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Housekeeping

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Ketones and aldehydes have double bonds that may fluoresce and glycine binds and ... Terminally differentiated cell. Stem cells used where turnover occurs rapidly ... – PowerPoint PPT presentation

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Title: Housekeeping


1
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)

2
Part 1
  • An Introduction to Stem Cells

3
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

4
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.

5
Stem Cells
  • The body uses stem cells when final
    differentiated state is incompatible with cell
    division

6
  • Stem cells used where turnover occurs rapidly
  • Lining of the gut, mouth, esophagus
  • Epidermal layer of skin
  • Blood forming tissues

7
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

8
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9
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

10
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)

11
From marrow, umbilical cord Sometimes in blood
stream
HSC
12
Part 2
  • Fibroblasts as stem cells, their differentiation
    into adipocytes, and potential clinical uses

13
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

14
  • Transformations are regulated by the composition
    of ECM, cell shape, hormones and growth factors

fibroblast
cartilage cell
Bone cell
Fat cell
Smooth muscle cell
15
Fibroblasts
  • Function
  • Undoubtedly, the most ubiquitous cell
  • Active cells
  • Forms collagen, collagenases, elastin and other
    fibers
  • Produces ground substance (ECM)

16
  • Appearance (in vivo)
  • Spindle shaped or possess wide cytoplasmic
    processes.
  • Cytoplasm looks bluish in typical histological
    stains
  • Nucleus has prominent nucleolus/nucleoli

17
White Adipocytes
  • Appearance
  • triglycerides accumulated in cytoplasm
  • non vesicle bound
  • nucleus pushed to the side.

18
  • Function
  • Uptake, synthesis, storage and mobilization of
    neutral lipid
  • used as fuel for cells, cushion, insulation
  • Location
  • under skin
  • around kidneys
  • in bones (adults)

19
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.

20
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21
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

22
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.

23
Differentiation of cells in culture
24
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

25
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

26
Dexamethasone
IBMX
27
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

28
Take home message from this image insulin
glucose pool of fatty acids fat cells
29
Fat, sugar and caffeine
30
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31
Oil Red O staining of lipid inclusions
This slide also seems to have a counterstain
which we will not have
32
Part 3
  • E63 Muscle Cell Line

33
Overview of Muscle Cell Types
34
Skeletal muscle morphology
35
Skeletal muscle development
  • Myoblasts
  • one nucleus each
  • lacking myofibrils
  • Myotubes
  • develop myofibrils
  • other organelles

Skeletal muscle is a multinucleated syncitium
36
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

37
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.
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