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Inducing gametic cells from stem cells, and viceversa

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Ally Hill March 2, 2009. Morgan Batson Biol 430. Robyn Sharma. Presentation Outline. Introduction. 1st Paper (Geijsen et al., 2004) germ cells from embryonic stem ... – PowerPoint PPT presentation

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Title: Inducing gametic cells from stem cells, and viceversa


1
Inducing gametic cells from stem cells, and
vice-versa
  • Ally Hill March 2, 2009
  • Morgan Batson Biol 430
  • Robyn Sharma

2
Presentation Outline
  • Introduction
  • 1st Paper (Geijsen et al., 2004)
  • germ cells from embryonic stem cells
  • 2nd Paper (Guan et al., 2006)
  • stem cells from testis cells
  • Significance
  • Conclusion

http//www.cartoonistgroup.com/store/add.php?iid1
0598
3
Stem Cells
  • Share 3 Characteristics
  • Unspecialized cells
  • Renew themselves for
  • long periods of time
  • Induced to form
  • specialized cells

http//hsb.iitm.ac.in/jm/ARCHIVES/July-Aug05/arti
cles_files/stem.html
4
Types of Stem Cells
  • Embryonic Stem (ES) Cells
  • inner cell mass
  • pluripotent
  • Adult Stem Cells
  • replenish dying cells
  • multipotent (normally)

5
Derivation of embryonic germ cells and male
gametes from embryonic stem cells
  • Letters to Nature
  • Niels Geijsen et al., 2004

6
What researchers hypothesized
  • primordial germ cells
    gametes
  • first blood lineages
  • 2. ES cells embryoid bodies
    sustain blood
    development
  • THEREFORE embryoid bodies might also support
    primordial germ cells?

1. Epiblast
7
Experiment 1
Pluripotency
Germ cell development
Germ cell specific, male gonad
(Geijsen et al., 2004)
8
Experiment 2
  • Cells SSEA1/Oct4 possible primoridal germ
    cells
  • Treated cells with retinoic acid

(Geijsen et al., 2004)
9
Results for Experiment 2
(Geijsen et al., 2004)
10
Experiment 3
  • SSEA1 cells manifested erasure of epigenetic
    imprints unique property of PGCs

(Geijsen et al., 2004)
11
Experiment 4
differentiation
  • PGCs
    male gametes?

(Geijsen et al., 2004)
12
Experiment 5
  • Immunostained cell populations for gene marker
    specific to male meiotic germ cells
  • Large population in EB, some haploid cells also
    found

(Geijsen et al., 2004)
13
  • 50 able to fertilize oocyte
  • 20 formed blastocyst

(Geijsen et al., 2004)
14
Conclusion
  • Embryoid bodies do support primordial germ line
    formation into haploid cells
  • Have ability to fertilize oocytes develop into
    blastocysts
  • Provides accessible model for germline studies

15
Pluripotency of spermatogonial stem cells from
adult mouse testis
  • Letters to Nature
  • Kaomei Guan et al., 2006

16
Introduction
  • Embryoid bodies ? functional male gametes
  • can we do the reverse?
  • Can we get embryonic stem (ES) cells from
    spermatogonial stem cells (SSCs) from adult male
    testis?
  • Stra8 marker of spermatogonia

17
Isolating Spermatogonial Stem Cells
  • Therefore proves accurate isolation of
    spermatogonial stem cells

(Guan et al., 2006)
18
Experiment 1
(Guan et al., 2006)
19
Experiment 1
Basic medium epiblast
I
II
LIF embryonic stem cell-like
LIF cultured cells grow like stem cells (30th
passage)
IV
(Guan et al., 2006)
20
Experiment 2
  • hanging drop method for embryoid body formation
  • Differentiated cells, loss of pluripotency markers

Stra8/GFP Loss of Oct3/4
(Guan et al., 2006)
21
Experiment 3
  • Can maGSCs become differentiated into 3 tissue
    layers?

Expression of mesoderm
(Guan et al., 2006)
22
Mesodermal Differentiation
(Guan et al., 2006)
23
Mesodermal Differentiation
myotubes
endothelial
Smooth muscle
(Guan et al., 2006)
24
Ectodermal Differentiation
Dopaminergic neuron
Neurofilament
Overlay
(Guan et al., 2006)
25
Endodermal Differentiation
Keratinized Epithelial Cells
Epithelial Cells
Bile Duct Cells
(Guan et al., 2006)
26
Are maGSCs pluripotent in vivo?
endoderm
mesoderm
ectoderm
Teratoma formation
(Guan et al., 2006)
27
Developmental Potentials
(Guan et al., 2006)
28
Conclusion
  • SSC can become sperm or pluripotent stem cells
    depending on their conditions
  • maGSCs gt 3 germ layers form teratomas
  • need LIF for SSCs to proliferate to maGSCs
  • In vivo interaction with SSCs and Sertoli cells
    ? spermatogenesis only
  • Absence of Sertoli cells? pluripotent stem cells
  • Capacity to form multipotent cells persists in
    adult mouse testis

29
Summary and Discussion
  • Generating synthetic egg sperm from stem
    cells
  • Create viable embryos
  • Treat infertility
  • Studies of human germ cells
  • Generating ES cells from testis
  • Fewer ethical issues
  • Adult capabilities for stem cell production
  • Individual cell-based therapy
  • Establishment of human maGSCs from testicular
    biopsies

30
Strengths Weaknesses
  • Strengths
  • verified stem cell capabilities through multiple
    experiments
  • Geijsen et al., 2004 systematic approach,
    logical steps
  • Weaknesses
  • Minimal explanation interpretation of their
    findings
  • ie. no possible reason given for why growth
    stopped at blastocyst stage
  • New research showing that maGSCs have some, but
    not all the characteristics of true pluripotent
    cells.

31
Future Directions
  • isolate stem cells from human testis (Conrad et
    al., 2008)
  • grow complete adult from synthetic sperm egg
    cells
  • transplant differentiated tissue from maGSCs to
    deficient individuals cure diseases
  • investigate controls for differentiation
  • investigate creating stem cells from other adult
    cells
  • Woltjen et al., 2009 (in press) manipulated
    skin cells

32
References
  • Conrad S, Renninger M, Hennenlotter J, Wiesner T,
    Just L, Bonin M, Aicher W, Buhring H-J, Mattheus
    U, Mack A, Wagner H-J, Minger S, Matzkies M,
    Reppel M, Hescheler J, Sievert K-D, Stenzl A and
    Skutella T. 2008. Generation of pluripotent
    stem cells from adult human testis. Nature.
    456 344 349.
  • Gaun K, Nayernia K, Maier LS, Wagner S, Dressel
    R, Lee JH, Nolte J, Wolf F, Li M, Engel W and
    Hasenfuss G. 2006. Pluripotency of
    spermatogonial stem cells from adult mouse
    testis. Nature. 440 1199 1203.
  • Geijsen N, Horoschak M, Kim K, Gribnau K, Eggan K
    and Daley GQ. 2004. Derivation of embryonic
    germ cells and male gametes from embryonic stem
    cells. Nature. 427 148 154.
  • Woltjen K, Michael IP, Mohseni P, Desai R,
    Mileikovsky M, Hamalainen R, Cowling R, Wang W,
    Liu P, Gertsenstein M, Kaji K, Sung H-K and Nagy
    A. 2009. piggyBac transposition reprograms
    fibroblasts to induced pluripotent stem cells.
    Nature. Advance online publication, March 1, 2009
    (DOI 10.1038/nature07863)

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