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