Ex-vivo expansion of Embryonic Stem Cell-derived Neural Stem Cells

2009

António Filipe SousaInstituto Superior Técnico

6/15/2009

Index

1. Introduction

• 1.1. Stem Cells.• 1.2. Neural Stem Cells.• 1.3. Isolation and Culture.• 1.4. Characterization of the NSC.• 1.5. Oxygen effect on NSC culture.

2. Results and Discussion

• 2.1. Expansion of NSC• 2.2. Differentiation of NSC• 2.3. Cellular markers expression study

3. Conclusion

1. Introduction

1.1. Stem Cells

■ Ability to self-renew by multiplying in an undifferentiated state.■ Ability to differentiate into the various cell types that constitute an organism.

Have the ability to specialize in all postembryonic tissues and organs, and also extraembryonic tissues;

Capable of giving rise to most tissues of an organism;

Can only differentiate into a limited range of cell types;

Other cells, named as progenitor cells, can divide a limited number of times before facing a change in their potency or undergoing differentiation.

1. Introduction

1.2. Neural Stem Cells

■ Types:

Embryonic;Fetal;Adult.

The isolation of NSC from the CNS was first described by Reynolds and Weiss (1992), by a process called neurosphere formation assay (NFA).

1.3. Isolation and culture

■ NSC are not purified and not directly identifiable;■ Neurospheres differentiate much more readily into astrocytes than neurons,■ Oxygen and nutrient diffusion limitations and waste accumulation.

Limitations

1. Introduction

Embryonic Stem (ES) Cells, can be cultured in adherent conditions.

ES cells can be readily converted to transient neuroepithelial cells.

Progenitor cells that can be expanded as stem cell lines (NS cells) by the action of growth factors.

These new cells only persist in FGF and EGF and proliferate in absence of other cells types.CGR8-NS cells, in this work!

1. Introduction

1.4. Characterization of the NSC

1.5. Oxygen effect on NSC culture

Study and compare the proliferation of mouse ES cell derived NSC, both in conditions of normoxia (20% of oxygen) and hypoxia (2% of oxygen).

Goal!

Flow Citometry

Extracellular marker: CD44Intracellular markers: Nestin, GFAP, TUJ1

2. Results and Discussion

2.1. Expansion of NSC

0 0.5 1 1.5 2 2.5 3 3.50.00E+00

1.00E+05

2.00E+05

3.00E+05

4.00E+05

5.00E+05

6.00E+05

NormoxiaHypoxia

Time (Days)

Num

ber

of

cell

s

Lag phase – NSC adaptation to the plate and culture medium.

Exponential growth phase.

Stabilization of NSC growth rate (not shown)

Under hypoxia cells FI is 1.7 fold higher than under normoxia

1. Introduction2. Results and Discussion

<50 50-100 >1000

2

4

6

8

10

12

2%20%

Number of cells / colony

% o

f C

olo

ny F

orm

ing

Unit

s

Plating a very low number of cells in a plate, will select the only ones that are the most multipotent and “really stem”.

Fig. Bright field Microscopic photographies of the clonal growth of cells in normoxia (a) and hypoxia (b).

a b

The lack of cell interactions between cells during this study, suggests that hypoxia can somehow substitute this type of cell-contacts effect and so the number of NSC increase.

2. Results and Discussion

2.2. Differentiation of NSC

Fig. Bright field Images obtained with an optical microscope, from a cell population obtained after neuronal (a) and astrocytes (b) differentiation.

a b

Can still differentiate into different lineages of cells, after culture in hypoxia!

3 days of culture. RHB-A media supplemented with 1% FBS (GIBCO)

7 days of culture. RHB-A media with 15ηg/mL FGF-2 and then switched to RHB-A without growth factors

2. Results and Discussion

2.3. Cellular markers

expression study

CD44 may be a candidate for separating multipotent NS cells from more differentiated cells.

Normoxia Hypoxia

72,9% with growth factors exchange and 76,6% with no exchange

59,4% with growth factors exchange and 72,6% with no exchange

This could mean that not changing the medium under hypoxic conditions may cause NS cells to differentiate

2. Results and Discussion

2.3. Cellular markers

expression study

In order to determined if the multipotency of the ES cell-derived NS cells is affected by hypoxic culture, the expression of the multipotency marker Nestin was determined during time in culture

Suggesting that hypoxia conditions have no effect on the multipotency of NS cells

Nestin expression > 90% - Cells maintain their multipotency and can still proliferate.

Nestin expression not 100% - some cells in culture may have differentiate into more committed neural progenitors.

2. Results and Discussion

2.3. Cellular markers

expression study

It was also made the study of the expression of GFAP and TUJ1 by the cells in culture, after their expansion in hypoxic conditions.

Fig. On the left it is a graphic showing expression of GFAP by the cells in culture for the differentiation into astrocytes. On the right it is seen the expression pattern of TUJ1 by the cells in culture for differentiation into neurons.

Optical observation

Analysis of flow

cytometry

results for cellul

ar marke

rs

NSC cultured in

hypoxic

conditions

maintain

their potent

ial

3. Conclusion

• Hypoxic conditions favor mouse ES cells-derived NSC expansion

• Multipotentiality is not affected, in hypoxia

• Culture under hypoxic conditions may be used for a more efficient large-scale production of CGR8-NS cells

• After culture in hypoxic conditions, cells can still differentiate into cells from the central nervous system, neurons and glial cells, in this work, astrocytes

António Filipe SousaInstituto Superior Técnico

6/15/2009

• Direct transplantation of these cells to the CNS can one day be a method of treatment for neurodegenerative diseases.