Download STEM CELLS

Regenerative Medicine
Regenerative medicine seeks to revolutionize the ways
we improve the length and quality of life by restoring,
maintaining, or enhancing tissue and organ function. It
involves the merging of several fields, including cell/tissue
engineering, biomaterials development, and stem cell
biology.
Source: NIH, 2003
Stem Cells
Stem cells have emerged as the starting material
of choice to produce cells and tissues to treat
degenerative,
diseases.
genetic,
and
immunological
Atomic bombings of Hiroshima and Nagasaki
1945
Hallmarks of a Blood-forming Stem Cell
McCulloch, E.A., Till, J.E. (1960) The radiation sensitivity of normal mouse bone marrow cells,
determined by quantitative marrow transplantation into irradiated mice. Radiation Research
13(1):115-125.
Till, J.E., McCulloch, E.A. (1961) A direct measurement of the radiation sensitivity of normal mouse
bone marrow cells. Radiation Research 14:213-22
STEM CELLS
General definition: special kind of cell that has a unique capacity to renew itself
and to give rise to specialized cell types (NIH, 2001).
Properties:
Self-renewal capacity
Multilineage differentiation ability
In vivo engraftment ability
STEM CELLS
What are stem cells?
Ability to self-renew by
multiplying in an
undifferentiated state
Ability to differentiate into
various cell types that
constitute an organism
Source: Bongso and Lee in Stem Cells - From Bench to Bedside, World Scientific Publishing Com, 2010
STEM CELLS - Classification
Source: www.stemcellresources.org
STEM CELLS - Classification
Differentiation Potential
Totipotent cells: have the capacity to specialize into extraembryonic membranes and
tissues, the embryo, and all postembryonic tissues and organs. They have the ability to
replicate indefinitely without losing their total potency.
Pluripotent cells: capable of giving rise to most tissues of an organism;
Multipotent cells: can differentiate into only a limited range of cell types;
Unipotent cells: can differentiate into one type of cells. ex: testis stem cells.
Totipotent Stem Cells
Totipotent stem cell – ex. Zygote (union of male and female germ cells). A single
totipotent stem cell has the ability to give rise to the whole organism.
Source: Stem Cell Information, NIH
The only totipotent cells are the fertilized egg and the cells produced by its cleavage in the first 4 days
(as shown by the ability of mammals to produce identical twins, triplets, etc.).
Totipotent Stem Cells
Mammalian Development
Figure 19-5 Molecular Biology of the Cell (© Garland Science 2008)
Pluripotent Stem Cells
Pluripotent Stem Cells
A single pluripotent stem cell has the ability to give rise to all types of cells that develop
from the three germ layers (mesoderm, endoderm and ectoderm) from which all cells of
the body arise.
 ESC: derived from a group of cells called the inner cell mass, which is part of the early (5day) embryo, called the blastocyst;
 EGC: derived from fetal tissue; specifically they are isolated from the primordial germ
cells of the gonadal ridge of the 5- to 10-week fetus. Later on, the gonadal ridge develops
into the testes or ovaries and the primordial germ cells give rise to eggs or sperm;
 ECC: isolated from teratocarcinomas, a tumor that occasionally occurs in the gonads.
Unlike the other two, they are usually aneuploid;
Pluripotent Stem Cells
Source: www.nature.com
Pluripotent Stem Cells
A single pluripotent stem cell has the ability to give rise to all types of cells that
develop from the three germ layers of the embryo
mesoderm, endoderm and ectoderm
from which all cells of the body arise
Unlike totipotent cells, pluripotent stem cells fail in its ability to differentiate into
extra embryonic membranes, namely the trophectoderm (the outer layer of the
developing blastocyst that will give rise to the placenta), and tissues namely the
trophoblast (extraembryonic tissue responsible for negotiating implantation,
developing into the placenta and controlling the exchange of oxygen and
metabolites between the mother and the embryo).
PLURIPOTENT STEM CELL TIMELINE
1981: Isolation of mouse
embryonic stem cells
(mESC) by Evans & Kaufman
1960s-1970s: Isolation of
human embryonic
carcinoma cells (hECC)
2006: Derivation of induced
pluripotent stem cells (iPSC) by
Yamanaka and co-workers
1998: Isolation of human
embryonic stem cells (hESC)
by Thompson and co-workers
and human embryonic germ
cells (hEGC) by Gearhart and
co-workers
EMBRYONIC STEM CELLS
Undifferentiated cells isolated from the inner cell mass of the blastocyst
Inner cell mass
Source: NIH, 2001
trophectoderm
Source: www.thetechherald.com/
5-day embryo:200 cells
Inner cell mass: 30 cells
Embryonic Stem Cells (ESC)
Undifferentiated cells isolated from a group of cells called the inner cell mass,
which is part of the pre-implantation embryo, called the blastocyst
(Source:http://www.stemcellresearchfoundation.org)
ESC CHARACTERISTICS
Long-term self-renewal capacity
Can be cultivated indefinitely (unlimited number
of
symmetrical divisions) without differentiation
Pluripotency
Capability to differentiate into all mature cell types that are originated from
the three primary germ layers of the embryo even after in vitro long term
culture
ectoderm (e.g. neuronal cells)
endoderm (e.g. pancreatic cells)
mesoderm (e.g. cardiomyocytes)
ESC PLURIPOTENCY
Pluripotent Stem Cells
Source: Stem cell Information, NIH
hESC – The Pioneers
hESC CULTURES
Human Embryonic Stem Cells
HUMAN EMBRYONIC STEM CELLS - Properties
Clonogenic cells, capable of undergoing an unlimited number of symmetrical divisions without
differentiating (long-term self renewal)
Pluripotent cells capable to give rise to cells from all 3 germ layers of the embryo (ectoderm,
mesoderm, endoderm) both in vitro (trough Embryoid Bodies or directed differentiation protocols)
Develop into teratomas when injected into SCID mice (immunocompromised mice)
Exhibit and maintain a stable, diploid karyotype;
Express specific transcription factors such as Oct-4, Sox2 and Nanog, surface markers such as Tra-160, Tra-1-81, SSEA-3 and SSEA-4, and Alkaline Phosphatase (AP)
Express high levels of Telomerase
Mouse and rat embryonic stem cells should also:
contribute to the formation of a healthy chimeric adult when injected into a blastocyst which is then
implanted in a female’s uterus (Chimeras: mixture of cells derived from both ESC donor and recipient
blastocyst in tissues and organs);
 enter the germline of these animals, that is, contribute to their pool of gametes;
hESC CHARACTERIZATION
Surface Markers and Transcription factors
hESC CHARACTERIZATION
Embryoid Bodies/Directed Differentiation
Huangfu D et al., Nature Biotech, 2008
hESC CHARACTERIZATION
Telomerase expression
On the end of the chromosomes there are noncoding repeating sequences named
telomeres;
Telomeres can be rebuilt using an enzyme called telomerase.
Source: Stem Cell Information, NIH, 2001
hESC lines express high
levels of telomerase
Source: www.sciencemag.org
hESC CHARACTERIZATION
Teratoma Formation in SCID mice
Mesoderm:
Mesoderm: bone
cartilage
Ectoderm: neural
epithelium
Mesoderm:
striated muscle
Mesoderm: fetal glomeruli
and renal tubules
Endoderm:
gut epithelium
Source: Odorico et al, Stem Cells, 2001
EMBRYONIC STEM CELLS - Chimera formation
Why embryonic stem cells?
Feature
Embryonic
Fetal
Adult (ex. Bone
marrow)
Isolation



Proliferation



Pluripotency

/

Differentiation
Potential



Ethical issues

/

Safety issues
(immunocompatibility,
teratoma formation)

?

Clinical numbers



Sources of human ESC
Available hESC lines;
Excess embryos from IVF clinics
Embryos created for research by IVF
Therapeutic cloning (or Somatic Cell Nuclear
Transfer, SCNT)
Therapeutic Cloning
European and International Regulation
Source: Elstner et al, SC Research , 2009
on ESC research
European and International Regulation
Source: Elstner et al, SC Research , 2009
on ESC research
Glossary
Blastocyst - a preimplantation mammalian embryo, i.e. at the end of cleavage and ready for
implantation into the uterine epithelium, made of hundred or more cells depending on species.
Consists of a continuous outer layer of cells, the trophectoderm, which give rises to the placenta, a
fluid filled cavity , the blastocoel, and a cluster of cells on the interior, the inner cell mass from which
ESC are isolated.
Cell passage – a round of cell growth and proliferation in culture. Passaging consists of removing cells
from one culture plate and replating them into new plates.
Embryoid bodies (EBs) – clumps of cellular structures arising from ESC in culture; they contain cells
from the 3 germ layers and are not part of normal development, only occurring in vitro.
Embryonic stem cells – pluripotent stem cells derived from the ICM upon explantation in culture,
which can differentiate in vitro into many different lineages and cell types and, upon injection into
blastocysts, can give rise to all tissues including the germline.
Embryonic germ cells – pluripotent cell line originating from transformed PGCs. In contrast to
pluripotent ESC, PGCs are unipotent in vivo, but become pluripotent in culture.
Embryonic carcinoma cells – pluripotent cell line originating from transformed primordial germ cells.
These cells are derived from teratocarcinomas.
Feeder layer – cells, usually fibroblasts, utilized in co-culture to maintain pluripotent stem cells.
These are incapable of division but provide physical support and soluble factors to the other cells.
Germline stem cells – unipotent cell line derived from mouse testes, which reconstitutes
spermatogenesis when transplanted into sterile recipients. Multipotent germline stem cells have the
potential to produce teratomas and chimeric animals.
Glossary
Inner cell mass – cells of the blastocyst that appear transiently during development and give rise to the
3 germ layers of the developing embryo.
Multipotency – ability of a cell to give rise to different cell types of a given cell lineage. These cells
include most adult stem cells.
Pluripotency - ability of a cell to give rise to all cells of the embryo.
Primordial germ cells - give rise to oocytes and sperm in vivo and to embryonic germ cells when
explanted in vitro.
SCID = severe combined immunodeficiency. These mice lack a functioning immune system (have
neither T cells nor B cells) and so cannot reject foreign tissue.
Telomerase – an enzyme composed of a catalytic protein component and an RNA template and that
synthesizes DNA at the ends of chromosmes and confers replicative “immortality” to cells.
Teratoma
• A teratoma is a type of tumor;
• The word teratoma comes from Greek and means roughly "monstrous tumor".;
• A teratoma is a tumor with tissue or organ components resembling normal derivatives of all three germ
layers,
• Teratomas typically contain gut-like structures such as layers of epithelial cells and smooth muscle;
skeletal or cardiac muscle; neural tissue; cartilage or bone; and sometimes hair or teeth.
Totipotency – ability of a cell to give rise to all cells of an organism, including embryonic and
extraembryonic tissues.
Glossary
Undifferentiated – not having changed to become a specialized cell type by the generation of
structures or production of proteins characteristic of a specialized cell.
Unipotency - capacity of a cell to sustain only one cell type or cell lineage. Ex. terminally
differentiated cells, certain adult stem cells such as testis stem cells.