1. 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

2. 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

3. 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

4. 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

5. Cells of adult plants remain totipotent: cloning a carrot Moore et al Figure 9.2 Wm C Brown Publishing 1 mm 3 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…

6. 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)

7. 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

8. 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

9. 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)

10. 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

11. Stem cells that resupply differentiated cells are pluripotent: example blood Hemopoetic stem cell: Divides to renew itself for lifespan of animal Can 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

12. 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

13. 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

14. 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?

15. Loss of dopamine-producing cells in the brain Goal: stem cell replacement Mouse embryonic stem cells -- cured mouse Parkinson’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.

16. 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

17. Two types of cloning: reproductive and therapeutic ECB 21-41 Reproductive cloning has been accomplished for large mammals, not humans Therpeutic cloning in humans reported two months ago Somatic nucleus must be reprogrammed to embryonic program by egg cytoplasm Somatic cell nuclear transplant (SCNT)

18. Q: other animal species cloned? A: Mice, pigs, cats, cows, mule, horse etc Reproductive cloning of Dolly the sheep

19. San Diego Zoo: frozen tissue Used dolly-type cloning, frozen nucleus implanted into a regular cow cell Banteng: endangered cow species

20. Q: human cloning?

21. 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.

22. 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 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

23. 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)

24. 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)

25. 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