ST

EM

CE

LL B

IOE

NG

INE

ER

ING

STEM CELL BIOENGINEERING

Cláudia Lobato da Silva

Research activities at

Centro de Engenharia Biológica e Química

Large-scale ex-vivo

expansion systems

Control of self-renewal

versus differentiation

Stem Cell

Bioengineering

Determining the molecular and cellular mechanisms of stem cell

expansion/differentiation and controlling culture conditions in an ex-vivo

microenvironment – THE BIOREACTOR.

ST

EM

CE

LL B

IOE

NG

INE

ER

ING

Stem Cell Bioengineering

ST

EM

CE

LL B

IOE

NG

INE

ER

ING

Stem Cell Expansion/Differentiation: what’s the need?

GOAL: increase the number of stem cells available (which is limited) while maintaining their in vivo engraftment potential and/or induce controlled cell differentiation into specific cell types.

WHAT FOR?

CELL THERAPY

GENE THERAPY

ALTERNATIVE TO ANIMAL MODELS

ST

EM

CE

LL E

XP

AN

SIO

N /

DIF

FE

RE

NT

IAT

ION

Ex-vivo expansion of hematopoietic stem and progenitor cells from human bone marrow and umbilical cord blood:- co-culture with mesenchymal stem cell-derived stroma;- culture in stroma-free conditions.

RE

SE

AR

CH

Main Research Topics

Ex-vivo expansion and neural differentiation of mouse embryonic stem cells and mouse neural stem cells

HE

MA

TO

PO

IET

IC C

ELL

EX

PA

NS

ION

CB

0

10

20

30

40

11 13 16 21 24 26Days

Fo

ld

CD

34+ C

ell

Ex

pa

ns

ion

CB

0

10

20

30

40

50

11 13 16 21 24 26

Days

Fo

ld

CD

34+C

D3

8- Ce

ll

Ex

pa

ns

ion

CB CD34+ CB CD34+CD38-CB

0

10

20

30

40

11 13 16 21 24 26Days

Fo

ld

CD

34+ C

ell

Ex

pa

ns

ion

CB

0

10

20

30

40

50

11 13 16 21 24 26

Days

Fo

ld

CD

34+C

D3

8- Ce

ll

Ex

pa

ns

ion

CB

0

10

20

30

40

11 13 16 21 24 26Days

Fo

ld

CD

34+ C

ell

Ex

pa

ns

ion

CB

0

10

20

30

40

50

11 13 16 21 24 26

Days

Fo

ld

CD

34+C

D3

8- Ce

ll

Ex

pa

ns

ion

CB CD34+ CB CD34+CD38-

BM

0

2

4

6

8

10

0 4 10 14

Days

Fold

CD

34

+ C

ell

Ex

pa

ns

ion

with stroma

without stroma

BM

0

20

40

60

80

100

0 4 10 14

Days

Fo

ld C

D3

4+C

D3

8- C

ell

Exp

ansi

on

with stroma

without stroma

BM CD34+ BM CD34+CD38-BM

0

2

4

6

8

10

0 4 10 14

Days

Fold

CD

34

+ C

ell

Ex

pa

ns

ion

with stroma

without stroma

BM

0

20

40

60

80

100

0 4 10 14

Days

Fo

ld C

D3

4+C

D3

8- C

ell

Exp

ansi

on

with stroma

without stroma

BM CD34+ BM CD34+CD38-

Ex-vivo Expansion of Hematopoietic Cells in Serum-free Medium

Lobato da Silva, C. et al, Experimental Hematology 33; 828-835 (2005)

PluripotentStem Cell

(PSC)

Stem Cell(SC)

MyeloidStem Cell

(MSC)

LymphoidStem Cell

(LSC)

CFU-G,M

CFU-MEG

BFU-E CFU-E

CFU-Eo

Granulocytes

Monocytes

Platelets

Erythrocytes

Eosinophils

T and B cells of theimmune system

Hematopoietic Stem Cell

(HSC)

Predictive Modelling

Pre

dict

ive

Mod

elin

g

Y

Xk

YdX

Xd

Xe XkXkPkXk

dt

Xd

• self-renewal term keX

• cell death term, kkX

• term accounting for the differentiation kd

X

• limitation factor

First-order kinetic including:

SCkSCkSCkHSCkSCkdt

SCdk

LySCd

MySCd

SCde

Kinetic modeling can provide a significant insight into the "limiting steps" involved in the hematopoietic hierarchy and into the influence of conditions affecting the expansion process.

Lobato da Silva, C. et al, Bioprocess Biosystems Eng., 25, 365-369 (2003)

PR

ED

ICT

IVE

MO

DE

LLIN

G

0.0E+00

5.0E+07

1.0E+08

1.5E+08

2.0E+08

2.5E+08

3.0E+08

0 5 10 15 20 25

Time (days)

To

tal

Via

ble

Cel

ls

Experimental Theoretical

0.0E+00

1.0E+08

2.0E+08

3.0E+08

4.0E+08

5.0E+08

6.0E+08

7.0E+08

0 5 10 15 20 25

Time (days)

To

tal

Via

ble

Cel

ls

Experimental Theoretical

BM + hu-ST BM - hu-ST

0.0E+00

1.0E+06

2.0E+06

3.0E+06

4.0E+06

5.0E+06

0 5 10 15 20 25

Time (days)

CD

34

+

Experimental Theoretical

0.0E+00

1.0E+06

2.0E+06

3.0E+06

4.0E+06

0 5 10 15 20 25

Time (days)

CD

34

+38

-

Experimental Theoretical

0.0E+00

1.0E+06

2.0E+06

3.0E+06

4.0E+06

0 5 10 15 20

Time (days)C

D 3

3+

Experimental Theoretical

CD34+ CD34+CD38- CD33+

Modelling

Lobato da Silva, C. et al, Bioprocess Biosystems Eng., 25, 365-369 (2003)BM

Mod

elin

g R

esul

ts

MO

DE

LLIN

G

EM

BR

YO

NIC

ST

EM

CE

LL E

XP

AN

SIO

N

Fernandes, A.M. et al, Journal of Biotechnology (accepted for publication) (2007)

Embryonic Stem Cell Expansionon microcarriers under stirred conditions

0.0E+00

1.0E+06

2.0E+06

3.0E+06

4.0E+06

0 1 2 3 4 5 6 7 8Time (days)

Via

ble

cel

ls/m

L

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8Time (days)

Fo

ld In

crea

se

DMEM/FBS medium o SF medium ■ DMEM/FBS medium □ SF medium

0.0E+00

1.0E+06

2.0E+06

3.0E+06

4.0E+06

0 1 2 3 4 5 6 7 8Time (days)

Via

ble

cel

ls/m

L

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8Time (days)

Fo

ld In

crea

se

DMEM/FBS medium o SF medium ■ DMEM/FBS medium □ SF medium

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 6 7 8Time (days)

Sp

ecif

ic g

row

th r

ate

(day

-1) DMEM/FBS medium o SF medium

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 6 7 8Time (days)

Sp

ecif

ic g

row

th r

ate

(day

-1) DMEM/FBS medium o SF medium

EM

BR

YO

NIC

ST

EM

CE

LL

CH

AR

AC

TE

RIZ

AT

ION

Fernandes, A.M. et al, Journal of Biotechnology (accepted for publication) (2007)

Embryonic Stem Cell Expansionon microcarriers under stirred conditions

MTT

DAPI

ALP

Day 2 Day 4 Day 6 Day 8

AC

KN

OW

LE

DG

EM

EN

TS

Acknowledgments

Collaborations:

University of Nevada, Reno, USA

Rensselaer Polytechnic Institute, Albany, USA

Institute of Molecular Medicine (IMM)

Crioestaminal - Saúde e Tecnologia S.A.

Financial Support:

Fundação para a Ciência e Tecnologia,

Ministério da Ciência Tecnologia e Ensino Superior