Lecture 24 Differentiation and stem cells
*Stem cells and differentiation in plants
Totipotency
Stem cells in animals
Therapeutic use
Cloning
Therapeutic
Reproductive
Therapeutic cloning in humans
Stem cells
Self-renewal
Differentiation
ECB 21-35
Stem cells undifferentiated cells that divide and give rise to cells that
differentiate into specialized cells of plant and animal tissues
Stem cells in plants are localized in “meristems”
Shoot apical
meristem
Shoot apical
meristem
MBoC (4) figure 21-111 and 112 © Garland Publishing
Shoot apical meristem
Root apical meristem
Lateral or axial meristems
Floral meristem
Cell fate in root is determined by position
endodermis
cortex
stele
Differentiation
renewal
Meristem
Cells leave meristem and enter files (colors) and differentiate into
specific fates (stele, endodermis, cortex etc.)
Cells of adult plants remain totipotent:
cloning a carrot
Regenerated adult plant…
1 mm3 fragments (“explants”)
from adult root…
Induce with hormones to initiate
shoot and root formation…
Culture “embroid” in liquid
culture, then agar…
Culture explants in liquid culture medium…
Cells “dedifferentiate” and begin to divide,
forming “callus” tissue…
Moore et al Figure 9.2
Wm C Brown Publishing
Move to soil…
Cells from young animal embryos are also totipotent
Embryonic stem cells
(ES cells)
ECB 21-40
Totipotent - capable of forming all differentiated cells of adult
Pluripotent - capable of forming more than 1 differentiated cell type
Cells of early mammalian embryos are “totipotent”
8-cell mouse
embryos
Aggregate in vitro
Adapted from MBoC (4)
figures 21-85 and 21-86
© Garland Publishing
Inject one cell from “red” 8cell embryo into “grey”
blastocyst
Culture in vitro
to “blastocyst”
Red blastomeres incorporate
into “inner cell mass” of
blastocyst
Implant into hormonallyprimed female for
gestation and birth
Tetraparental
“chimeric” pup
Implant into hormonallyprimed female for gestation
and birth
Descendants of red cells in all
tissues of resulting chimeric
pup, including germline
Totipotency “lost” during development and differentiation (~16 cells in mouse)
Differentiation occurs in three stages
•
Fertilized animal eggs and early embryonic cells can give rise to all the
different cell types of the body, they are considered “totipotent.”
– Identical twins
•
Cell fates become progressively restricted during development, a process
called “differentiation.”
•
Differentiation occurs in three stages
– Specification
• Fate is not absolute
• Cell identity subject to change
– Determination
• Fate is fixed, and cannot change in response to environment
– Differentiation
• Changes in cell structure and function
How do cells lose totipotency?
• Gross DNA rearrangement or loss (rare?)
– B-lymphocytes (make antibodies) splice genes encoding IgG HC
– Mammalian erythrocytes (red blood cells) enucleate
• Terminal differentiation (some tissues/cells)
– Loss of cell division capacity: muscle, neurons, others
• Altered gene expression (most common)
– Transcriptional regulation by transcription factors,
– Reversible, in principle (with difficulty)
Differences in gene expression make all cell
types of organism unique
ECB 8-15
Genes A, B , C, D
smooth muscle transcribes A, B
hepatocytes A, C
Lymphocytes B, C, D
35,000 -40,000 genes allow nearly infinite combinations to define cell type
Stem cells that resupply differentiated cells are
pluripotent: example blood
Blood cells must be renewed
but not capable of cell division
(red blood cells lack a nucleus)
ECB 21-39
Hemopoetic stem cell:
Divides to renew itself for lifespan of animal
Can form a limited number of cell types (pleuripotent)
But not differentiated
Bone marrow contains hemopoietic stem cells for blood cells
X-irradiation stops production
of blood forming cells…
Inject bone marrow from healthy
donor of different MHC “tissue type”…
Lethal without treatment…
Irradiated host survives after
bone marrow transplant…
New blood cells have MHC type of
marrow donor…
MBoC (4) figure 22-34 © Garland Publishing
Lecture 24 Differentiation and stem cells
Stem cells and differentiation in plants
Totipotency
Stem cells in animals
Therapeutic use
Cloning
Therapeutic
Reproductive
Therapeutic cloning in humans
Stem Cells -- therapeutic use?
• Embryonic stem cells donated embryos
from In Vitro Fertilization clinics
• 4-5 days old (blastocyst stage)
• cultured cells grow in petri plates
(30 cells --> millions after ~6 months
• Conduct research to try to induce them to differentiate into
specialized cell type of interest
• Great potential for therapeutic uses:
-inject patient with stem cells that are induced to
differentiate into defective cell, tissue
Parkinson’s disease
Loss of dopamine-producing cells in the brain
Goal: stem cell replacement
Mouse embryonic stem
cells -- cured mouse
Parkinson’s disease
(model system)
Hope for treatment of diabetes, osteoarthritis etc.
Using embryonic stem cells from patient would eliminate risk of rejection
Federal Regulations
G.W. Bush: August 2001:
federally-funded research - can only use
previously isolated ES cells
(~17 lines in use, most in private laboratories)
2 issues with ES cells:
1. The source
2. The potential to clone humans
Two types of cloning: reproductive and therapeutic
Somatic cell nuclear transplant (SCNT)
Somatic nucleus must be
reprogrammed to embryonic
program by egg cytoplasm
ECB 21-41
Reproductive cloning has been accomplished for large mammals, not humans
Therpeutic cloning in humans reported two months ago
Reproductive cloning of Dolly the sheep
Q: other animal
species cloned?
A: Mice, pigs, cats, cows,
mule, horse etc
Banteng:
endangered cow species
San Diego Zoo: frozen tissue
Used dolly-type cloning,
frozen nucleus implanted into a regular cow cell
Q: human cloning?
Rhesus Monkey
model for primate cloning, no success!
Problems in mitosis following nuclear transplant
Regular fertilized
egg. Green =
centrosome protein
Tripolar spindle
In primates, removal of nucleus also removes most of the spindle proteins.
Aberrant cell division--> gross chromosomal segregation defects.
Existing ES lines created by in vitro fertilization
In vitro fertilization (IVF): use normal human egg/sperm for
fertilization followed by lab culture until young embryo and then
implant into female
Rather than implant, these embryos can be used to isolate ES cells
About half of embryos made by IVF yield ES cell lines
But no success with nuclear transplant method until recently……..
Hwang et al., Evidence of a Pluripotent Human Embryonic Stem Cell
Line Derived from a Cloned Blastocyst. ScienceExpress 12 Feb 2004
Go to Marriot library, and log onto
http://www.sciencemag.org/cgi/rapidpdf/1094515v1
Experimental procedure for therapeutic
human cloning
Somatic cell nuclear transplant (SCNT)
Cumulus cells from ovary (2N)
20 blastocyst
embryos
242 eggs from
16 women:
Voluntary donors
Electrofusion of cells
Poke hole in eggs and gently extrude
spindle
No needles!
ECB 21-41
1 ES line
(much lower than 50% of blastocysts using IVF)
Images of enucleation and ES colonies
Spindles
before
enucleation
After:
spindles
outside
egg
Light microscopy of human ES cell colonies
Immunofluorescence for nestin
(marker of ES cells)
Karyotype (2N)
Human ES cells cause teratomas in
immunodeficient mice
Teratoma = cancerous tissue containing lots of different cell types
pigmented
retinal
epithelium
Neuroepithelial rosset
ostoid island
showing bony
differentiation
cartilage
glandular epithelium
with smooth muscle
and connective tissue
Shows
pleuripotency of
human ES line