Housekeeping

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Housekeeping

• Importance of scrupulousness in culture room
• Cell adhesion variations
• Confluence formula on p. 44 should have a capital
  E
• Quiz results
• Ketones and aldehydes have double bonds that may
  fluoresce and glycine binds and blocks those
• Use corrected lab sheet
• Pre-lab on p. 56p. 22 has E63 specifics, but you
  will still seed what TA says to seed.
• Post-lab on p.58 (formula on p. 56)

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Part 1

• An Introduction to Stem Cells

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4 types of stem cells

• Embryonic stem cells - Stem cells taken from
  human embryos
• Fetal stem cells- Stem cells taken from aborted
  fetal tissue
• Umbilical stem cells - Stem cells take from
  umbilical cords
• Adult stem cells - Stem cells taken from adult
  tissue

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Definition of a Stem Cell

• Capable of self renewal
• Capable of multilineage differentiation
• In vivo, capable of functional reconstitution of
  a given tissue
• In tissues that use stem cells, if the stem cells
  lose their ability to regenerate and provide
  further pool of stem cells, the tissue will
  degenerate.

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Stem Cells

• The body uses stem cells when final
  differentiated state is incompatible with cell
  division

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• Stem cells used where turnover occurs rapidly
• Lining of the gut, mouth, esophagus
• Epidermal layer of skin
• Blood forming tissues

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Stem Cell Development

• Development of stem cells begins after exposure
  to a morphogen
• Substance that influences the development of form
• Once they begin to change, researcher can then
  determine which of four basic tissue types it
  will develop into.
• Epithelia, neural, muscle, or connective

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Potential of stem cells

• Pluripotent
• Give rise to most cells of the body
• Mesenchymal cells fit this category
• Totipotent
• Can generate the totality of cell types that
  comprise an organism
• Restricted in use to describe the potential of
  fertilized eggs and blastomeres of the early
  embryo

• Unipotent
• Gives rise to only one type of cell
• Germ cells best example
• Sperm and oocytes
• Oligopotent
• Give rise to only a few types of cells
• Epidermal cells
• Multipotent
• Give rise to many types of cells
• Intestinal stem cells

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Adult stem cells

• Hematopoietic stem cells (HSCs)
• Blood and bone marrow
• Connective tissue stem cells
• Fat, bone, cartilage--fibroblasts
• Some epithelial stem cells
• Skin, sensory reception
• Neuronal stem cells (NSCs)
• Muscle stem cells (MSCs)

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From marrow, umbilical cord Sometimes in blood
stream
HSC
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Part 2

• Fibroblasts as stem cells, their differentiation
  into adipocytes, and potential clinical uses

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Fibroblasts are Unique

• The family of connective tissue cells include
• Fibroblasts
• Cartilage cells (chondrocytes)
• Bone-forming cells (osteocytes)
• Fat cells (adipocytes)
• Smooth muscle cells
• Fibroblasts are able to transform into any of the
  other member of the family, sometimes reversibly

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• Transformations are regulated by the composition
  of ECM, cell shape, hormones and growth factors

fibroblast
cartilage cell
Bone cell
Fat cell
Smooth muscle cell
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Fibroblasts

• Function
• Undoubtedly, the most ubiquitous cell
• Active cells
• Forms collagen, collagenases, elastin and other
  fibers
• Produces ground substance (ECM)

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• Appearance (in vivo)
• Spindle shaped or possess wide cytoplasmic
  processes.
• Cytoplasm looks bluish in typical histological
  stains
• Nucleus has prominent nucleolus/nucleoli

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White Adipocytes

• Appearance
• triglycerides accumulated in cytoplasm
• non vesicle bound
• nucleus pushed to the side.

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• Function
• Uptake, synthesis, storage and mobilization of
  neutral lipid
• used as fuel for cells, cushion, insulation

• Location
• under skin
• around kidneys
• in bones (adults)

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Adult Adipose is a source of stem cells

• White adipocytes removed from body
• Cultured in presence of de-differentiating
  factors (especially low serum)
• cells revert to fibroblasts
• Cells given various factors to stimulate
  production other cell types
• New cells replaced into damaged area in vivo
• Still a long way to go before this more widely
  used.

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Fat used to repair skull

• December 2004 in Germany on 7 year old girl
• Limited amount of bone can be taken from iliac
  crest
• Fat-derived stem cells might play a role in
  augmenting the repair process
• Anecdotal evidence of potential application

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Differentiation

• Differentiation is the process by which
  scientists preprogram the stem cells to become
  specific cells.
• Embryonic and fetal stem cells have the potential
  to morph into a greater variety of cells than
  adult stem cells do.
• But, the factors required to coax ES and FS cells
  into newer cells are also less well known
• Major ethical questions

• Differentiated cells are then injected into the
  area of the body being targeted for tissue
  regeneration.
• When stem cells come into contact with growth
  chemicals in the body, the chemicals program the
  stem cells to grow into the tissue surrounding
  it.

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Differentiation of cells in culture
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How differentiation occurs in cultured cells

• Spontaneously
• Will happen with our E63s
• Cell-cell interaction
• Add chemical inducer of differentiation
• As with the fibroblast/adipocyte
• Common inducers for many cell types include
• Retinoic acid
• DMSO
• DEX
• Withdraw factors required for pluripotence
• Change the DNA

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Inducing differentiation in 3T3s

• Dexamethasone-
• a glucocorticoid (stress hormone)
• a common inducer of differentiation
• Isobutylmethylxanthine-
• in the caffeine family
• causes glycogen stores to become glucose
• increases levels of cAMP
• Insulin-produced by islet of Langerhans cells in
  pancreas in response to spike of glucose
• Pool of glucose and triglycerides
• From our serum supplemented media

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Dexamethasone
IBMX
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Brief interpretation of previous image

• Dexamethasone and IBMX will encourage the cells
  to uptake triglycerides from the serum.
• Only used for induction
• Insulin prevents triglyceride liberation from the
  cells.
• Continued for differentiation

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Take home message from this image insulin
glucose pool of fatty acids fat cells
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Fat, sugar and caffeine
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Oil Red O staining of lipid inclusions
This slide also seems to have a counterstain
which we will not have
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Part 3

• E63 Muscle Cell Line

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Overview of Muscle Cell Types
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Skeletal muscle morphology
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Skeletal muscle development

• Myoblasts
• one nucleus each
• lacking myofibrils

• Myotubes
• develop myofibrils
• other organelles

Skeletal muscle is a multinucleated syncitium
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E63s

• A cell line created here at U of I from rats
• Stephen Kaufmans lab
• Grows in Horse Serum rather than Newborn Calf
  Serum
• Use the appropriate media!
• Use Kanamycin rather than Penicillian/Streptomycin
  
• Grown in 8-10 CO2

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Additional Exercises for Today

• You need more adipocytes for making lysates next
  week, so you will start another induction today.
• As usual, you will make a 10cm stock dish so you
  can seed a new dish of 3T3s for a control against
  your adipocytes on the return day.
• You will need E63s at full differentiation for
  making lysates next week, so you will seed one
  6cm dish so it will have a chance to fuse, and
  seed a 10cm so you have a stock to make a control
  dish on your return day.