The end of the beginning for pluripotent stem cellsPeter J. Donovan* & John Gearhart†

*Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania†The Institute of Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland

Jason Ip

Graduate Student

“If we do the work that we can do in this country, the work that we

will do when John Kerry is president, people like Christopher

Reeve will get up out of that wheelchair and walk again”

-Senator John EdwardsOctober 12, 2004

Background Pluripotent stem cells

def. cells in a stem cell line capable of differentiating into several different final differentiated types

First recognized in teratocarinomas

Primordial germ layers The ectoderm, endoderm,

and mesoderm, are the three major cell lineages

Formed during gastrulation (cell migration resulting in cleavage)

Background The three types of embryonic

tissue Embryonic stem (ES) Embryonic germ (EG) Embryonal carcinoma (EC)

Attributes of EC, ES, and EG cells Transcription factor Oct4 Alkaline Phosphatase Telomerase

…Upregulation sustains pluripotency

Agenda

The Science of Pluripotency Developmental potential The basic biology of human development Embryonic stem cells vs. adult stem cells

Bringing stem cells to the clinic Expansion and differentiation Safety considerations in cell-based therapies The future of stem cells

The Science of Pluripotency

Developmental potential

Assessment in three independent assays

1. In vitro differentiation in a Petri dish

2. Differentiation into teratomas or teratocarcinomas within histocompatible mice

3. In vivo differentiation within blastocoel cavity of a pre-implantation embryo

Developmental potential

Directing differentiationManipulation of cellular environment

Growth of cells at high density Growth of cells on different types of feeder cells Addition of growth factors Growth on crude or defined ECM substrates …differentiation varies and lacks robustness

Suspended three dimensional aggregation Development of embryoid bodies

Developmental potential Embryoid bodies

Capable of differentiating into any of the three primordial germ layers

Germ layer cells are multipotent, as opposed to pluripotent

The basic biology of human development Pluripotent stem cells can…

Aid in deciphering developmental gene-expression

Survival Proliferation Differentiation Migration

Lend insight to tumorigenesis and genetic diseases

Embryonic stem cells vs. adult stem cells Ethical consideration

What marks the beginning of life? Main differences

The number of potential derivatives; embryonic > adult

FeasibilityLack of publication on adult stem cell

research (add more?)

Bringing stem cells to the clinic

Successful transplantations of mouse ES cells1. Cardiomyoctes form stable and functional

grafts2. Glial precursors interact with host neurons to

replenish lost myelin in the brain and spinal cord

3. Embryoid bodies differentiate into neurons in the spinal cord, promoting motor recovery

4. Insulin-producing cell line implanted into mice resulted in normalized glycemia

Successful transplantations of mouse ES cells Rats with motor injury and stroke treated with

neuronal cells derived from human EC cells resulted in partial recovery of motor function

•••

?

Successful transplantations of mouse ES cells

•••

Transplanted cells are replacing lost cells such as

neurons or glia

Transplanted cells providing factors facilitating the

regeneration of host cells

Cell-based therapies may be useful in abating the effects of injury and disease

Stem cell expansion and differentiation Requirements leading to clinical therapy

1. Growth in large quantities

2. Controlled homogeneous differentiation

3. Histocompatibility Limitations

1. Stem cell survival in long-term culture

2. Stem cell genetic mutations

Stem cell expansion and differentiation Possible solutions

Progenitor cells Advantages

Derived from embryo bodies Easier to grow and expand Possess normal karyotype

Disadvantages Limited self-renewal capability Can be unipotent or

multipotent Example:

Neural progenitors can be formed from human ES cells in high density culture, become neurons

Stem cell expansion and differentiation Genomics

Microarray technology can reveal expression of growth factors, growth-factor receptors, and cell-adhesion molecules

Expression profiles allow for optimal conditioning of stem cell growth environment

Safety considerations in cell-based therapies Three key safety issues:

1. Histocompatibility

2. Tumorigenesis

3. Infection from serum-containing culture

Safety considerations in cell-based therapies Histocompatibility

Immune suppression Slows immune response Nonspecific

Tolerance induction Antigen-induced Specific

Embryo-derived compatible cells Therapeutic Cloning (somatic cell nuclear transfer) Genetic recombination of existing stem cell lines to match

patient

Safety considerations in cell-based therapies

Genetic RecombinationTherapeutic cloning

Safety considerations in cell-based therapies Tumorigenesis

Conflicting arguments Imprinted genetic loci are erased in EG cell lines EG cell lines behave normally in chimeras

Key questions How can cells be ensured to migrate to designated

sites? At what stage of differentiation should

transplantation occur? (hmmm?)

Safety considerations in cell-based therapies Infection from serum-

containing culture Infection caused by blood-borne

bacteria Serum contains necessary

growth factors Human ES cells require fetal

calf serum or conditioned medium via mouse feeder cells for growth

The future of stem cells

Mouse ES cells have contributed much to our understanding of embryogenesis

ProspectsStem cell therapeuticsDeeper understanding of human growth and

development Treatment on non-human primates likely

to be a next step before use in the clinic

References

http://www.biology-online.org/dictionary.asp http://www.csa.com/discoveryguides/stemcell/images/pluri.jpg http://en.wikipedia.org/wiki/Image:Cloning_diagram_english.svg http://alignmap.com/wp-content/Graphics/JohnEdwards(098).jpg http://upload.wikimedia.org/wikipedia/commons/d/d4/Cell_differentiation.gif http://www.bio.miami.edu/dana/pix/gastrulation.jpg http://abcnews.go.com/Health/wireStory?id=4313450 http://www.brown.edu/Courses/BI0032/adltstem/stem-cell.gif http://www.wormbook.org/chapters/www_germlinegenomics/

germlinegenomicsfig1.jpg http://static.howstuffworks.com/gif/stem-cell-therapeutic.gif http://regentsprep.org/Regents/biology/units/reproduction/crossingover.gif http://omegascientific.com/catalog/images/fetal-bovine-fam.jpg