Todays Outline

1
Todays Outline

• Review SC behavior(s)
• The SC environs
• The Spermatogonial stem cell
• Tricks of the (SC) trade using SSCs as prime
  example
• Papers for Jan 28th Background for mammalian
  SSCs and their niche

2
Lets review properties of stem cells

• They have extensive proliferative potential
• They can generate many progeny

• Can generate one or more differentiated cell
  types

• Yet, they self-renew
• They themselves remain undifferentiated

• They sense tissue damage/stress, and alter the
• balance between self-renewing differentiative
  divisions

These properties make them key players in tissue
homeostasis
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How are these properties controlled?

• Intrinsically internal, autonomous control
  programs

• Extrinsically control is vested in
  neighboring,
• differentiated cells their extracellular
  environment

This controlling environment is called The Stem
Cell Niche
Identify the Niche Identify the controlling
signals!
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Todays Outline

• Review SC behavior(s)
• The SC environs
• The Spermatogonial stem cell
• Tricks of the (SC) trade using SSCs as prime
  example
• Papers for Jan 28th Background for mammalian
  SSCs and their niche

5
History Lesson Schofields niche hypothesis

• Several experimental findings that are
  inconsistent with the view that the spleen
  colony-forming cell (CFU-S) is the primary
  haemopoietic stem cell are reviewed. Recovery of
  CFU-S, both quantitatively and qualitatively, can
  proceed differently depending upon the cytotoxic
  agent or regime used to bring about the
  depletion.
• The virtual immortality of the stem cell
  population is at variance with evidence that the
  CFU-S population has an age-structure which has
  been invoked by several workers to explain
  experimental and clinical observations.
• To account for these inconsistencies, a
  hypothesis is proposed in which the stem cell is
  seen in association with other cells which
  determine its behaviour. It becomes essentially a
  fixed tissue cell. Its maturation is prevented
  and, as a result, its continued proliferation as
  a stem cell is assured.
• Its progeny, unless they can occupy a similar
  stem cell niche, are first generation
  colony-forming cells, which proliferate and
  mature to acquire a high probability of
  differentiation, i.e., they have an
  age-structure. Some of the experimental
  situations reviewed are discussed in relation to
  the proposed hypothesis.

Blood Cells 4, 725 (1978).
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Thought question
What happens if a stem cell finds itself out of
its Niche?
Well return to this question later.....
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How does one find the niche?
First identify the stem cells Easy or
tough? What methodologies to use?
Lineage-tracing LRCs? Verify by
transplantation / repopulation
Do stem cells occupy a consistent position in
the tissue? If so, characterize those environs
(cell markers for neighbor cells expression
of signals from those cells or on ECM)
Re-introduce stem cells, show that they home to
this defined environment, that it supports
stem cell behavior
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Wait a minute....who cares about the
niche dont plenty of SCs live in a dish?
Actually.....No
While some do.....

• ES cells internal, autonomous control program
  LIF

• Neural Stem cells (NSCs) FGF

• iPS cells .......

• Such cells are derived been selected (to live
  without a niche)
• added growth factors
• supplied matrix / substratum
• etc,...

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Development
Many cells
Many types of cells
One cell
Undifferentiated to Specialized /
Differentiated
Toti-potent to restricted potential
This progress toward differentiation is inexorable
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Stem cells maintain their undifferentiated state

• These cells block that inexorable process
• (they self renew, are long-lived
• provide for tissue homeostasis in vivo)

They require a proper niche to do so
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Evidences that Stem Cells wish to differentiate

• A major issue with hESC lines
• Difficulty of growing them
• Strong tendency to differentiate

• The HSC known for 50 yrs purified for 30
  yrs
• A major issue is expanding them
• ie, maintaining SC-ness while in culture
• Strong tendency to differentiate

The niche is important
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Thought question
What happens if a stem cell finds itself out of
its Niche?

• Differentiation
• Altered developmental potential

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Neural stem cells (NSCs) can be formed from CNS
cells
e.g., Johe et al, 1996
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Single cells correct culture conditions
generate undifferentiated cells
3 weeks later
Single cell
5 days later
14 days later
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These cells are multipotential
MAP2 (1) Neurons
phase
NSCs can adopt one of three fates ....at odds
with the Developmental Biology of the CNS
Conflicts with lineage-tracing experiments
GFAP (0.1) Astrocytes
O4 (0.7) oligodendrocytes
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Do NSCs with these capacities truly exist in vivo?
Tri-potential NSCs are generated by culturing
conditions that cause deregulation of normal
neural tube D/V patterning
Gabay et al, 2003 Mukoyama et al, 2006
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Why do we care whether culturing alters SC
potential?

• Stem or progenitor cells for many(?) adult
  tissues
• They exist in very low number
• A critical issue for some therapeutic
  approaches
• Can one mobilize the enodogenous tissue
  stem cell pool for repair?
• If youve identified SC potential fates only
  by isolating them and growing in culture....
• if some fates are simply a function of
  culturing....
• Then what you mobilize endogenously may not do
  what you intend
• -- Even so, these SCs can be quite useful for
  other therapeutic approaches

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The Niche a definition

• A subset of a tissues cells
• (including extracellular substrates)
• that houses one or more stem cells
• that controls their self-renewal
• progeny production in vivo

Information flow Niche cells gt Stem cells gt do
their thing
There is also feedback Stem cells gt Niche cells
gt do their thing
Spradling et al, 2001
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(Theoretical) mechanics of a niche

• They support either of two types of
• Stem cell renewal mechanisms
• If a lineage mechanism prevails
• If a population mechanism prevails

Spradling et al, 2001
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Asymmetric divisions Beautiful paradigms

• Asymmetric divisions
• If a lineage mechanism prevails

Yu et al, 2006
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How hard is it to find the niche?
A rough HSC timeline60 yrs
1980s
1990s
1945
1960s
1988
1990s
1950s
1992
2003
2005
Bone marrow T-plants
A second HSC niche defined
Lethal doses of radiation
Markers for mouse HSCs
The HSC niche defined?
Markers for human HSCs
Peripheral blood for HSCs
Animal models of radiation
Till McCulloch, Bone Marrow
Umbilical Cord Blood T-plants
Calvi et al, 2003 Zhang et al Kiel et al, 2005
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Hematopoesis An example stem cell-maintained
system
Till McCulloch Siminovich et al, 1963
Spangrude et al, 1988
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Hematopoesis An example stem cell-maintained
system
Till McCulloch Siminovich et al, 1963
Spangrude et al, 1988
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An HSC niche (?)
adapted from Yin Li, 2005
Taichman Emerson, 1998 Calvi et al, 2003
Zhang et al, 2003 Kiel et al, 2005
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An HSC niche (?)
Calvi et al, 2003 Zhang et al Kiel et al, 2005
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For the HSC system, there are still
controversies...
Identifying the stem cells -- One methodology
relies on the presumption that SCs are slow
cycling -- Compared purified HSCs, by
transplantation / repopulation with Label
Retaining Cells (LRCs) -- Found little
correspondence.....
Kiel et al, 2007 Nature 449(7159) 238-42.
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The emergence of the vascular niche as a common
theme?
NSCs in the subventricular zone
Palmer et al, 2000 Shen et al, 2004 from
Doetsch, 2003
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Todays Outline

• Review SC behavior(s)
• The SC environs
• The Spermatogonial stem cell
• Tricks of the (SC) trade using SSCs as prime
  example
• Papers for Jan 28th Background for mammalian
  SSCs and their niche

29
Spermatogenesis
Stem Cells
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faculty.southwest.tn.edu/rburkett/AP2_r7.jpg
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Transplantation / repopulation A Spermatogonial
Stem Cell Assay

• StepA Donor marked by LacZ / GFP
• Step C Recipients are chemically depleted of
  germ cells

Brinster Zimmerman, 1994 from Brinster, 2002
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Reconstituted spermatogenesis!
Remove the host testis after two months Voilá!
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Spermatogonial Stem Cell Assay

• Step E / F Germline transmission!

Brinster, 2002 after Brinster Zimmerman, 1994
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• A technical / nomenclature point
• ASingle Undifferentiated spermatogonia
• on basement membrane in tubules
• Gonia divide with incomplete cytokineses
• Apaired
• Aaligned
• Generating chains of gonial cells
• As gonial chains differentiate further,
• they move away from basement membrane
• (internally)

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Where is the niche?
Week 1
Week 2 - 4
Day 1
Cells seed to tubule basement membrane
SSC divisions generate gonial chains
Cells in lumen
gt 1 month
Fully reconstituted
Lateral spreading symmetric divisions of SSCs
produce more SSCs
Nagano et al, 1999
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But....where is the niche?
Week 1
Week 2 - 4
Cells seed to tubule basement membrane
Divisions generate gonial chains along
basement membrane
In the testis Theres a lot of tubule w/
basement membrane Therere a lot of Sertoli
cells interstitial cells.... Your papers for
Jan 28th will get you closer.....
Nagano et al, 1999
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Once you have a functional assay for Stem Cells

• Estimate SC numbers in the donor tissue ()
• Discriminate between asymmetric symmetric SC
  divisions
• Characterize the niche
• Purify SCs
• Establish culture conditions to expand them
• Genetically modify SCs
• much more (re Yoshidas work Jan 28th)

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Spermatogonial Stem Cell AssayModifications
Step B Cells can be infected with Lentivirus
Cells can be FACS sorted 200 x
enrichment in SSCs
Brinster, 2002
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Enrichment of SSCs
adapted from Shinohara et al, 2000
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FACS Enrichment of SSCs
Take cryptorchid, then sort for Thy1, MHC-
Kubota et al, 2003
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Todays Outline

• Review SC behavior(s)
• The SC environs
• The Spermatogonial stem cell
• Tricks of the (SC) trade using SSCs as prime
  example
• Papers for Jan 28th Background for mammalian
  SSCs and their niche

Niche-delivered self-renewal signals SC
Longevity SC Aging Natural SC replacement
Dedifferentiation back to SC fate
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Stem cells
A (male) flys view
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(No Transcript)
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Meiosis Differentiation
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Wild-type Testis
Skipped in lecture
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How does one Determine that Stem Cells Maintain
a Tissue/Organ?
Take advantage of the fundamental properties of
a stem cell

• Self-Renewal

• Longevity

We can do lineage tracing.....
Skipped in lecture
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Skipped in lecture
Lineage Tracing
If a Gonialblast is marked, and you wait a few
days (i.e., a few cell divisions)..
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Skipped in lecture
The mark is chased away
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Skipped in lecture
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Skipped in lecture
The mark remains, and a marked family is produced
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Skipped in lecture
Stem Cells for the Germ-line Lineage
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Skipped in lecture
Stem Cells for the Somatic (Cyst) Lineage
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Skipped in lecture
Self-renewing Divisions Maintain Spermatogenesis
Two Stem Cell Lineages
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Fruitfly Stem cell biology

• Some Description of the System

• Approaches

Mutational Genetics - Select a Phenotype
Genomics
Skipped in lecture
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Skipped in lecture
Meiosis Differentiation
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Hyper-proliferation mutants
Skipped in lecture
A problem restricting Self-Renewal to one cell or
restricting the extent of Amplification
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Defective stem cell maintenance mutants
Skipped in lecture
The Jak - Stat Pathway
Erika Matunis, Johns Hopkins SOM
Minx Fuller, Stanford
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Jak/STAT signaling pathway
Skipped in lecture
Arbouzova and Zeidler, 2006
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The Testis Stem Cell Niche
Erika Matunis, Johns Hopkins
Minx Fuller, Stanford
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Oriented divisions push daughter cells away
from Niche
Model Distal daughter removed from the
influence of the self-renewing chemokine
Yamashita et al, 2003
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A Beginning Pathways affecting the Balance
between Self-Renewal and Differentiation
Skipped in lecture
Extrinsic control from hub Jak/Stat pathway
Kiger et al Tulina Matunis, 2001
Intrinsic control(?) Hub/GSC interface Yamashita
et al, 2003
Extrinsic control from cyst cells
Egf-R/Ras/Raf/MapK Pathway Kiger et al Tran et
al, 2000 Schulz et al, 2002
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Aging of Stem Cell Populations?
Can derive several features of SCs from lineage
tracing experiments....

• Marks a stem cell...
• See what happens over time

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GSC Half-life 2 Weeks
T (½) 14 days
Percent of Testes with Marked GSC
3
5
7
14
21
Age, in days (N)
Wallenfang et al, 2006
64
Does half-life account for the number of GSCs
present as testes age?
Actual number of stem cells/testis
of GSCs
Predicted from half-life data
Age, in days
How are stem cells preserved?
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Are stem cells being replenished over time?
Skipped in lecture
Day 7
Curious Phenomenon Testes that retain a marked
GSC fill with marked progeny
Wallenfang et al, 2006
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Stem cells are being replenished

• Revealed by lineage-tracing

3 days after marking
21 days after marking
Wallenfang et al, 2006
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Open question How might this occur? Are the
normal radial divisions shifted?
Centrosomes Somatic cells DNA
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While GSC numbers are maintained,GSCs cycle less
frequently

• S-phase index by BrdU pulse labeling
• Flies at different ages
• Count BrdU GSCs / total

Skipped in lecture
Wallenfang et al, 2006
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GSC cycling decreases with age
Skipped in lecture
0.3
0.2
S Phase Index
0.1
14
0
21
35
63
0 vs 35day 35 vs 63 day are significant
Age (days)
Wallenfang et al, 2006
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In aging tests, niche expression of
theself-renewing chemokine decreases....
Skipped in lecture
Upd / OS expressed in hub cells
Hub expression decays
STAT activated in fewer cells
STAT activated in surrounding tier
Boyle et al, 2007
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Todays Outline

• Some tricks of the (SC) trade
• using SSCs as example
• GSC longevity they have a half-life
• Natural GSC replenishment switch from
  asymmetric to symmetric divisions?
• GSC aging slowed cycling other affects?
• Dedifferentiation of TA cells back into SCs!
• Matunis seminar, March 3rd

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Todays Outline the section just finished --

• Some tricks of the (SC) trade using SSCs as
  example
• GSC longevity they have a half-life
• GSC replenishment switch from asymmetric to
  symmetric divisions?
• GSC aging slowed cycling other affects?
• Dedifferentiation of TA cells back into SCs!
• Matunis seminar, March 3rd

Aging of HSCs Chambers et al, 2007
Same behaviors for SCs in the fly ovary Todd
Nystul, February 5th
Keep these behaviors in mind while reading
assigned papers for January 28th Discussion
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Todays Outline

• Review SC behavior(s)
• The SC environs
• The Spermatogonial stem cell
• Tricks of the (SC) trade using SSCs as prime
  example
• Papers for Jan 28th Background for mammalian
  SSCs and their niche

74

• Remember
• ASingle Undifferentiated spermatogonia
• on basement membrane in tubules
• Gonia divide with incomplete cytokineses
• Apaired
• Aaligned
• Chains of gonial cells
• As gonial chains differentiate further,
• they move away from basement membrane
• (internally)

75

• A marker expressed in
• undifferentiated gonia
• AS
• Apaired
• Aaligned

A testis cross-section of chains will appear
differently compared to a whole mount tubule
Ngn3-GFP in whole tubules
Ngn3 tubule cross sections
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Some candidate regulators

• GDNF (extrinsic)
• GDNF /- germ cell loss
• Excess GDNF expression excess early-stage
  gonia

From where is GDNF normally expressed? Sertoli
cells (all of them?)
GDNF is being used to culture preps enriched for
SSCs (Brinster lab)
Meng et al, 2001 Yomogida et al, 2003
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Some candidate regulators

• PLZF (intrinsic)
• PLZF - /- germ cell loss
• PLZF expressed in chains

WT

• What does PLZF regulate?
• How is its expression regulated?

Anti-PLZF
plzf-
Buass et al, 2004 Costoya et al, 2004
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Conceptual Issues at the heart of these
papers GSC longevity GSC replenishment Dediffer
entiation back to stem cell behavior Where is
the niche?
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• Technical aspects of papers
• Lineage tracing
• Cre / LoxP technology
• Hormone-inducible Cre (Cre-ER)
• -- Cre-mediated excision leads to LacZ
  expression
• -- Tamoxifen treatment adjusted to generate
  constant level of partial labeling
• Transplantation / repopulation assays
• -- prepping hosts chemical depletion of SSCs
  (busulfan)
• Discriminating host from donor cells
• -- A Ubiquitin-eGFP trasngene also used
• (triple t-genic lines)

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Skipped in lecture
(rough) Stem cell / Niche status
Stem cell type
in vivo existence
Niche location
Defined signals
(derived)
N/A
N/A
ESCs
EGCs
(derived)
N/A
N/A
NSCs
Likely derived
Emerging
No
Very suggestive Controversial
HSCs
Definitive
Emerging
Epithelial
Definitive
Very suggestive
Emerging
Intestinal
Definitive
Suggestive?
Emerging
Spermatogonial
Definitive
Emerging
Emerging?
Mesenchymal
.............(Steve does not know
enough....)....................
Suggestive
Muscle Sattelite cell
Definitive, yes?
Emerging?
Steves relatively uninformed opinion I
suggest we assemble a table to fill in thru the
course....with citations for the evidences