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Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3DPorous Polymer Scaffold for Liver Tissue Engineering

Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan; Wolff, Anders; Emnéus,Jenny; Aspegren, Anders; Dufva, Martin

Publication date:2014

Link back to DTU Orbit

Citation (APA):Hemmingsen, M., Muhammad, H. B., Mohanty, S., Wolff, A., Emnéus, J., Aspegren, A., & Dufva, M. (2014).Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3D Porous Polymer Scaffold forLiver Tissue Engineering. Poster session presented at 3rd International Conference on Tissue Science &Regenerative Medicine, Valencia, Spain.

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a

Perfused 3D Porous Polymer Scaffold for Liver Tissue Engineering

*Technical University of Denmark, 2800 Kgs. Lyngby, Denmark, **Cellectis AB, Gothenburg, Sweden.

Website: www.nanobio4trans.eu e-mail: mette.hemmingsen@nanotech.dtu.dk

Mette Hemmingsen*, Haseena Bashir Muhammad*, Soumyaranjan Mohanty*, Anders Wolff*,

Jenny Emneus*, Anders Aspegren**, Martin Dufva*

Introduction and Aim

A huge shortage of liver organs for transplantation has motivated the research field of tissue engineering to develop bioartificial liver tissue and even

a whole liver. The goal of NanoBio4Trans is to create a vascularized bioartificial liver tissue, initially as a liver-support system. Due to limitations of

primary hepatocytes regarding availability and maintenance of functionality, stem cells and especially human induced pluripotent stem cells (hIPS

cells) are an attractive cell source for liver tissue engineering. The aim of this part of NanoBio4Trans is to optimize culture and hepatic differentiation

of hIPS-derived definitive endoderm (DE) cells in a 3D porous polymer scaffold built-in a perfusable bioreactor. The use of a microfluidic bioreactor

array enables the culture of 16 independent tissues in one experimental run and thereby an optimization study to be performed.

Funding provided by EU Program HEALTH.2012.1.4-2 [Medical technology for transplantation and bioartificial organs], Contract:304842

Bioreactor Array System for Tissue Culture

Our approach for engineering liver tissue is to

culture and differentiate hIPS-derived DE cells

in a 3D porous polymer scaffold housed in a

perfusable bioreactor to ensure supply of

oxygen and nutrients and removal of waste.

Single bioreactor

Porous polymer scaffold

Bioreactor array

Outlet vials

LEGO® motor

8 channel

micropumps

Inlet vials

Pressure inlet to avoid

bubble formation

Issues at Flow Conditions

Time

Conc. of

added

factors

At Flow Conditions

Time

Conc. of

added

factors

At Static Conditions

.

Concentration of Added

Signaling Factors

One important parameter when optimizing conditions for flow cultures is the flow rate. The flow rate is

a balance between ensuring sufficient supply of nutrients and oxygen, but at the same time avoiding

to much shear stress onto the cells and removal of cell-to-cell signaling factors. Besides testing

different flow rates, we apply different scaffold designs with a different flow profile. Another issue to

consider is the concentration of added signaling factors. At flow the concentration of added factors is

constant, while at static conditions in a batch culture, the concentration decreases between each

exchange of medium. Thus, a lower concentration than the one optimized for batch cultures might be

better at flow conditions.

Gene Expression of Liver Markers

0

0.5

1

1.5

2

2.5

3

3.5

Ge

ne

exp

ress

ion

[r.

u.]

ALB

0

200

400

600

800

1000

1200

1400

1600

Gen

e ex

pre

ssio

n [

r.u

.]

AFP

0

1

2

3

4

5

6

Gen

e ex

pre

ssio

n [

r.u

.]

CYP3A5

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0.004

Gen

e ex

pre

ssio

n [

r.u

.]

CYP3A7

0

2

4

6

8

10

12

14

16

Ge

ne

exp

ress

ion

[r.

u.]

HNF4A

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0.004

0.0045

Gen

e ex

pre

ssio

n [

r.u

.]

CAR

0

1

2

3

4

5

Ge

ne

exp

ress

ion

[r.

u.] ALB

0

500

1000

1500

2000

2500

3000

3500

Ge

ne

exp

ress

ion

[r.

u.] AFP

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Ge

ne

exp

ress

ion

[r.

u.] CYP3A5

0

0.00005

0.0001

0.00015

0.0002

0.00025

Ge

ne

exp

ress

ion

[r.

u.] CYP3A7

0

5

10

15

20

Ge

ne

exp

ress

ion

[r.

u.] HNF4A

0

0.005

0.01

0.015

0.02

0.025

Ge

ne

exp

ress

ion

[r.

u.] CAR

A) Conditioned medium collected from a parallel static

batch culture increased the gene expression of liver

markers. Analysed at day 9 and day 14 after the DE stage.

Similar gene expression level of the hepatocyte nuclear transcription factor 4α was observed at

perfusion cultures compared to conventional static cultures, whereas a decreased expression was

seen for the transcription factor CAR and the CYP enzymes CYP3A5 and CYP3A7. Furthermore,

expression of albumin and α-fetoprotein was almost knocked down. However, expression of most of

the markers was increased by the use of A) conditioned medium or B) medium with a two times lower

concentration of the added signalling factors than those optimized for conventional batch cultures.

No

rma

l m

ed

ium

C

on

ditio

ne

d m

ed

ium

B) Differentiation medium with a two times lower concentration of

added signalling factors than those optimized for conventional

batch cultures (2x dil. medium) resulted in increased expression of

most liver markers at day 14 after the DE stage.

Conclusion

The results suggest that the flow conditions affect cell-to-cell signalling necessary for liver differentiation and/or functionality, as well as the

concentration of added signalling factors in the differentiation medium has to be adapted to the different environment at flow culture with

constant renewal of the culture medium.

Cell morphology

A hepatocyte-like cell morphology was obtained with

polynucleated cells and ”bile-canaliculi”-like structures

(shown by the red arrows). Imaging at day 14 of

differentiation after DE stage. Live stained cells A) and

Höchst stained cells B) in the 3D scaffold. C) Phase contrast

image of differentiated cells in a 2D perfusion chamber.

A)

B)

C)

Inlet

Outlet

Flow Rate

1. Supply of nutrients 2. Removal of waste

1. Shear stress 2. Removal of cell-

cell signaling factors

6 mm

5 mm

The bioreactor array enables culture/differentiation

of 16 independent tissues in one experimental run.