Lecture 24 Differentiation and stem cells
*Stem cells and differentiation in plants
Totipotency
Stem cells in animalsTherapeutic use
CloningTherapeuticReproductive
Therapeutic cloning in humans
Stem cells
Stem cells - undifferentiated cells that divide and give rise to cells that
differentiate into specialized cells of plant and animal tissues
Self-renewal
Differentiation
ECB 21-35
Stem cells in plants are localized in “meristems”
MBoC (4) figure 21-111 and 112 © Garland Publishing
Shoot apical meristem
Root apical meristem
Lateral or axial meristems
Floral meristem
Shoot apical meristem
Shoot apical meristem
Cell fate in root is determined by position
Meristem
renewal
Differentiation
Cells leave meristem and enter files (colors) and differentiate into specific fates (stele, endodermis, cortex etc.)
endodermis
cortex stele
Cells of adult plants remain totipotent: cloning a carrot
Moore et al Figure 9.2 Wm C Brown Publishing
1 mm3 fragments (“explants”) from adult root…
Culture explants in liquid culture medium…Cells “dedifferentiate” and begin to divide, forming “callus” tissue…
Induce with hormones to initiate shoot and root formation…
Culture “embroid” in liquid culture, then agar…
Move to soil…
Regenerated adult plant…
Cells from young animal embryos are also totipotent
Totipotent - capable of forming all differentiated cells of adult
Pluripotent - capable of forming more than 1 differentiated cell type
ECB 21-40
Embryonic stem cells (ES cells)
Cells of early mammalian embryos are “totipotent”
Totipotency “lost” during development and differentiation (~16 cells in mouse)
8-cell mouse embryos
Culture in vitro to “blastocyst”
Aggregate in vitro
Implant into hormonally-primed female for gestation and birth
Tetraparental “chimeric” pup
Red blastomeres incorporate into “inner cell mass” of blastocyst
Inject one cell from “red” 8-cell embryo into “grey” blastocyst
Implant into hormonally-primed female for gestation and birth
Descendants of red cells in all tissues of resulting chimeric pup, including germline
Adapted from MBoC (4) figures 21-85 and 21-86© Garland Publishing
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, Dsmooth muscle transcribes A, Bhepatocytes A, CLymphocytes 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
Hemopoetic stem cell:Divides to renew itself for lifespan of animalCan form a limited number of cell types (pleuripotent)But not differentiated
Blood cells must be renewed but not capable of cell division (red blood cells lack a nucleus)
ECB 21-39
Bone marrow contains hemopoietic stem cells for blood cells
Inject bone marrow from healthy donor of different MHC “tissue type”…
X-irradiation stops production of blood forming cells…
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 animalsTherapeutic use
CloningTherapeuticReproductive
Therapeutic cloning in humans
• 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
Stem Cells -- therapeutic use?
Loss of dopamine-producing cells in the brain
Goal: stem cell replacement
Mouse embryonic stemcells -- cured mouseParkinson’s disease(model system)
Parkinson’s disease
Using embryonic stem cells from patient would eliminate risk of rejection
Hope for treatment of diabetes, osteoarthritis etc.
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
Federal Regulations
Two types of cloning: reproductive and therapeutic
ECB 21-41
Reproductive cloning has been accomplished for large mammals, not humansTherpeutic cloning in humans reported two months ago
Somatic nucleus must be reprogrammed to
embryonic program by egg cytoplasm
Somatic cell nuclear transplant (SCNT)
Q: other animal species cloned?
A: Mice, pigs, cats, cows, mule, horse etc
Reproductive cloning of Dolly the sheep
San Diego Zoo: frozen tissueUsed dolly-type cloning, frozen nucleus implanted into a regular cow cell
Banteng: endangered cow species
Q: human cloning?
Problems in mitosis following nuclear transplant
Rhesus Monkey model for primate cloning, no
success!
Tripolar spindle
Regular fertilized egg. Green =centrosome protein
In primates, removal of nucleus also removes most of the spindle proteins.
Aberrant cell division--> gross chromosomal segregation defects.
In vitro fertilization (IVF): use normal human egg/sperm for fertilization followed by lab culture until young embryo and then implant into femaleRather than implant, these embryos can be used to isolate ES cells
About half of embryos made by IVF yield ES cell lines
Existing ES lines created by in vitro fertilization
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
ECB 21-41
Somatic cell nuclear transplant (SCNT)
Cumulus cells from ovary (2N)
Poke hole in eggs and gently extrude spindle
No needles!
Electrofusion of cells
242 eggs from 16 women:Voluntary
donors
20 blastocyst embryos
1 ES line(much lower than 50% of blastocysts using
IVF)
Images of enucleation and ES colonies
Spindlesbefore
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
glandular epithelium with smooth muscle and connective tissue
Neuroepithelial rosset
pigmented retinal epithelium
ostoid island
showing bony
differentiation
cartilage
Shows pleuripotency of human ES line
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