GERM CELLS DERIVED FROM EMBRYONIC STEM CELLS - PowerPoint PPT Presentation

Loading...

PPT – GERM CELLS DERIVED FROM EMBRYONIC STEM CELLS PowerPoint presentation | free to view - id: 212950-ZDc1Z



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

GERM CELLS DERIVED FROM EMBRYONIC STEM CELLS

Description:

Capable of developing into many (pluripotent) or any (totipotent) cell types ... Digested EB genome with PvuII to run through gel ... – PowerPoint PPT presentation

Number of Views:712
Avg rating:5.0/5.0
Slides: 54
Provided by: cbal9
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: GERM CELLS DERIVED FROM EMBRYONIC STEM CELLS


1
GERM CELLS DERIVED FROM EMBRYONIC STEM CELLS
  • PRESENTED BY CB ALLARD AND ANNA YU

2
INTRODUCTION
  • EMBRYONIC STEM CELLS
  • PRIMORDIAL GERM CELLS
  • NORMAL GERM CELL DEVELOPMENT
  • OUTLINE OF EXPERIMENTS

3
1. EMBRYONIC STEM CELLS
  • Overview
  • Typically derived from blastocyst in vitro
  • Can be maintained indefinitely
  • Pluripotency/Totipotency
  • Capable of developing into many (pluripotent) or
    any (totipotent) cell types
  • Oct-4 expression indicates pluripotency
  • Embryoid bodies develop when LIF is removed

4
1. EMBRYONIC STEM CELLS
  • Implications
  • Organ regeneration/transplants
  • Studies of diseased tissue and mutation effects
    on development
  • Unforeseen medical benefits
  • Ethical concerns
  • Experiments require eggs from donors

5
2. PRIMORDIAL GERM CELLS
  • Germ cells are derived from PGCs
  • Differentiate from the proximal epiblast
  • Detectable by staining for alkaline phosphatase
  • Late gastrulation PGCs migrate to gonads, then
    differentiate into egg or sperm

6
2. PRIMORDIAL GERM CELL
  • Migrate to genital ridge, which develops into
    gonad and induces PGCs to develop into germ cells

7
3. NORMAL GERM CELL DEVELOPMENT
  • Sex of the germ cell is determined by induction
    from the gonad, not from germ cell genes (recall
    sperm may carry X chromosome)
  • Follicles female somatic gonad cells which
    surround developing oocyte and synthesize
    estradiol

8
3. NORMAL GERM CELL DEVELOPMENT
  • Females diploid germ cells arrest at meiosis
    prophase I until sexual maturity undergo final
    meiotic divisions prior to fertilization
  • Males diploid germ cells arrest in G1 enter
    meiosis 7-8 days after birth
  • Meiosis markers are indicators of
  • germ cell development

9
(No Transcript)
10
3. NORMAL GERM CELL DEVELOPMENT
  • IMPRINTING
  • Germ cells must be highly specialized, but also
    be capable of starting over at zygote stage
  • A female eggs haploid genotype may find itself
    in a male sperm in the next generation.
  • Hence, alterations made during specialization
    are erasable in next generation

11
  • IMPRINTING
  • Normal development of embryo requires haploid
    genome from each parent, not just diploidy
  • Genome remembers which parent it came from
    using methylation markers (recall sex chromosomes
    insufficient an X genome can come from a male)
    which shut off certain genes
  • Imprinting remains during development but must be
    erased in the germ cell line

12
4. OVERVIEW OF EXPERIMENTS
  • Allowed stem cells to differentiate into embryoid
    bodies (EBs)
  • Detected and isolated PGC-like entities from EBs
  • PGCs differentiated into gamete-like cells
    (oocytes and sperm)
  • Various markers used to identify PGCs, oocytes,
    and sperm

13
(No Transcript)
14
Visualizing germ line expression of gcOct4-GFP
  • Oct-4 gene expression specific to germ line and
    epiblast in EBs
  • Deleted epiblast promoter region, leaving the
    germ cell promoter intact ? only expressed in
    germ cell and not in epiblast
  • Inserted GFP gene in place of Oct4 gene

15
  • 4. Transfected ES cells with gcOct4-GFP and
    cloned
  • 5. gcOct4-GFP only expressed in found germ cells

16
Derivation of embryonic GCs and male gametes from
ES cells(Geijsen et al 2003)
  • ES cells experimentally induced to differentiate
    into EBs
  • Quantified gene expression and looked for germ
    line markers
  • Isolated RNA from ES cells, EBs at different
    times of development, and testis (control)
  • Amplified by RT-PCR

17
Gene expression markers
  • 1. pluripotent ES cells (undifferentiated) and
    PGCs
  • Oct4SSEA1 surface antigens
  • 2. Mature germ cells stella fragilis (Fgls)
  • 3. Expression in germ line and not in soma
  • Dazl Rnh2
  • Piwil2 Tdrd1
  • Rnf17 Tex14

18
Undifferentiated embryonic stem cells and
primordial germ cells
Germ cell markers
Germ line markers (not in soma)
control
19
Which genes expressed where?
  • All marker genes expressed in undifferentiated ES
    cells
  • From ES to EB - decreased expression of almost
    all marker genes
  • Exception rare population of cells expressed
    Oct-4 and SSEA1 as EBs developed
  • Oct-4/SSEA1 cells
  • remainder of undifferentiated ES cells?
  • OR PRIMORDIAL GERM CELLS???

20
Rare population of Oct-4/SSEA1 cells
undifferentiated ES cells or germ cells?
  • Retinoic acid differentiates between ES cells and
    germ cells
  • ES cells stop dividing and differentiate
  • germ cells proliferate

21
Oct-4/SSEA1 cells from EBs proliferated ? most
likely PGCs
22
Further evidence that Oct-4/SSEA1 cells were
PGCs
  • Stained for alkaline phosphatase in SSEA1 EBs
    cells
  • Small population of SSEA1 showed positive
    staining
  • Positive stained cells surrounded by moving cells
    resembling migratory PGCs

23
Erasure of epigenic imprints?
  • PGCs are the only cells that show erasure of
    imprints
  • Igf2r has region DMR2 that is hypermethylated
    only on the maternal allele
  • Hypothesis if the cells in question are PGCs,
    then methylation should not be detected at DMR2

24
Erasure of epigenic imprinting?
  • Leukemia inhibitory factor, stem cell factor, and
    basic fibroblast growth factor placed in culture
    (support development of embryonic germ cells)
  • Digested EB genome with PvuII to run through gel
  • Put in Mlu1 (methylation sensitive enzyme) does
    not digest methylated genes

25
By day 10 all imprinting signals erased in Igf2r
DMR2 region
26
Quick summary
  • Rare population of cells had Oct4 and SSEA1
    expression after ES cell differentiation
  • Rare population of cells proliferated in RA
  • Rare population of cells stained positive for
    alkaline phosphatase
  • Rare population of cells showed epigenic erasure
  • RARE POPULATION OF CELLS DISPLAY IN VIVO
    PRIMORDIAL GERM CELL CHARACTERISTICS

27
Are the PGCs in a defined region within the
embryoid body?
  • Immunohistochemical analysis of 7 day old EBs
  • CD41 labels haematopoietic cells
  • SSEA1 labels germ cells
  • Both cell populations found very close together,
    as in vivo

28
Do PGCs differentiate into normal gametes?
  • Detected Sry in day 5 of EB development
  • Day 11, strong upregulation of acrosin and haprin
  • No expression of ZP1, 2, or 3 (female
    gametogenisis genes)

29
PGCs showed characteristic gene expression of
sperm development
30
Do these male germ cells undergo proper meiosis?
  • FE-J124 antibody that binds male meiotic germ
    cells
  • Hoechst 33342 fluorescent dye that binds to DNA
    for quantification purposes

31
Inefficient meiosis of EB derived male germ cells
  • EB derived FE-J1 germ cells had lower proportion
    haploidy (36) than cells from adult mice testes
    (68)

32
Are EB derived haploid cells biologically
competent as sperm cells?
  • Isolated FE-J1/Oct-4 cells from day 20 EBs
  • Intracytoplasmic injections into oocytes at two
    independent labs - same results
  • one in five developed into blastocysts
  •  

33
Derivation of Oocytes from Mouse Embryonic Stem
Cells(Hubner et al 2003)
  •  allow ES cells to differentiate
  • Identify cells with oocyte characteristics
  • gcOct4-GFP and c-kit expression marks early germ
    cells (gcOct4-GFP expressions Oct4 expression)
  • Vasa post-migratory germ cells
  • SCP3 and DMC1 meiosis specific markers

34
Separated into four populations of cells based on
expression of gcOct4-GFP and ckit
35
Formation of follicular structures
  • cell aggregates collected by centrifugation and
    then cultured
  • some looked like early ovarian follicles
  • about 20 of those follicles produced oocytes
    larger than 40um

36
Do follicle-like structures produce estradiol?
  • Natural follicles make estradiol
  • Hypothesis if detect estradiol in culture, then
    follicle like structures are probably follicles
  • Result found estradiol by day 12, peaked at day
    20

37
Characterization of Oocytes
  • Day 26, detected oocyte-like cells released from
    somatic cells

38
  • Hypothesis if oocyte specific gene markers (ZP1,
    ZP2, ZP3, Fig alpha, GDF-9) are found in the
    cells released, they may be oocytes
  • Results all expressed except ZP1, which may
    explain weak zona prone to detaching

39
Meiosis detection?
  • After 16 days, expressed proteins typical of
    meiosis
  • SCP3/COR1 staining in stage before leptotene
    (first phase of prophase I)

40
Surprise blastocyst like structure derived from
ES cells
  • follicular outgrowths similar to oocytes that
    have been parthenogenetically activated
  • May be artifact of lab techniques
  • None survived to birth after implantation

41
Summary of the experiments
  • ES cells, somatic cells, germ cells, gametes all
    have different RNA and protein markers
  • These differences make it possible to distinguish
    between different cell types

42
Summary of sperm derivation
  • Oct-4/SSEA1 cells proliferated in retinoic
    acid stained positive for alkaline phosphatase ?
    most likely PGCs (rather than undifferentiated ES
    cells)
  • Showed erasure of epigenic imprints, similar to
    in vivo germ cells
  • PGCs showed characteristic gene expression of
    male gamete development (Sry, Gcnf, acrosin,
    haprin)
  • Inefficient meiosis about half the amount of
    cells meiotic compared to testis cells
  • Fertilized donor eggs 20 developed into
    blastocysts

43
Summary of oocyte derivation
  • Oct4, c-kit, Vasa detection implied germ cell
    development
  • Formation of follicle-like structures, 20
    developing into 40um oocytes
  • Estradiol production by follicles
  • oocyte-like cells released from somatic cells
    expressed oocyte specific gene markers (ZP2, ZP3,
    Fig alpha, GDF-9)
  • Meiosis detection
  • Blastocyst like structures arose without
    fertilization or parthogenic activation

44
DISCUSSION
  • POTENTIAL MEDICAL BENEFITS
  • POTENTIAL RESEARCH BENEFITS
  • ETHICAL IMPLICATIONS
  • LIMITATIONS/FURTHER RESEARCH

45
1. POTENTIAL MEDICAL BENEFITS
  • Fertility treatment
  • Understand causes of infertility
  • Use synthetic sperm to treat male infertility
  • Germ-cell tumors
  • Understanding the causes
  • Designing treatment
  • Recipients for nuclear transplants, gene therapy,
    etc.

46
1. POTENTIAL MEDICAL BENEFITS
  • Nuclear transplants into stem cell-derived oocytes

47
2. POTENTIAL RESEARCH BENEFITS
  • Understanding imprinting and human germ cell
    development
  • A limitless supply of eggs could be derived from
    a single line of ES cells
  • Use of eggs to produce diseased tissue for study

48
2. POTENTIAL RESEARCH BENEFITS
  • Study of sex chromosomes effects on development
  • Oocytes were derived from male stem cells
    (diploid XY)
  • Study effects on development of a YY zygote
  • Potential to study meiosis in a YY oocyte

49
2. POTENTIAL RESEARCH BENEFITS
  • TRISTEM
  • Company in London, UK
  • Claims to have developed technique to
    successfully transform white blood cells into
    stem cells
  • Proof of pluripotency but not yet of totipotency
  • If true, potential to derive all types of cells
    of an individuals own genotype potential for
    fertility treatment, organ regeneration (without
    rejection), cancer therapy, etc.
  • Bypasses ethical concerns too

50
3. ETHICAL IMPLICACTIONS
  • Stem cell research uses human tissues, but
  • Donor consent is given
  • only for acceptable purposes what is acceptable
    though?
  • Avoid inappropriate commercialization

51
3. ETHICAL IMPLICATIONS
  • 4. Embryo having the potential to become an
    autonomous being - person has a right to life
  • Can a blastocyst be considered as a person?
  • Is a cell worth the same respect as an embryo?
  • 5. Medical advancement worth the consequences of
    cloning?
  • 6. human cloning wasteful of embryos and fetuses
    (cloned embryos have had poor success of
    surviving to adulthood)

52
3. ETHICAL IMPLICATIONS
  • 7. autonomy (right of self government)
  • 8. Sex selection is legal in Canada and USA for
    non-medical reasons
  • 9. Designer babies

53
4. Limitations
  • 1. In vitro vs. in vivo
  • inefficient meiosis in sperm
  • parthenotes formed by uninduced/unfertilized eggs
  • nuclear transfer experiments in oocytes may be
    questionable, given their weak membrane
  • 2. Future studies
  • oocytes whether they show erasure of epigenic
    imprints
  • Can ES cell derived germ cells fertilize each
    other and will it grow into normal embryo?
  • 3. Legislature? Implications of doing such
    research (illegal cloning? Designer babies?)
About PowerShow.com