNS
- - - - Structural changes observed during the development of a particular organ
    - - Small Scale
        
        Induction, Adult trans-differentiation, & some mesenchymal-epithelial interaction
        
        Mesenchyme is embryonic tissue which develops in to skeletal and connective tissues
        
        Mesechymal-Epithelial Interaction
        
        Develops almost all organs
        
        Play a key role in adult tissue homeostasis
        
        Involved in morphogenesis & organogenesis of tissues & organs
        
        Can be exploited to improve tissue function in bone marrow, skin, liver, & bladder
        
        Bone Marrow Transport
        
        Pre-clinical studies show that MSC cultured ex-vivo can promote recovery of hematopoietic cells & reduce the risk of graft failure
        
        MSC can induce paracrin effect
        
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      - Large Scale
        
        Gastrulation
        
        Where the embryo reorganizes from a simple ball of cells into a multilayered structure called the gastrula
    - - 2 different cell types interact
        
        Re-organize to form a tissue outline
        
        Several different ways of interaction
        
        Diffusion of soluble signals
        
        Contact w/ insoluble ECM
        
        Contact btwn membrane-bound receptors
      - 4 Types
        
        Negative
        
        Collective of cells restrict potential of each other
        
        Instructive
        
        Change cell type of responder cell due to interaction
        
        Responder cell will not do anything w/o instructor cell
        
        Salivary glands are an example
        
        Mesenchyme turns into neural precursors
        
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        Permissive
        
        Responder cell contain all potential needed, but require an environment (e.g. ECM)
        
        Reciprocal
        
        Tissues signal each other to grow (e.g. kidney morphogenesis)
        
        4 Steps
        
        Loose mesenchymal cells interact w/ epithelium ureter bud
        
        Leads to elongation & iterative branchin of uretic bud
        
        Leads to inducuction & differentiation of nephrons at uretic bud
        
        Mesenchymal cells begin to condense around tip & undergo mesenchymal to epitheleal conversion
        
        Forms the vesicle
        
        Vesicle forms the comma-body
        
        Gamma body decelops to form S-body
        
        S-Body differentiates to form
        
        Glomerulus
        
        Tubules
    - - Initiation
        
        Initial arrangement & structure of cells
        
        Can have a tremendous impact on morphogenesis process
      - Spatio-temporal Process
        
        The coordinated activity of multiple cell types over space & time
      - Final State
        
        Quasi-stable state where no further morphogenic process occurs
    - - Number of cells that participate
        
        <1000 cells
      - Physics dimensions
      - Time scales of the process
        
        Hours to days
      - Magnitude of the biomechanical forces at work
      - Energy Expenditure