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… Move to soil…

Cells “dedifferentiate” and begin to divide,
forming “callus” tissue…
Moore et al Figure 9.2
Wm C Brown Publishing
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 Adapted from MBoC (4)
figures 21-85 and 21-86
embryos © Garland Publishing

Aggregate in vitro
Inject one cell from “red” 8-
cell embryo into “grey”
blastocyst

Culture in vitro
to “blastocyst”

Red blastomeres incorporate

into “inner cell mass” of
blastocyst

Implant into hormonally-

primed female for
gestation and birth Implant into hormonally-
primed female for gestation
and birth
Descendants of red cells in all
Tetraparental tissues of resulting chimeric
“chimeric” pup 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 Inject bone marrow from healthy
of blood forming cells… 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 =
Tripolar spindle centrosome protein

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 1 ES line
No needles! (much lower than 50% of blastocysts using IVF)
ECB 21-41
 Images of enucleation and ES colonies

After:
Spindles
spindles
before
enucleation 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
Neuroepithelial rosset
epithelium

ostoid island
showing bony cartilage
differentiation

Shows
pleuripotency of
glandular epithelium
with smooth muscle human ES line
and connective tissue