Properties of all stem cells Two characteristics define a stem cell: Self Renewal and Differentiation. ES cells can differentiate into all known cell types of the organism - pluripotent Adult SCs differentiate into a limited number of functional/differentiated progeny – multipotent Any claim of the isolation of a Stem Cell must be supported by evidence of self-renewal and differentiation of the same cell. Stem cells are euploid, normal diploid.

Embryonic Stem Cells [hES] versus human Adult Stem Cells hES originate from the ICM of preimplantation blastocysts. hES cells are pluripotent: ES can differentiate into all cell types, including germ cells Are found in, and have been grown from bone marrow, heart, brain, liver, skin, prostate,etc Adult SC are merely multipotent. Adult SC can differentiate into a limited number of functional cells

hES versus Adult Stem Cells ES cells are immortal ES cells self-renew symmetrically = exponentially In vivo, ES cells are tumorigenic, giving rise to teratomas. Adult SC are mortal, their self-renewal is limited to ~30-40 doublings Adult SC self-renew asymmetrically, cannot be grown to infinite numbers Adult SC are not tumorigenic in vivo

Adult Stem Cells *Adult Stem Cells = Organ SCs = Somatic SCs = Post-implantation SCs. *At implantation ES cell self-renewal switches from symmetric to asymmetric. *The molecular basis for this switch is not known.

Weaknesses of current techniques used to grow hES cells Ethical considerations Moral attitudes The law (other)

Weaknesses of current results with ES cells The efficiency of converting hES cells to functional cells suitable for transplantation is low Problems of graft rejection have not been solved in any system Ethical problems may not be relieved by political changes for many years.

Amniotic Fluid Stem Cells Amniotic fluid harbors adult stem cells that can be grown in culture and differentiated in at least 4 directions, Harvesting amniotic fluid is not contentious ethically nor medically Hundreds of thousands of donors are available to cover most possible transplantation backgrounds

MAFSC cells grown from AF samples AES-7 9/04/02

Confluent MAFSC Cultures

NANOG SOX2 OCT4 REX Expression of Stem Cell-Associated Genes by Adherent MAFSC Cells RT-PCR of Stem Cell-associated genes from AF adherent colonies. Lanes 1,3,4 & 6: RNA from cells at 38 days post-plating. Lanes 2 & 5: RNA from cells at 31 days post-plating. Lane 7: Control: no RNA added.

MAFSC Differentiation into adipocytes and osteoblasts c: Fat cells, Oil Red O staining. d: Osteogenic diff., von Kossa staining Hum Reprod. 19:1450, 2004

MAFSC Differentiation into neuronal cells

Neural differentiation of MAFSC Cells MAFSC cells were treated for 15 hours with neural induction medium, fixed with paraformaldehyde: stained with Gills’s haematoxylin (panels A and B), incubated with a primary antibody specific for  -Tubulin III (panels C and D), and GFAP (panels E and F) respectively followed by a secondary FITC-conjugated antibody. Twenty-four hours before neuronal induction, the subconfluent culture medium was replaced with a preinduction medium consisting of DMEM with 20% FBS and 1mM β-Mercaptoethanol (BME). Preinduction medium were removed, the cells were washed with PBS and transferred to a neuronal induction serum-free medium composed of DMEM with 2% dimethylsulfoxide (DMSO), 200 μM butylated hydroxyanisole (BHA), 25 mM KCl, 2 mM valproic acid, 10 μM forskolin, 1 μM hydrocortisone and 5 μg/ml insulin. After 15 hours of induction treatment, the cells were fixed with 4% paraformaldehyde for immunofluorescence analysis.

What are MAFSC Cells Good For? New York Times, November 18, 2006: “Stem Cell Experiment Yields Heart Valves” CHICAGO, Nov. 17 (AP) — Scientists for the first time have grown human heart valves using stem cells from the fluid that cushions babies in the womb, offering an approach that may be used to repair defective hearts. The idea is to create new valves in the laboratory while a pregnancy progresses and have them ready to implant in a baby with heart defects after it is born.

What are MAFSC Cells Good For? The Swiss experiment follows recent successes at growing bladders and blood vessels and suggests that people may one day be able to grow their own replacement heart parts, in some cases, even before they are born. It is one of several tissue engineering advances that could lead to homegrown heart valves that are more durable and effective than artificial or cadaver valves. “This may open a whole new therapy concept to the treatment of congenital heart defects,” said Dr. Simon Hoerstrup of the University of Zurich. Dr. Hoerstrup led the work, which was presented Wednesday at an American Heart Association conference. However:

Weakness of current techniques used to grow Human Adult Stem Cells All post-implantation stem cells are mortal, whether grown from prostate, heart, liver, lung, brain, skin or Amniotic Fluid. The cells senesce after ~40 doublings Thus, one cannot (yet) grow a large number of stem cells from the amniotic fluid….. This has been known as the Achilles Heel of the Adult Stem Cell field. What is one to do?