Download What is a stem cell? - Scheid Signalling Lab @ York University

Stem cell biology basics
By the end of this lecture you will:
1. Understand where stem cells come from
2. Understand the different types of stem cells
3. Understand what stem cells do
4. Understand what stem cell tech has to offer
A life story…
What is a stem cell?
Stem cell
SELF-RENEWAL
(copying)
Identical stem cells
Stem cell
DIFFERENTIATION
(specializing)
Specialized cells
Why self-renew AND differentiate?
1 stem cell
1 stem cell
Self renewal - maintains
the stem cell pool
4 specialized cells
Differentiation - replaces dead or damaged
cells throughout your life
Where are stem cells found?
embryonic stem cells
blastocyst - a very early
embryo
tissue stem cells
fetus, baby and throughout life
Types of stem cell:
1) Tissue stem cells
Tissue stem cells:
Where we find them
surface of the eye
skin
testicles
brain
breast
intestines (gut)
bone marrow
muscles
Tissue stem cells:
What they can do
blood stem cell
differentiation
found in
bone marrow
MULTIPOTENT
only specialized types of blood cell:
red blood cells, white blood cells,
platelets
Types of stem cell:
2) Embryonic stem cells
How are embryonic stem cells grown in the
laboratory?
• Growing cells in the laboratory is known as cell culture.
• Human embryonic stem cells are isolated by transferring the
inner cell mass into a plastic laboratory culture dish that
contains a nutrient broth known as culture medium.
• The inner surface of the culture dish is typically coated with
mouse embryonic skin cells that have been treated so they
will not divide. This coating layer of cells is called a feeder
layer
= sticky surface to which they can attach, also release
nutrients into the culture medium.
Embryonic stem (ES) cells:
Where we find them
blastocyst
cells inside
= ‘inner cell mass’
embryonic stem cells taken from
the inner cell mass
outer layer of cells
= ‘trophectoderm’
fluid with nutrients
culture in the lab
to grow more cells
Over the course of several days, the ESC proliferate and begin
to crowd the culture dish.
Cells are removed gently and plated into several fresh culture
dishes. The process of replating the cells is repeated many
times and for many months = subculturing. Each cycle of
subculturing referred to as a passage.
Embryonic stem cells that have proliferated in cell
culture for six or more months without differentiating,
are pluripotent, and which appear genetically normal
are referred to as an embryonic stem cell line.
Once cell lines are established FREEZE CELLS!
What laboratory tests are used to identify embryonic stem
cells?
– growing and subculturing the stem cells for many months.
This ensures that the cells are capable of long-term selfrenewal.
– Scientists inspect the cultures through a microscope to see
that the cells look healthy and remain undifferentiated.
– Using specific techniques to determine the presence of
surface markers that are found only on undifferentiated
cells.
– presence of a protein called Oct-4, which is typically made
by undifferentiated cells. Oct-4 is a transcription factor,
helps turn genes on and off at the right time, which is an
important part of the processes of cell differentiation and
embryonic development.
– Karyotyping = examining the chromosomes under a
microscope. This is a method to assess whether the
chromosomes are damaged or if the number of
chromosomes has changed.
Embryonic stem (ES) cells:
What they can do
differentiation
embryonic stem cells
PLURIPOTENT
all possible types of specialized cells
Embryonic stem (ES) cells:
Challenges
skin
neurons
embryonic stem cells
blood
?
liver
What laboratory tests are used to identify embryonic stem
cells?
– growing and subculturing the stem cells for many months.
This ensures that the cells are capable of long-term selfrenewal.
– Scientists inspect the cultures through a microscope to see
that the cells look healthy and remain undifferentiated.
– Using specific techniques to determine the presence of
surface markers that are found only on undifferentiated
cells.
– presence of a protein called Oct-4, which is typically made
by undifferentiated cells. Oct-4 is a transcription factor,
helps turn genes on and off at the right time, which is an
important part of the processes of cell differentiation and
embryonic development.
– Karyotyping = examining the chromosomes under a
microscope. This is a method to assess whether the
chromosomes are damaged or if the number of
chromosomes has changed.
Pros and cons of each type
Embryonic
Cell lines last and last and
last
Pluripotent
Easy to find
Ethical issues - when does
life begin?
Adult
Cell lines do not last
Not pluripotent
Hard to locate
No ethical issues
Types of stem cell:
3)Induced pluripotent (iPS)
stem cells
Sir John Gurdon
Induced pluripotent stem cells (iPS cells)
‘genetic reprogramming’
= add certain genes to the cell
cell from the body
induced pluripotent stem (iPS) cell
behaves like an embryonic stem cell
differentiation
culture iPS cells in the lab
Advantage: no need for embryos!
all possible types of
specialized cells
24 candidate genes based on hypothesis that they play pivotal role in maintaining ES cell pluripo
Fbx15
Fbx15 is specifically expressed in mouse ES cells, but not required = ES cell marker
gene
Homologous recombination of neomycin r+ cassette into Fbx15 gene via homologous
Retroviral infection of 24 factors into mouse embryonic fibroblasts (MEFs) derived
from Fbx15 βgeo/ βgeo embryos
0 Colonies when only single factor was transduced
Many colonies when all 24 factors were transduced simultaneously
29 colonies, 6 assayed, 4 of which expressed ES cell morph
-round shape, large nucleoli and scant cytoplasm
-similar growth rates (doubling time)
-Similar on a genetic level
- Some combination of the 24 transduced factors induced expression of ES cell marker
genes in MEF culture
-Removal of individual factors to test their contribution to neomycin r+ colonies
-Chose factors which resulted in 0 colonies after 10 days and fewer after 16 days
iPS-MEF3 cells were not healthy
RT-PCR for ES cell marker genes
Global gene expression analyses by DNA microarrays – iPS-MEF4/10 more like ES then MEF
Preliminary Conclusion:
4 factors were sufficient to induce neomycin resistance and ES cell like morphology:
Oct3/4
Sox2
Klf4
c-Myc
iPS-MEF3 cells are substantially different then iPS-MEFS4/10
iPS-MEFS4/10 are similar but not identical to ES cells:
- Similar genetically
- Similar morphologically
What About Pluripotency?
Teratoma formation is a key indicator of pluripotency
cells from all three germ layers are formed
Histology of teratomas derived from iPS-MEF3 cells = undifferentiated cells
Histology of teratomas derived from iPS-MEF4 cells
Trophoblast
Endoderm
Mesoderm
Ectoderm
RT-PCR of marker genes from teratomas
In vitro embryoid body formation
Differentiation of embryoid bodies
Mesoderm
Endoderm
Ectoderm
iPS-MEF4/10 embryoid bodies began to differentiate into cells from all three germ layers
Conclusion: the four selected factors could induce
pluripotent cells from EMBRYONIC fibroblasts
Can we induce pluripotency on ADULT cells?
Repeated experiments in another cell type:
-tail-tip fibroblasts (TTFs) from Fbx15 βgeo/ βgeo which constitutively express
GFP
iPS-TTFgfp4
RT-PCR ES cell marker
In vitro differentiation
Teratoma histology
Similar results:
- Neomycin resistant colonies
- ES cell like morphology
- ES cell markers
- Teratoma formation
- Histology = three germ layers
-Microinjection of iPS-TTFgfp clone into mouse blastocyst
-Implant into a foster mother
-Embryos positive for contribution of iPS-TTFgfp cells up to E13.5
-Histology showed GFP positive cells in all three germ layers
Conclusion: the four selected factors could reprogram
adult- terminally differentiated cells to a pluripotent state
So why these four factors?
Oct3/4 - Mouse embryos that are Oct-4-deficient or have low expression levels
of Oct-4 fail to form the inner cell mass, lose pluripotency and differentiate into
trophectoderm. Essential transcription factor in maintaining pluripotency (Boyer
et al., 2005; Loh et al., 2006)
Sox2 - transcription factor that is essential for maintaining self-renewal and
pluripotency in undifferentiated embryonic stem cells (Boyer et al., 2005; Loh et
al., 2006).
c-Myc – may induce global histone acetylation, thus allowing Oct3/4 and Sox2
to bind their specific target loci.
Klf4 - Represses p53 directly, p53 represses Nanog durring ES cell
differentiation -> indirectly activates Nanog
Discussion
- Only ~0.02% of transfected cells became ES-like cells, why so low?
- Source of iPS cells: contamination? ~0.067% of mouse skin cells are stem
cells
- Cells induced by 3 factors were nullipotent
- Microarray analysis showed cells were not of stem cell origin
- Levels of 4 factors required may have a very specific and narrow range
- Although mRNA levels were overexpressed in iPS cells, protein levels were similar
to ES cells
- iPS cells can differentiate in vitro and in vivo despite presence of vectors
- Mechanism exists to regulate at protein level
- Limited capacity of iPS cells to integrate into normal tissue in vivo ie. No chimera
pups
- Posttranslational modification differs between iPS and ES cells ie. Methylation
- Are these cells caught in an in-between state?
RT-PCR ES cell marker
Western Blot
Discussion
- Only ~0.02% of transfected cells became ES-like cells, why so low?
- Source of iPS cells: contamination? ~0.067% of mouse skin cells are stem
cells
- ruled out by repeating experiments with bone marrow stroma with high levels of
SC
- Cells induced by 3 factors were nullipotent
- Microarray analysis showed cells were not of stem cell origin
- Levels of 4 factors required may have a very specific and narrow range
- Although mRNA levels were overexpressed in iPS cells, protein levels were similar
to ES cells
- iPS cells can differentiate in vitro and in vivo despite presence of vectors
- Mechanism exists to regulate at protein level
- Limited capacity of iPS cells to integrate into normal tissue in vivo ie. No chimera
pups
- Posttranslational modification differs between iPS and ES cells ie. Methylation
- Are these cells caught in an in-between state?
The future of IPS
1. IPS will offer a pool of genetically viable stem cells
for individualized medicine
2. May cure the worst of degenerative diseases
-MS
-Diabetes
-Parkinsons
-Paralysis
3. Cells for testing medicine
4. No ethical issues