Lecture 24 Differentiation and stem cells Stem cells

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 MBo. C (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 mm 3 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 MBo. C (4) figures 21 -85 and 21 -86 © Garland Publishing 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 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 Ig. G 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… MBo. C (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. Science. Express 12 Feb 2004 Go to Marriot library, and log onto http: //www. sciencemag. org/cgi/rapidpdf/1094515 v 1

Experimental procedure for therapeutic human cloning Somatic cell nuclear transplant (SCNT) Cumulus cells from ovary (2 N) 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 (2 N)

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