Kasper C., van Griensven M., P?rtner R. (Eds.) Bioreactor Systems for Tissue Engineering II: Strategies for the Expansion and Directed Differentiation of Stem Cells

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Preface

First of all, the editors of this special volume would like to thank all the authors for

their excellent contributions. We would also like to thank Prof. Dr. Thomas Scheper

as well as Dr. Marion Hertel and Ingrid Samide from Springer for providing the oppor-

tunity to compose this volume and Springer for organizational and technical support.

Tissue engineering represents one of the major emerging ﬁelds in modern bio-

technology. Tissue engineering combines different disciplines ranging from bio-

logical and material sciences to engineering and clinical disciplines. The aim of

tissue engi neering is the development of therapeutic approaches to substitute

diseased organs or tissues or improve their function. Stem cells are early progeni-

tors that may substitute diseased tissues or provide cues for endogenous healing.

Stem cells are present in virtually all tissues. The ﬁrst chapters describe different

sources of stem cells including isolation and expansion. The use of fetal tissues

and umbilical cord is discussed as they come from immunoprivileged sites and

are considered to be early stem cells. The use of adipose-derived stem cells is dis-

cussed as a readily available autologous source. Subsequently, newer techniques for

“manufacturing” stem cells from somatic cells using “induced pluripotent stem cell”

technology are discussed and described in two chapters. The following chapter deals

with bioreactor cultivation of stem cells. Speciﬁc tissues such as cartilage and

endothelial precursors built the bridge to the last chapters. In those chapters, clinical

applications are the focus of interest. It covers a wide range of clinical applications

from veterinary orthopedics and human bone diseases until cardiologic applications.

This small overview indicates that we have tried to cover the area of stem cells

from isolation, expansion up to clinical applications. The road has been walked

already for a substantial distance. Howeve r, we are still at the beginning of this

exciting new technology.

We hope that this state-of-the-art book is helpful to your research. Please enjoy

reading it, as much as we enjoyed preparing it.

Summer 2010 Cornelia Kasper

Ralf Po

rtner

Martijn van Griensven

Contents

Alternative Sources of Adult Stem Cells: Human Amniotic Membrane .... 1

Susanne Wolbank, Martijn van Griensven, Regina Grillari-Voglauer,

and Anja Peterbauer-Scherb

Mesenchymal Stromal Cells Derived from Human Umbilical Cord

Tissues: Primitive Cells with Potential for Clinical and Tissue

Engineering Applications ..................................................... 29

Pierre Moretti, Tim Hatlapatka, Dana Marten, Antonina Lavrentieva,

Ingrida Majore, Ralf Hass, and Cornelia Kasper

Isolation, Characterization, Differentiation, and Application

of Adipose-Derived Stem Cells ............................................... 55

rn W. Kuhbier, Birgit Weyand, Christine Radtke, Peter M. Vogt,

Cornelia Kasper, and Kerstin Reimers

Induced Pluripotent Stem Cells: Characteristics and Perspectives ...... 107

Tobias Cantz and Ulrich Martin

Induced Pluripotent Stem Cell Technology in Regenerative

Medicine and Biology ........................................................ 127

Duanqing Pei, Jianyong Xu, Qiang Zhuang, Hung-Fat Tse,

and Miguel A. Esteban

Production Process for Stem Cell Based Therapeutic Implants:

Expansion of the Production Cell Line and Cultivation

of Encapsulated Cells ........................................................ 143

C. Weber, S. Pohl, R. Poertner, Pablo Pino-Grace, D. Freimark,

C. Wallrapp, P. Geigle, and P. Czermak

Cartilage Engineering from Mesenchymal Stem Cells .................... 163

C. Goepfert, A. Slobodianski, A.F. Schilling, P. Adamietz, and R. Po

rtner

xiii

Outgrowth Endothelial Cells: Sources , Characteristics and Potential

Applications in Tissue Engineering and Regenerative Medicine ......... 201

Sabine Fuchs, Eva Dohle, Marlen Kolbe, and Charles James Kirkpatrick

Basic Science and Clinical Application of Stem Cells

in Veterinary Medicine ...................................................... 219

I. Ribitsch, J. Burk, U. Delling, C. Geißler, C. Gittel, H. Ju

lke, and W. Brehm

Bone Marrow Stem Cells in Clinical Application: Harnessing

Paracrine Roles and Niche Mechanisms ................................... 265

Rania M. El Backly and Ranieri Cancedda

Clinical Application of Stem Cells in the Cardiovascular System ....... 293

Christof Stamm, Kristin Klose, and Yeong-Hoon Choi

Index .......................................................................... 319

xiv Contents

Adv Biochem Engin/Biotechnol (2010) 123: 1–27

DOI: 10.1007/10_2010_71

Springer-Verlag Berlin Heidelberg 2010

Published online: 17 March 2010

Alternative Sources of Adult Stem Cells:

Human Amniotic Membrane

Susanne Wolbank, Martijn van Griensven, Regina Grillari-Voglauer,

and Anja Peterbauer-Scherb

Abstract Human amniotic membrane is a highly promising cell source for tissue

engineering. The cells thereof, human amniotic epithelial cells (hAEC) and human

amniotic mesenchymal stromal cells (hAMSC), may be immunoprivileged, they

represent an early developmental status, and their application is ethically uncontro-

versial. Cell banking strategies may use freshly isolated cells or involve in vitro

expansion to increase cell numbers. Therefore, we have thoroughly characterized

the effect of in vitro cultivation on both phenotype and differentiation potential of

hAEC. Moreover, we present different strategies to improve expansion including

replacement of animal-derived supplements by human platelet products or the

introduction of the catalytic subunit of human telomerase to extend the in vitro

lifespan of amniotic cells. Characterization of the resulting cultures includes phe-

notype, growth characteristics, and differentiation potential, as well as immuno-

genic and immunomodulatory properties.

Keywords Adipogenesis, Expansion, hTERT, Human amniotic cells, Immor-

talization, Immunomodulation, Immunophenotype, Mesenchymal markers,

Osteogenesis, Platelet lysate, Stem cell markers, Telomer ase

S. Wolbank, M. van Griensven (*)

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstraße

13, 1200 Vienna, Austria

Austrian Cluster for Tissue Regeneration, Vienna, Austria

e-mail: Martijn.van.Griensven@trauma.lbg.ac.at

A. Peterbauer-Scherb

Red Cross Blood Transfusion Service of Upper Austria, Krankenhausstrasse 7, 4020 Linz, Austria

R. Grillari-Voglauer

Department of Biotechnology, Institute of Applied Microbiology, University of Natural Resources

and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria

Contents

1 Introduction . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.1 Stem Cell Characteristics of Amnion-Derived Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.2 Expansion and Cryoconservation of Amnion-Derived Cell: Towards Cell Banking . . 4

2 Stem Cell Characteristics and Immunomodulatory Potential of Human

Amnion-Derived Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.1 Isolation of Separate Populations of hAEC and hAMSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.2 Stem Cell Characteristics and Immunomodulation of hAEC and hAMSC . . . . . . . . . . . 8

3 Phenotypic Shift and Reduced Osteogenesis During In Vitro Expansion

of Human Amnion Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.1 Shift in Surface Antigen Expression During Cultivation of hAEC . . . . . . . . . . . . . . . . . 10

3.2 Osteogen ic Dif ferent iatio n Poten tial of h AEC Decr ease s

upon In Vitro Cultivation ..................................................... 12

4 Platelet Lysate for FCS-Free Expansion and Cryoconservation of Amnion-Derived Cells . . . 12

5 hTERT Induced Extension of In Vitro Life Span of Amnion-Derived Stem Cells . . . . . . . 14

6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

6.1 Stem Cell Characteristics and Immunomodulatory Potential of Human

Amnion-Derived Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

6.2 Phenotypic Shift and Reduced Osteogenesis During In Vitro Expansion of

Human Amnion Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

6.3 Platel et Ly sate for F CS-Free Expansion and Cr yoconse rvation of

Amnion-Derived Cells ......................................................... 22

6.4 hTERT Induced Extension of In Vitro Life Span of Amnion-Derived Stem Cells . . . 22

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Abbreviations

7-AAD 7-Amino-actinomycin D

ALPL Alkaline phosphatase gene

AP Alkaline phosphatase

AR Alizarin red

ASC Adipose-derived stem cells

BGLAP Bone gamma-carboxyglutamate protein

BMPR1B Bone morphogenetic protein receptor 1B

BMPR2 Bone morphogenetic protein receptor 2

BMSC Bone marrow mesenchymal stem cells

BrdU 5-Bromo-2-deoxy uridine

CBFA1 Core binding factor alpha

DMSO Dimethylsulfoxide

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme-linked immunosorbent asssay

FCS Fetal calf serum

FOI Fold of induction

hAEC Human amniotic epithelial cells

hAMSC Human amniotic mesenchymal stromal cells

2 S. Wolbank et al.

HPRT Hypoxanthine-guanine phosphoribosyltransferase

hTERT Human telomerase reverse transcriptase

Lep Leptin

MLR Mixed lymphocyte reaction

MSC Mesenchymal stem cells

O-Kit Mesenchymal stem cell osteogenic stimulatory kit

OO Oil red O

PBMC Peripheral blood mononuclear cells

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PD Population doubling

PDpT Population doubling post transduction

PHA Phytohemagglutinin

PL Platelet lysate

PPARg Peroxisome proliferator-ac tivated receptor gamma

PRP Platelet-rich plasma

pT Post transduction

RT-PCR Reverse transcriptase polymerase chain reaction

SA-b-gal Senescence associated b-galactosidase

SC Stem cells

TA Telomerase activity

TGF-b Transforming growth factor beta

TRAP Telomeric repeat ampliﬁcation protocol

vK von Kossa

1 Introduction

Various cell sources have been proposed for regenerative medicine, each having

their advantages and drawbacks. Since mature cell types are rarely available in

sufﬁcient quality and amounts, research has focused on undifferentiated stem cells.

Embryonic stem cells are characterized by pluripotency and an unlimited self-

renewal capacity [1]. The major drawback of these cells is their high tumorigenic

potential. Additionally, their generation is associated with major ethical concerns.

In contrast, recovery of adult stem cells is not ethically restricted, tumorigenic

conversion was observed only in sparse cases [2], and autologous application is

possible. In 1999, Pittenger found that bone marrow not only contained hemato-

poietic stem cells but also mesenchymal stem cells (MSC) [3]. However, important

limitations of bone marrow mesenchymal stem cells (BMSC) are their limited

proliferation capacities, their low frequency, and donor site morbidity. Further-

more, decreased differentiation potential with donor age has been reported [4].

During recent years, human adult MSC from various sources including adipose

tissue, muscle, connective tissue, skin, placenta, blood, cord blood, synovium,

Alternative Sources of Adult Stem Cells: Human Amniotic Membrane 3

periosteum, and perichondrium have been established as promising tools in regen-

erative medicine [5–11]. The ﬁrst successful cell based therapies for diseases such

as myocardial infarction, multiple sclerosis, amyotrophic lateral sclerosis, graft-

versus-host-disease, osteogenesis imperfecta, and Crohn’s ﬁstula have been con-

ducted [12–17].

1.1 Stem Cell Characteristics of Amnion-Derived Cells

Placenta derived cells, in particular those from amniotic membrane, have been

described to combine qualities from both embryonic and adult stem cells, with a

differentiation capacity to derivatives of all three germ layers, and a lack of

tumorigenicity [18, 19]. Amniotic membrane is the innermost of the fetal mem-

branes and consists of a single layer of epithelial cells residing on a basement

membrane, overlying a stromal layer. Human amniotic epithelial cells (hAEC) and

human amniotic mesenchymal stromal cells (hAMSC), respectively, can be released

separately from these two layers by differential enzymatic digestion [19–21]. Both

of these cell types have been described to express markers of mesenchymal

and embryonic stem cells [18, 19, 22–25]. What makes these cells especially

attractive is that large amounts can be isolated from an uncontroversial material

that is usually discarded after birth. Most importantly, immunosuppressive char-

acteristics of amniotic cells might render allogeneic application possible [19, 24 ,

26]. Furthermore, their fetal origin may provide amniotic cells not only with stem

cell potential but also with an immunoprivileged status [27]. Human amnion is

widely used in surgery and wound treatment for burned skin, decubitus ulcers, and

in ophthalmology [28, 29]. When transplanting amniotic membrane intracorneally

or under the kidney capsule, no rejection but only a mild cell-mediated reaction was

observed [27].

All these characteristics would make amniotic cells ideal candidates for tissue

engineering and their application in regenerative medicine. For this purpose, cells

can theoretically be used directly after isolation, or after in vitro cultivation, the

latter of which permits a gain in cell numbers, but important disadvantages are

increases in the risk of contamination with pathogens, accumulation of mutations,

and loss of differentiation potential and functionality.

1.2 Expansion and Cryoconservation of Amnion-Derived

Cell: Towards Cell Banking

To clarify the effect of in vitro culture on the quality of amnion-derived cells, a

thorough characterization comparing these cells before and after cultivation has

been performed.

4 S. Wolbank et al.

Applicability of these cells for allogeneic transplantation and stem cell based

therapies could further be boosted by standardized collection, quality control, and

careful selection of functional and safe cell banking products. However, in order to

provide sufﬁcient stem cell numbers for cell banking and cell based therapies, their

limited replicative potential has to be overcome. Regarding this aim, we followed

two strategies: (1) optimization of the expansion medium using human derived

growth supplements instead of fetal calf serum (FCS) and (2) introduction of the

catalytic subunit of human telomerase.

1.2.1 Effect of In Vitro Expansion on Amnion-Derived Stem Cells

As a consequence of the adaptation processes to the artiﬁcial cell culture environ-

ment and/or the possible enrichm ent of clones that have a growth advantage

in vitro, the phenotype of cells may change during cultivation. Such alterations

during in vitro cultivation have been described for BMSC [3] and in detail for

adipose-derived stem cells (ASC) [30–32].

We systematically analyzed the surface antigen expression proﬁle of hAEC

directly after isolation and in the course of in vitro cultivation, with a focus on

mesenchymal and embryonic stem cell markers and investigated possible func-

tional consequences of in vitro cultivation regarding their osteogenic and adipo-

genic differentiation potential.

1.2.2 Strategies to Circumvent Growth Limitation In Vitro: Use of Platelet

Lysate During Expansion and Cryopreservation

Culturing mammalian cells usually involves expansion in cell culture medium

supplemented with FCS. Furth ermore, FCS is also a crucial component of cryo-

preservation media. While FCS is the golden stand ard to supplement research cell

culture media, its application for cell based therapies should be minimized as it

bares the risk for transmission of pathogens including prions, viruses and zoonoses

[33]. Immunological in vitro reac tions to FCS after cultivation have already been

demonstrated [34, 35]. In addi tion to the reported disadvantages of FCS there is a

predicted shortage in FCS in the next few years resulting in 50% increased purchase

prices [36].

Hence, well-screened human sources for growth factors would be favorable for

cell therapy. As such, platelets may offer a viable alternative to FCS. They contain

in their a-granules growth factors including platelet derived growth factor, basic

ﬁbroblast growth factor, insulin-like growth factor, and transforming growth factor

beta (TGF-b)[33] whereby TGF-b1 is the most abundant [37]. These growth

factors play important roles during wound healing by exerting above all mitogenic

activity.

Platelet derived products – such as platelet lysate (PL) or platelet-rich plasma

(PRP) – have already been proposed as culture supplement for several cell types

Alternative Sources of Adult Stem Cells: Human Amniotic Membrane 5