6. TWO BASIC CHARACTERISTICS:
• 1. Self-renewal
• Grow and proliferate indefinitely by mitosis to
create populations of identical cells
• 2. Differentiation
• Differentiate into adult cells and tissue types of
the body
• Controlled by key signals
7. POTENCY
• Some stem cells possess greater ability to
differentiate than others (potency)
• Totipotent
• Can form all adult body cell types
• Can also form the specialized tissues needed for
development of the embryo
• Ex. placental cells
• Pluripotent
• Have the potential to eventually differentiate into all
of the 220 human adult cell types
10. BLASTOCYST
• Inner Cell Mass
• Source of human embryonic stem cells (hESCs)
• Help to develop the embryo
• Pluripotent
11. HESCS
• The first successful isolation and culturing of hESCs occurred in 1998 by James Thomson
at the University of Wisconsin
• Also, in 1998 John Gearheart and colleagues at John Hopkins Universiy isolated
embryonic germ cells (primitive cells that form the gametes) from human fetal tissue
12. HESCS
• When hESCs are isolated, scientists use a
holding pipette that applies a brief
suction to “hold” the blastocyst in place
• A glass micropipette is then inserted into
the blastocyst to gently remove cells from
the inner mass
• These cells are then cultured in the lab
• Work is derived from stem cell research in
other animals such as monkeys, sheep,
cows and mice
13. HESCS
• Initially, the main source of hESCs was leftover embryos from assisted reproductive
technologies such as IVF
• Excess IVF embryos are frozen at ultra-low temperatures, destroyed, or donated to
research
• An estimated over 600,000 of IVF-generated embryos are stored in clinic within the
U.S. alone
16. HESCS
• hESCs avoid senescence in part because they express high levels of telomerase
• Some stems cells have been maintained for over 3 years and over 600 rounds of
division without apparent problems
• Cultured cells that can be maintained and grown successfully are called cell lines
• Stem cells grow rapidly and can be frozen for long periods of time and still retain
their properties
17. STIMULATING HESCS
• Directed differentiation - can be coaxed
into different types of cells in vitro
• Regenerative medicine
• Major focus – what controls the
pluripotency of stem cells into discrete
cell types
• Includes growth factors, hormones,
small proteins
18. NANOG
• TGF – β, bone morphogenic proteins
(BMPs) and other growth
differentiation factors act on a gene
for a transcription factor called
Nanog
• Nanog is a key protein the maintains
hESCs in an undifferentiated,
pluripotent state
21. ASCS
• ASCs reside in differentiated tissues of the
body
• Appear in small numbers
• Can be isolated
• Ex. brain, intestine, hair, pancreas, fat
22. ASCS
• Do not require the destruction of an embryo
• Harvested by fine-needle biopsy
• Possibly even from cadavers
• Abundant in adipose tissue
23. ASCS
• ASCs from one tissue can differentiate into another different specialized cell type
• Ex. ASCs from muscle tissue could be used to develop a blood cell
• Some studies have shown that ASCs may not be as pluripotent as hESCs
26. AMNIOTIC FLUID
• Stem cells can be isolated from amniotic fluid
• Have been differentiated into neurons, muscle cells, adipocytes, bone, blood vessels
and liver cells.
• Not entirely clear is these are truly different from hESCs or ASCs
27. PLACENTAL
• Stem cells from cord tissue or cord
blood
• Easily accessibility
• Painless to both mother child
• Pluripotent
• Decrease chances of rejection
29. NUCLEAR REPROGRAMMING
• Producing pluripotent cells without
destroying embryos
• Nuclear reprogramming of somatic
cells
• Once cells differentiate their fate IS
reversible
30. NUCLEAR REPROGRAMMING
• The basic idea:
• Take a differentiated, adult cell
• Alter gene expression patterns
• Reprogram cell to an early stage in the
differentiation pathway
• Push cells backwards to undifferentiated,
pluripotent state
31.
32.
33. INDUCED PLURIPOTENT CELLS
• The use of transcription-factor genes to induce reprogramming creates induced
pluripotent cells (iPSCs)
• Success in cows, dogs, horses, humans, mice, primates, pigs, rats and sheep
• Revolutionary process with immense potential
34.
35. IPSCS
• In 2006, Shinya Yamanaka of Kyoto University created the first
iPSCs
• Used retroviruses to deliver four transgenes
• Expression of these four genes “reprogrammed” mouse
fibroblasts to an earlier stage
• Show many properties of hESCs, including self-renewal and
pluripotency
• Appear indistinguishable for hESCs
• Also has led to the development of a new way to clone adult
mammals
36.
37. IPSCS
• Researchers are trying to produce viral vector-free iPSCs to avoid random
integration into the genome
38. IPSCS
• In 2010, researchers at Mount Sinai School of Medicine demonstrated that fetal skin
cells in amniotic fluid could be readily reprogrammed into iPSCs with greater
efficiency than other somatic cells
• Skin cells from amniotic fluid were cultured and tranfected with plasmids
• Showed gene-expression patterns characteristic of stem cells and telomerase
activity
• Capable of differentiation both in vivo and in vitro
39. TRANSFECTION
• Transfection is the process of deliberately introducing
nucleic acids into cells
• Often used for non-viral methods in eukaryotic cells
• May also refer to other methods and cell types,
although other terms are preferred - transformation is
more often used to describe non-viral DNA transfer in
bacteria and non-animal eukaryotic cells
• In animal cells, transfection is the preferred term as
transformation is also used to refer to progression to a
cancerous state (carcinogenesis) in these cells
40. IPSC CHALLENGES
• 1. Are relatively inefficient to produce
• (only about 1 in 1,000 somatic cells
exposed to most reprogramming
approaches becomes an iPSC)
41. IPSC CHALLENGES
• 2. Require constant feeding to
maintain viable cell lines
• 3. Show low viability compared
to other cell types once they
have been stored frozen
43. IPSC CHALLENGES
• 5. May retain an epigenetic memory that cannot be fully erased by reprogramming
44. IPSC CHALLENGES
• 6. May be prone to
single-nucleotide
polymorphisms, copy-
number variations, and
other mutations
45. IPSC CHALLENGES
• 7. Occasionally show spontaneous
differentiation into mature cell types
when in culture
• 8. Can sometimes be difficult for
directing differentiation into
particular cell types
