characterization of embryoid bodies formed with different protocols...

Characterization of embryoid bodies formed with different protocols

20111117Han-Ni Cheng

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Outline• Introduction

• Formation of embryoid bodies

• Characteristics of assay

- histology

- germ layer markers expression

- efficiency of differentiating

• Conclusion2

Introduction

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Embryonic stem (ES) cells are known to

maintain high proliferation, differentiation,

and self-renewal capacities.

4Fig. 1. Blastocysts contain pluripotential stem cells.

Human - 6~7 dayMouse - 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

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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 glandMesoderm muscle, blood vessels, reproductive → system, urinary system , skeletal system.

Major Problems of during EB formationAgglomeration

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2hr8hr

(Dang et al., 2001)

• 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

Formation of embryoid bodies

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Formation of embryoid bodies

• Static Suspension culture (SSC)

• Hanging drop (HD)

• Entrapment

• Bioreactor (Stirred suspension

system)9

Static Suspension culture

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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

Hanging drop

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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

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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)

Commercial - AggreWell

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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.)

Bioreactor

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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 hrs15–20 rpm

Single ES cellsoxygenator membrane

Characteristics of assay

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Histology

16Fig. 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(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

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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)

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α-fetoprotein (AFP)


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)

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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

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(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.


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(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.

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).

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Conclusion

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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

The End

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Diagram depicting important steps for the conversion of hESCs tocardiomyocytes. Early mesoderm differentiates via pre-cardiac mesodermand committed cardiac progenitors further to functional cardiomyocytes(CM). Early and late cardiomyocytes are mainly discriminated based ontheir electrophysiological properties and structural organization. Typicalmarkers for each step are indicated as well as some functional features ofthe differentiated cells.

(Vidarsson et al., 2010)

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(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.

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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)

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•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

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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)

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)

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(R&D systems web)

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.

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(R&D systems web)

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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)


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SSC

HD

D1 D2 D3 D4 D5 D6 D7Brachyury

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)

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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)

characterization of embryoid bodies formed with different protocols...

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