1. Characterization of
embryoid bodies formed
with different protocols
20111117
Han-Ni Cheng
1

2. Outline
• Introduction
• Formation of embryoid bodies
• Characteristics of assay
- histology
- germ layer markers expression
- efficiency of differentiating
• Conclusion
2

3. Introduction
3
Embryonic stem (ES) cells are known to
maintain high proliferation, differentiation,
and self-renewal capacities.

4. 4Fig. 1. Blastocysts contain pluripotential stem cells.
Human - 6~7 day
Mouse - 3.5 day
(Terese et al., 2001)
Cleavage stage embryo Cultured
blastocyst
Isolated inner cell mass
Mouse fibroblast
feeder cells
Established ES cell cultures
Cells dissociated
and replated
Mouse fibroblast
feeder cells

5. 5
 For most ES cell lines, this occurs via the
formation of three-dimensional aggregates
called embryoid bodies (EBs).
Ectoderm →nervous system, reproductive tract
etc.
Endoderm →digestive system, respiratory system
and most gland
Mesoderm →muscle, blood vessels, reproductive
system, urinary system , skeletal
system.

6. Major Problems of during EB formation
Agglomeration
6
2hr8hr
(Dang et al., 2001)

7. • Agglomeration of EBs that may have
negative effects on proliferation and
differentiation in the mouse model. (Dang et
al., 2001)
• Because of agglomerated large EBs revealed
extensive cell death and eventually large
necrotic centers due to mass transport
limitations. (Nir et al., 2003) 7

8. Formation of
embryoid bodies
8

9. Formation of
embryoid bodies
• Static Suspension culture (SSC)
• Hanging drop (HD)
• Entrapment
• Bioreactor (Stirred suspension system)
9

10. Static Suspension culture
10
Single ES cells
37 ℃
CO2 4.5~5%
Cultured 24~48 hrsSingle ES cellsES colonies
0.25%Trypsin, 20~30 sec
1000 rpm for 3~5 mins
Count the cell number
Fig. 2. A rough flow chart about suspension culture.
(Gomes et al., 2010)
Petri dish

11. Hanging drop
11
top bottom
3~4 ml PBS
37 ℃
CO2 4.5~5%
Cultured 24~48 hrs
Single ES cells
Fig. 3. A rough flow chart about hanging drop.
(Ao et al., 2011)
Petri dish

12. 12
Entrapment
Single ES cells
37 ℃
CO2 4.5~5%
Cultured 24~48 hrs
96 well dish
104~105 ES/well
Fig. 4. A rough flow chart about entrapment for
formation of EBs. (Ao et al., 2011)

13. Commercial - AggreWell
13
Fig. 5. AggreWell™ contains microwells to make uniform cell
aggregates.
(A) AggreWell™400 plates contain microwells 400 μm in diameter.
Photo taken at 40x magnification.
(B) AggreWell™800 plates contain microwells 800 μm in diameter.
Photo taken at 40x magnification. (StemCell Tech.)

14. Bioreactor
14
Fig. 6. Bioreactors of stirred
suspension system: (A)slow
turning lateral vessel (STLV)
and (B)high aspect rotating
vessel (HARV).
(Rungarunlert et al., 2009)
STLV culture(A)
central gas transfer cord
HARV culture(B)
Cultured 12~24 hrs
15–20 rpm
Single ES cells
oxygenator membrane

15. Characteristics of assay
15

16. Histology
16
Fig. 7. Histological analysis of EBs.
B C
D E
A
(Mogi et al., 2009; Nir et al., 2003)
B
HD
STLV HARV
SSC SSC

17. 17(Nir et al., 2003)
Fig. 8. RT-PCR analysis demonstrated the generation of tissues
derived from the three germ layers in the two system cultures in the
human model.
Germ layer markers expression

18. 18
Germ layer markers expression
Fig. 9. RT-PCR analysis ectoderm layer markers expression in different
size by the entrapment. (A)RT-PCR analysis. (B) The quantification of
relative gene expression. (Park et al., 2007)
0
(A)
(B)
(B)

19. 19
α-fetoprotein (AFP)
Germ layer markers expression
Fig. 10. RT-PCR analysis demonstrated mouse EB endoderm mark (α-
fetoprotein, AFP) expression for day 4 and day 8 in vitro. (A)RT-PCR
analysis. (B) The quantification of relative gene expression. ** indicate
p < 0.01 as compared to 200 μm EBs.
(Choi et al., 2010)
(A) (B)

20. 20
Efficiency of differentiating
Fig. 11. Illustration of the cumulative percentage of EBs containing
contracting area derived from STLV, hanging drop and suspension
culture. Scale bar=500 μm
D
(Rungarunlert et al., 2009)
A B C

21. 21
Efficiency of differentiating
(Choi et al., 2010)
Fig. 12. The beating frequency of EBs. EB beating frequency refers to
the number of concave microwells containing beating EBs divided by
the total number of concave microwells. Error bars are standard
deviation and Scale bars are 500 μ m.

22. Efficiency of differentiating
22
(Choi et al., 2010)
Fig. 13. Quantitative analysis of neurite numbers and lengths from
EBs retrieved from the entrapment, showing that larger EBs had a
greater number of neurites than smaller EBs. Error bars are
standard deviation; ** indicate p < 0.01 as compared to 200 μm EBs.

23. Summary
• Small (100 μm) aggregates showed increased
expression of the ectodermal marker
compared to that in large (500 μm)
aggregates, which had an increased
expression of mesodermal and endodermal
markers.
• Large EBs (500/1000 μm) had batter
differenting efficiency than small EBs (200
μm).
23

24. Conclusion
24

25. Conclusion
• It's better to chose the best protocol which
consider experiment design and lab
condition.
• It appears that the size of EBs exerts a
stronger influence on their differentiation
potential than the method by which EBs are
derived. 25

26. The End
26

27. 27
Diagram depicting important steps for the conversion of hESCs to
cardiomyocytes. Early mesoderm differentiates via pre-cardiac mesoderm
and committed cardiac progenitors further to functional cardiomyocytes
(CM). Early and late cardiomyocytes are mainly discriminated based on
their electrophysiological properties and structural organization. Typical
markers for each step are indicated as well as some functional features of
the differentiated cells.
(Vidarsson et al., 2010)

28. 28
(Choi et al., 2010)
Differentiation of GOlig-mESC into OPCs. (A) Scheme showing the protocol of the
embryoid body (EB)-based and small molecule-driven differentiation. At D8, the
EBs were disaggregated and plated. The cells were passaged once per week when
they became confluent. (B) Morphology of day 4 EBs.

29. 29
Cost Unity Quantity Technicality
SC Low Low Random Low
HD Middle Middle Limited High
Ent. Middle High Limited Middle
Bioreactor High Middle to
High
Random Low
Table 1. Comparison of current in vitro cell culture systems for production of EBs
and other cell types. Suspension culture (SC). Hanging drop (HD). Entrapment
(Ent.).
(Dang et al., 2001; Rungarunlert et al., 2009; Xu et al., 2001)

30. 30
•Ngn2=Neurogenin 2
•HES1=Transcription factor HES-1 is a protein
that in humans is encoded by the HES1 gene.
•ASCL1=Achaete-scute homolog 1 is a protein
that in humans is encoded by the ASCL1 gene.
Ectoderm markers

31. 31

32. 32
Nestin
It is expressed in stem cells of the central
nervous system (CNS) but not in mature CNS
cells.
A class VI intermediate filament protein.
Intermediate filaments within cells; characteristic
of primitive neuroectoderm formation.
(R&D systems web)

33. AFP(α-fetoprotin)
A major plasma protein produced by the yolk
sac and the liver during fetal life.
A marker of the visceral endoderm. (Hogan et
al., 1981)
33
(R&D systems web)

34. Brachyury
Member of the T-box family of transcription
factors.
Brachyury is required in the early determination
and differentiation of mesoderm.
Brachyury is essential for the formation of the
posterior body in all vertebrates.
34
(R&D systems web)

35. 35
Germ layer markers expression
Phase DAPI AFP
Fig. 11. Immunofluorescence assay of primate EB endoderm mark (α-fetoprotein,
AFP) expression in vitro. Scale bar = 150 μm.
(Park et al., 2007)

36. Germ layer markers expression
36
SSC
HD
D1 D2 D3 D4 D5 D6 D7
Brachyury
Fig. 9. RT-PCR analysis mesoderm layer marker Brachyury expression in the two
system cultures in the mouse model. There had no diameter data.
(Mogi et al., 2009)

37. 37
Quantitative analysis of EB diameter distribution. EBs retrieved from concave
microwells after culturing for 4 days in vitro were more homogeneous in size
and their sizes were significantly regulated by microwell widths (200, 500, and
1000 mm).
(Choi et al., 2010)