Stem Cells in ResearchPromises and Pitfalls

Denise Inman, PhDUniversity of Washington

Department of Neurosurgery

Overview

• What are stem cells?– How do embryonic and ‘adult’ stem cells

differ?– How are different types of stem cell lines

created?

• Stem cells in research and medicine

• Alternatives to the embryo

Early DevelopmentFertilized egg

Totipotentstem cells

Totipotent: Can become any cell in body or placenta

Fate Decision

Pluripotentstem cells Blastocyst

Pluripotent: Can become any cell in body

ImplantationGastrulation

Fate Decision

Multipotentstem cells

Primary Germ CellsEndoderm (inner)Mesoderm (middle)Ectoderm (outer)

Multipotent: Can become any cell within a specific germ layer or cell lineage

Embryonic stem cells come from inner cell mass of blastocyst.

Embryonic Stem Cell Characteristics

•Not committed to a specific fate•Pluripotent—can differentiate into specialized cell types

•Self-renewing

endoderm mesoderm ectoderm

Courtesy of James Thomson, U. Wisconsin-Madison

Is that an Embryonic Stem Cell?

SCID Mouse:Severe CombinedImmunoDeficiency

Embryonic stem cells injected into a SCID mouse will grow into teratomas, tumors of the germ cell layers.

Individual ESCs under the correct conditions will make many different cell types.

The true potential of stem cells can only be assessed retrospectively

Stem Cells: From Embryonic to Adult

http://www.brown.edu/Courses/BI0032

Embryonic stem cells are those removed from the blastocyst before the fate decision from pluripotentiality to multipotentiality.

Adult stem cells are those multipotential cells that persist in fully developed tissues. These cells never differentiated into the mature cell types of the tissues in which they reside.

Adult Stem Cells

• Multipotential – Make cells within a specific lineage

• Not differentiated

• Rare

• Self-replicating

Neural stem cells in culture. One cell is extending a process.

Adult Stem Cells – Bone Marrow

NIH: stemcells.nih.gov/ info/basics/basics4.asp

Major repository of adult stem cells-Hematopoeitic-Mesenchymal

Give rise to immune system cells

Constant turnover

Stem Cell PhenotypeFate dictated by environment…

Neural stem cellsNeuronsAstrocytesOligodendrocytes

Oligodendrocyte Progenitor Cells

Stem cells placed in brain become neurons…

Stem cells placed in spinal cord become glial cells…

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Re-cap: What are stem cells?

• Embryonic and adult stem cells– Obtained at different developmental stages– Different potential

• Pluripotent versus Multipotent

– Sensitive to environment

Overview

• What are stem cells?– How do embryonic and ‘adult’ stem cells differ?– How are different types of stem cell lines created?

• Stem cells in research and medicine– How do scientists work with stem cells?

• In situ labeling• Primary culture• Cell lines

– Promises and perils of stem cells

• Alternatives to the embryo

Cell Lines

• Cells under propagation

• All cells are genetically identical

• Can be frozen and stored

Plate

Exponential Growth

Remove from plateReplate at lower density

Culturing Embryonic Stem CellsObtain stem cells from

1. Remove inner cell mass2. Put cells in dish with feeder layer3. Cells divide

Somatic Cell Nuclear Transfer

Fertilized egg

Blastocyst

+Sperm Oocyte

In Vitro Fertilization

Oocyte without nucleus

Inject nucleus from adult somatic cell

Blastocyst

Origins of ES Cell Lines

• Excess IVF embryos

• Therapeutic Cloning (somatic cell nuclear transfer) – Donor oocyte+somatic cell nucleus– Cells have characteristics of nuclear donor – Lines representing different diseases– Individualized lines: non-immunogenic to

donor

New England Journal of Medicine, Wellcome Trust

Somatic Cell Nuclear Transfer

Removing the egg nucleus before transferring a somatic cell nucleus

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Challenging: In cloned cell lines, about 4% of genes function abnormally, owing to departures from normal activation or expression of certain genes

-Imprinting, methylation state

Limited success: ~25 percent of nuclear transfers led to a blastocyst; 35 percent of blastocysts led to establishment of cell lines

Patient-specific embryonic stem cells derived from human SCNT blastocysts. Science 308(5729):1777-1783, 2005.

hES Cell Lines in the US

• Most, if not all, of the stem cell lines are contaminated with mouse feeder layer proteins.

• These cells will never be used in clinical application.

• Considerable biological variability across cell lines.

• Increased culturing can cause ES cells to accumulate epigenetic and genetic changes, altering their ability to form different types of cells.

Promises and Perils of Stem Cells

• Embryonic stem cells in therapy

• Cloning

• Adult stem cells in therapy

• Beyond cell replacement

• Beyond the embryo

What’s at stake?

What can ESCs do for you?

• Theoretically– Replace damaged, diseased cells– Gene therapy

• Genetically manipulated hES cells might serve as vectors to carry and express genes in target organs following transplantation in the course of gene therapy

Why Clone?

• Human protein production– Produce human protein-based medicine

in milk from transgenic cows• α-1-antitrypsin for cystic fibrosis

• Transplants without immune response – Organ rejection or graft-vs-host disease

Therapeutic and Reproductive Cloning

Therapeutic Cloning

How Promising are Adult Stem Cells?

• Bone marrow transplants– Hematopoeitic stem cell transfer

• Difficulty maintaining control once in vivo– Niche dictates phenotype– Plasticity

Adult Stem Cell Clinical Trials

• Bone marrow stem cells from self or allogeneic (sibling) transplant – after chemotherapy for myeloma,

glioma, leukemia, lymphoma, neuroblastoma, lung cancer

– sickle cell anemia, liver disease, autoimmune disorders, vascular disease

• Mesenchymal stem cells for myocardial infarction

Potential Beyond Cell Replacement

• Exploring disease mechanisms – study how basic cellular mechanisms are

disrupted or changed by disease proteins

• Drug discovery– High-throughput assays will identify targets.

For example, using mouse ES cell-derived neural cells for an assay to screen Alzheimer's disease

• Genetic screening • Toxicology testing

Overview

• What are stem cells?– How do embryonic and ‘adult’ stem cells

differ?– How are different types of stem cell lines

created?

• Stem cells in research and medicine

• Alternatives to the embryo

Beyond the Embryo

• The President’s Council for Bioethics– White Paper published May 2005– http://bioethics.gov/reports/white_paper/

text.html

ESCs without the E• De-differentiation

– Requires aid of special cytoplasmic factors obtained from oocytes (or from pluripotent embryonic stem cells)

Muscle cells

Multipotentprogenitors

•Obtainable from any adult•Immunocompatible•Some success with muscle, liver, blood

Issues:How far back can dedifferentiation go?

ESCs without the E

• Remove single cell from 6-8 cell embryo– Spin-off of preimplantation diagnosis

Issues:Is there harm in removing a cell?Could a cell line be established with one cell?Is cell at this stage totipotent?

Remove cell Establish cell line

Embryo

ESCs without the E

• Removal from dead embryo– Early IVF embryos (roughly 4-8 cells)

that have spontaneously died. Normal-appearing blastomeres in cleavage-arrested, mosaic embryos.

Issues: Can markers of organismic death be found? Can pluripotent stem cells be derived from dead embryos? If so, will they be chromosomally (and otherwise) normal?

Parthenogenesis

• Biochemically trick a human oocyte into thinking it has been fertilized.

• Treated eggs divide to the blastocyst stage (50-100 cells), at which point stem cells can presumably be derived.

• The “parthenogenetic” (that is, unfertilized but still developing) blastocyst-like entity is assumed by most to lack the potential for development as a human being.

Oocyte Blastocyst

Establish cell line

ESCs without the E

• Bio-engineered embryo-like artifacts– Embryos engineered to lack the essential

elements of embryogenesis but still capable of some cell division and growth

Altered Nuclear Transfer

Remove altered nucleus to oocyte

Somatic cell

OocyteCell Division Blastocyst

Embryo

ESCs without the E

• De-differentiation

• Single cell removal from embryo

• Removal from dead embryo

• Parthenogenesis

• Bio-engineered embryo-like artifact

Creative thinking, possible solutions to an ethical dilemma. Research has yet to determine if one or more of these proposals are possible.

Recent Research

• RNAi was used to change expression of a gene in a hESC line. – Stem Cells 23(3):299–305, 2005

• hESCs driven to develop into motor neurons. – Nature Biotechnology 23:215-221,

2005.

Recent Research

• Mesenchymal stem cells injected into rat heart increased pumping capacity and vessel growth after heart attack. – Journal of Clinical Investigation 115:326–338,

2005.

• “Stembrids” were made —one ESC was enucleated and then given the nucleus from an adult somatic cell. – Not shown that the resulting “stembrid” would

be immunologically acceptable to the adult somatic cell donor.

Summary

• Stem cells • Embryonic vs. Adult• IVF, SCNT• Therapeutic cloning and immune matching

• Much scientific progress, but therapies are not yet directly translated from research

• Greatest potential contribution from mechanistic studies in ESCs

• Embryonic alternatives need more development

Conclusion

• Stem cells are complicated: scientifically, ethically, legally. The best way to approach them is with education.

• Working with stem cells is one of the most important opportunities of our time.