Somatic, Germ and Stem cells
1) Distinctions between somatic and
germ cells.
2) Establishment of somatic cell types.
3) Establishment of the germ line.
4) Fundamental differences in germ cell
establishment between organisms.
5) Where stem cells fit in….
Cell types
• Two basic classes of cells in animals
– somatic cells
– germ cells
Somatic cells - almost everything we see
and use, skin, muscle, blood, etc.
Germ cells - the cells that are responsible for
reproduction, propagation of the species.
Stem cell - neither fish nor fowl. Can in some
cases do both.
Germ vs. Somatic cells
1) These two classes of cells are
established in different ways during
development.
2) There are two fundamentally
different processes by which germ
cells are generated in different
organisms.
Establishment of somatic cells
Gastrulation generates the three
classes of somatic cells
Three classes of somatic cells
• Ectoderm (outside)
• Endoderm (inside)
• Mesoderm (in between)
– frequently called the 3 germ layers
Establishment of the germline
• Two different ways in different organisms
1) Preformation (segregation)
2) Epigenesis (recreation)
Extavour and Akam, Development 130:5896 (2003)
Establishment of the germline
• Preformation
– Germ cells determinants (germ plasm) are set
aside before or at fertilization and remain
segregated throughout the lifespan of the
organism.
C. elegans
Drosophila
Germline formation in C. elegans
In worms, the point of sperm
entry defines the posterior pole
of the egg.
a) A fertilized egg with evenly
distributed P-granules
b) At pronuclear migration and
fusion, P-granules move to the
posterior, where the sperm
nucleus was located.
c) After first cell division all Pgranules in Posterior cell.
d) At 4-cell stage all P-granules
are in Posterior P1 cell
Germline formation in C. elegans
6 Founder Cells
+98 die
+14 die
Small posterior cell goes
germline
+1 dies
Germline formation in C. elegans
8-cell
DAPI staining
of nuclei
24-cell
Antibodies to
P-granules
Preformation
• C. elegans, Drosophila and many other model
organisms. Some amphibians, birds.
• Segregation of mRNAs and proteins required for
germ cell formation and function (germ plasm).
–
–
–
–
–
–
–
bruno (RNP-type binding protein, regulates translation)
gld-1 (RNA binding protein)
mex-1 (zinc-finger DNA-binding protein)
vasa (DEAD-box RNA helicase, interacts with eIF5b)
germ cell less (transc. repressor, binds E2F)
cycB (B-type cyclin, interacts with CDK1, ubiquitous)
tudor (Tudor domain containing, part of germ granules)
Epigenesis of germline in mammals
• Major distinction, no preexisting germ
plasm in oocytes. Primordial germ cells
are generated during development.
• Seen in all mammals (so far), many other
species.
• Shared feature with Preformation: germ
cells are "set aside" before other cell
types.
Epigenesis of germline in mouse
Developmental Cell 2: 537
(2002) Zhao and Garbers,
Male Germ Cell Specification
and Differentiation
Blue = embryonic
white = extraembryonic
Embryonic
Ectoderm
Embryonic
Mesoderm
Embryonic
Endoderm
Epigenesis of germline in mouse
BMP-4, -8b
BMP receptor
BioEssays 27.2, Matsui and Okamura, Mechanisms
of germ-cell specification in mouse embryos. 2005
Primordial germ cells
•
•
•
•
Generated by epiblast.
Separate from the 3 somatic cell layers.
Migrate to gonadal ridge. (Molyneaux et al. Dev. Biol. 240:488 (2001).
Populate ridge to form germ cells of gonad.
• In vitro, PGCs can differentiate into Embryonal Germ
Cells (EGC). (Donovan et al. Cell 44:831 (1986), Matsui
et al. Cell 70:841 (1992)).
• This differentiation is similar to the "reprograming" of
somatic nuclei seen in Oocytes and ES cells.
• EGCs are one of only a few pluripotent stem cells
currently known. Can contribute to all cell types in
embryos.
Human Embryonal Germ Cells
Human EGCs
Mouse ES cells
Shamblott et al. PNAS 95:13726 (1998)
Cells were grown in DMEM supplemented with 15% fetal bovine serum, 0.1mM nonessential amino
acids), 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml of penicillin,
100 ug/ml of streptomycin, 1,000 units/ml of human recombinant leukemia inhibitory factor (hrLIF), 1
ng/ml of human recombinant basic fibroblast growth factor (hrbFGF), and 10 mM forskolin.
Stem Cell Biology
1)
2)
3)
4)
5)
Definition of stem cells
Types of stem cells
Derivation of stem cells
Functions of stem cells
Promise of stem cells
Stem Cell Biology
1)
Definition of stem cells
Self-renewal
(expansion?)
Generation of
more restricted
progeny (lineagecommitment)
Types of stem cells
1) What defines a type of stem cell?
A) Where and how it was derived?
B) What is its POTENTIAL?
2) Potentiality
How many types of cells can the stem cell
generate?
Totipotent -> pluripotent -> unipotent
Types of stem cells
adult
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
Embryonal Stem Cells (ES)
Embryonal Germ Cells (EG) embryonic
Epiblast stem cell (EpiSC)
Induced Pluripotent Stem Cells (iPSCs)
Hematopoietic Stem Cells (HSC)
Intestinal "crypt" stem cell
Cardiac and skeletal muscle stem cells
Neuronal/glial stem cell
Epidermal stem cell
Corneal stem cells
Mammary gland stem cells
Others…..
Embryonal Stem (ES) cells
• Derived from inner cell mass of blastocysts
Nature. 1984 309(5965):255-6. Formation of germ-line chimaeras from embryo-derived
teratocarcinoma cell lines.
Bradley A, Evans M, Kaufman MH, Robertson E.
Embryonal Stem (ES) cells
• Normally cells go on to form embryo of
mouse
– Ectoderm, mesoderm, endoderm (3 germ layers)
– Germ cells, germline cells
• In culture, cells can be manipulated and
used to create novel mice
• Name Embryonic Stem Cell coined by Gail
Martin.
Properties of ES cells
• Pluripotent
– Germ line and all somatic tissues, no
extraembyronic tissues
• Immortal?
• Can be genetically modified
The Nobel Prize in Physiology or Medicine 2007 was
awarded jointly to Mario R. Capecchi, Sir Martin J.
Evans and Oliver Smithies "for their discoveries of
principles for introducing specific gene modifications
in mice by the use of embryonic stem cells".
Generation of chimeric mice
Properties of ES cells
• Pluripotent
– Germ line and all somatic tissues
Can differentiate in vitro into many cell types.
• Immortal?
• Can be genetically modified
• Genes essential for ESC pluripotency
–
–
–
–
–
Oct4
Niwa et al. Mol. Cell Biol. 22 (2000)
Nanog Mitsui et al. Cell 113 (2003)
Sox2
Avilion et al. Genes Dev. 17 (2002)
FoxD3 Hannah et al. Genes Dev. 16 (2002)
Stat3 signaling important (LIF)
Questions about ES cells
• How do they maintain pluripotency?
• How can we consistantly differentiate
them in vitro?
• How similar are human ES cells to
mouse?
• Can they (or something like them) be
generated without using embryos
(human)? Answered in 2006.
All fundamental questions about the
differentiated state.
More than one pluripotent embryoderived stem cell
?
?
?
?
Thursday reading
2009
Adult stem cells
•
•
•
•
•
•
•
•
•
Hematopoietic Stem Cells (HSC)
Intestinal "crypt" stem cell
Cardiac and skeletal muscle stem cells
Neuronal/glial stem cell
Epidermal stem cell
Corneal stem cells
Mammary gland stem cells
Embryonal germ cells
Others…….
Hematopoietic Stem Cells
McCulloch
A
younger
me
Hematopoietic Stem Cells
• Discovered in 1961 (Till and McCulloch, Rad.
Res. 14:213 (1961))
• Rescue of lethal irradiation of mice
– 950 rads kill 100% of mice (3-30 days).
– Mice die from anemia.
– Injection of bone marrow from unirradiated
mice rescues the irradiated mice.
– How to quantify, identify and isolate rescuing
cells?
Hematopoietic Stem Cells
• Rescue of lethal irradiation of mice
– Irradiate recipient mice
– Injection of bone marrow from non-irradiated mice
– Looked at hematopoietic organs of recipients
Hematopoietic Stem Cells
Hematopoietic Stem Cells
1) First quantification of "stem" cells.
2) Demonstration of different
differentiated cell types from one
cell.
3) Started race to purify HSC (1961).
Essentially completed in 1991
Hematopoietic Stem Cells
1) Finding appropriate markers.
lineage markers
2) Developing better assays.
competition assay vs. rescue
3) Cell sorting protocols.
FACS
4) Better understanding of
"reconstitution".
"Short-term"vs. "Long-term"
Properties of Hematopoietic
Stem Cells
1) Thy1lo, c-kit+, Sca-1+.
2) Negative for B220, Mac-1, Gr-1, CD3, 4, 8,
Ter119.
3) Are present as 0.007% of bone marrow.
4) Can do long-term reconstitution of all blood
cell types with single (or small numbers) of
cells.
5) Are resistant to killing with S-phase drugs or
labeling with BrdU.
6) Last several lifetimes.
Properties of Hematopoietic
Stem Cells
Weissman
Lessons from the search for
the Hematopoietic Stem Cell
1) Develop quantitative assays.
2) Find appropriate markers.
3) Develop in vitro assays, culture
systems.
4) Define lineage of cells of interest.
Types of stem cells
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
Embryonal Stem Cells (ES)
Embryonal germ cells (EGC)
Epiblast Stem Cells (EpiSC)
Induced Pluripotent Stem Cells (iPSC)
Hematopoietic Stem Cells (HSC)*
Intestinal "crypt" stem cell
Olfactory neuron stem cell
Cardiac and skeletal muscle stem cells
Neuronal/glial stem cell
Epidermal stem cell
Corneal stem cells
Mammary gland stem cells
Others…..
http://stemcells.nih.gov/info/glossary.asp
Epigenetic landscape
(Waddington, 1957)
Fig. 1. The developmental potential and epigenetic states of cells at different stages of development. A modification of C. H.
Waddington’s epigenetic landscape model, showing cell populations with different developmental potentials (left) and their
respective epigenetic states (right). Developmental restrictions can be illustrated as marbles rolling down a landscape into one of
several valleys (cell fates). Colored marbles correspond to different differentiation states (purple, totipotent; blue, pluripotent; red,
multipotent; green, unipotent). Examples of reprogramming processes are shown by dashed arrows.
Development 136, 509-523 (2009) doi:10.1242/dev.020867
Epigenetic reprogramming and induced pluripotency
Konrad Hochedlinger1 and Kathrin Plath2
Promise of stem cells
•
•
•
•
•
•
•
•
•
•
•
•
•
Diabetes
Spinal cord injuries
Stroke
Heart Disease
Neurodegenerative diseases
Multiple Sclerosis
Arthritis
Asthma, Emphasema
Spina Bifida
Cerebral Palsey
Cancer
Kidney failure
Amputations
Comparison of stem cell
features
ES, EG, EpiSc, iPSC
• Pluripotent
• Fast growing
• Easily cultured,
purified
• Genetically
manipulable
• Can be differentiated
in vitro
Adult stem
• Multipotent or
unipotent
• Slow growing
• Difficult to
culture, purify
• Hard to
manipulate
• Can often
differentiate
For Thursday
Reprogramming of somatic cells
into iPSCs
• Download papers and handouts
• Read papers carefully and be ready to
discuss the original paper in class
• Fill out Figure Facts sheets and e-mail
them before class on Thursday
• We'll discuss how this work has
progressed at the end of class on
Thursday.