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Animal Models for Stem Cell Therapy 1st Edition Bruno
Christ Digital Instant Download
Author(s): Bruno Christ, Jana Oerlecke, Peggy Stock (eds.)
ISBN(s): 9781493914524, 1493914529
Edition: 1
File Details: PDF, 11.13 MB
Year: 2014
Language: english
Methods in
Molecular Biology 1213

Bruno Christ
Jana Oerlecke
Peggy Stock Editors

Animal Models
for Stem Cell
Therapy
METHODS IN M O L E C U L A R B I O LO G Y

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 Animal Models
for Stem Cell Therapy

Edited by

Bruno Christ, Jana Oerlecke, and Peggy Stock

Department of Surgery, University of Leipzig, Leipzig, Germany
Editors
Bruno Christ Jana Oerlecke
Department of Surgery Department of Surgery
University of Leipzig University of Leipzig
Leipzig, Germany Leipzig, Germany

Peggy Stock
Department of Surgery
University of Leipzig
Leipzig, Germany

ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-4939-1452-4 ISBN 978-1-4939-1453-1 (eBook)
DOI 10.1007/978-1-4939-1453-1
Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014946352

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Preface

Initially, it was the biology of the embryonic stem cell and its unique features to develop
into multiple lineages that gave us insight into the molecular nature of developmental pro-
cesses leading to specified tissues and organs. Yet, soon we understood that there was a
much broader spectrum of applicability of stem cells in basic and applied sciences but
increasingly also in clinical use. This is today of high medicinal and socioeconomic interest
facing an increasing shortage of organs for transplantation, which is very often the only
remaining therapeutic option. In recent years the therapeutic potential of both embryonic
and adult stem cells has been investigated intensively in animal models of human diseases
giving hope for clinical feasibility of stem cell therapy in the near future. However, there is
a mandatory need to understand the way stem cells work under specified disease conditions,
which is as diverse as the diversity of the diseases itself. Therefore, it is evident that stem
cells from their nature as multi- or pluripotent cells may respond in a different way depend-
ing on the microenvironment of a specific disease and the affected tissue. This way of
response may comprise direct impact on tissue regeneration by differentiation of a stem cell
into the healthy cell of the tissue targeted but also paracrine effects such as secretion of
growth factors and/or cytokines affecting the diseased tissue to support its self-regenerative
capacity just to mention two major routes of action. This, however, implies that before
clinical translation of stem cell therapy it is warranted to study effects but as well side effects
of transplanted stem cells such as their fate, tumorigenic potential, tissue persistence, sys-
temic impact, and biodistribution etc. in animal models of human diseases.
This book addresses exemplified disease models of hepatic, cardiovascular, and neuro-
logical diseases as well as diseases of the connective and contractile tissue. We included also
a part describing the impact of stem cells on immunological diseases but also their potential
to modulate the host immunological response, which particularly makes mesenchymal stem
cells an attractive tool to avoid or at least reduce conventional immunosuppression in allo-
geneic stem cell transplantation settings or even to use them as immunosuppressant in, e.g.,
solid organ transplantation. The contents of the book are prepared to cover a wide range of
diseases and application of different kinds of stem cells such as embryonic and adult stem
cells but also reprogrammed tissue cells (iPS), which are the types of cells most frequently
discussed in the context of applied sciences and medicine. Hence, the description of the
animal disease models and their use in stem cell transplantation in this book covers interest
of basic scientists and clinicians to assess the biological as well as the therapeutic potential
of stem cell therapy. The standardization of protocols and assays is mandatory for future
implementation into regulatory documents presenting the results from different individual
cell types in different animal models in order to prepare a preclinical study in vivo as required
for later approval by regulatory bodies.

Leipzig, Germany Bruno Christ

Jana Oerlecke
Peggy Stock

v
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I INTRODUCTION
1 Stem Cells: Are We Ready for Therapy?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Insa S. Schroeder
2 Aurigon’s Point of View on the Safety Assessment of Cell-Based Therapies:
An Experience Based on the Participation in 15 ATMPs Projects. . . . . . . . . . . 23
Emmanuelle Cornali

PART II LIVER DISEASES

3 Preconditioning of the Liver for Efficient Repopulation
by Primary Hepatocyte Transplants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Petra Krause, Margret Rave-Frank, Hans Christiansen,
and Sarah Koenig
4 Age-Dependent Hepatocyte Transplantation for Functional
Liver Tissue Reconstitution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Peggy Stock
5 Treatment of NASH with Human Mesenchymal Stem Cells
in the Immunodeficient Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Sandra Winkler and Bruno Christ
6 The In Vivo Evaluation of the Therapeutic Potential
of Human Adipose Tissue-Derived Mesenchymal Stem Cells
for Acute Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Takeshi Katsuda, Hayato Kurata, Rie Tamai, Agnieszka Banas,
Tsuyoshi Ishii, Shumpei Ishikawa, and Takahiro Ochiya
7 A Mouse Model of Liver Injury to Evaluate Paracrine and Endocrine
Effects of Bone Marrow Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . 69
Chiung-Kuei Huang, Soo Ok Lee, Jie Luo, RongHao Wang,
Qiang Dang, and Chawnshang Chang
8 Animal Models to Test hiPS-Derived Hepatocytes in the Context
of Inherited Metabolic Liver Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Mathilde Dusséaux, Sylvie Darche, and Helene Strick-Marchand
9 Support of Hepatic Regeneration by Trophic Factors
from Liver-Derived Mesenchymal Stromal/Stem Cells . . . . . . . . . . . . . . . . . . 89
Suomi M.G. Fouraschen, Sean R.R. Hall, Jeroen de Jonge,
and Luc J.W. van der Laan

vii
viii Contents

PART III CARDIOVASCULAR DISEASES

10 Assessment of Functional Competence of Endothelial Cells
from Human Pluripotent Stem Cells in Zebrafish Embryos . . . . . . . . . . . . . . . 107
Valeria V. Orlova, Yvette Drabsch, Peter ten Dijke,
and Christine L. Mummery
11 Stem Cell Therapy for Necrotizing Enterocolitis: Innovative Techniques
and Procedures for Pediatric Translational Research . . . . . . . . . . . . . . . . . . . . 121
Jixin Yang, Yanwei Su, and Gail E. Besner
12 Exploring Mesenchymal Stem Cell-Derived Extracellular Vesicles
in Acute Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Stefania Bruno and Giovanni Camussi
13 Angiogenic Properties of Mesenchymal Stem Cells in a Mouse Model
of Limb Ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Leonardo Martins, Priscila Keiko Matsumoto Martin, and Sang Won Han
14 Methods to Assess Intestinal Stem Cell Activity in Response
to Microbes in Drosophila melanogaster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Philip L. Houtz and Nicolas Buchon

PART IV CONNECTIVE AND CONTRACTILE TISSUE

15 Muscle Pouch Implantation: An Ectopic Bone Formation Model . . . . . . . . . . 185
Greg Asatrian, Le Chang, and Aaron W. James
16 Bone Defect Repair in Mice by Mesenchymal Stem Cells. . . . . . . . . . . . . . . . . 193
Sanjay Kumar
17 Generation of Osteoporosis in Immune-Compromised Mice
for Stem Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Reeva Aggarwal, Vincent J. Pompili, and Hiranmoy Das
18 Application of Stem Cells for the Treatment of Joint Disease in Horses . . . . . . 215
Walter Brehm, Janina Burk, and Uta Delling
19 Adipogenic Fate Commitment of Muscle-Derived Progenitor Cells:
Isolation, Culture, and Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Anne-Marie Lau, Yu-Hua Tseng, and Tim J. Schulz
20 Skeletal Muscle Stem Cells for Muscle Regeneration . . . . . . . . . . . . . . . . . . . . 245
Johnny Kim and Thomas Braun

PART V NEUROLOGICAL DISEASES

21 Bone Marrow Stromal Stem Cells Transplantation in Mice
with Acute Spinal Cord Injury. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Virginie Neirinckx, Bernard Rogister, Rachelle Franzen,
and Sabine Wislet-Gendebien
 Contents ix

22 Histological Characterization and Quantification of Cellular

Events Following Neural and Fibroblast(-Like) Stem Cell Grafting
in Healthy and Demyelinated CNS Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Jelle Praet, Eva Santermans, Kristien Reekmans, Nathalie de Vocht,
Debbie Le Blon, Chloé Hoornaert, Jasmijn Daans, Herman Goossens,
Zwi Berneman, Niel Hens, Annemie Van der Linden, and Peter Ponsaerts
23 Improvement of Neurological Dysfunctions in Aphakia Mice,
a Model of Parkinson’s Disease, after Transplantation
of ES Cell-Derived Dopaminergic Neuronal Precursors . . . . . . . . . . . . . . . . . . 285
Sangmi Chung, Jisook Moon, and Kwang-Soo Kim
24 Methods for Assessing the Regenerative Responses of Neural Tissue . . . . . . . . 293
Steven W. Poser, Maria Adele Rueger, and Andreas Androutsellis-Theotokis
25 Mesenchymal Stem Cell-Based Therapy in a Mouse Model of Experimental
Autoimmune Encephalomyelitis (EAE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Annie C. Bowles, Brittni A. Scruggs, and Bruce A. Bunnell
26 Analysis of the Neuroregenerative Activities of Mesenchymal
Stem Cells in Functional Recovery after Rat Spinal Cord Injury. . . . . . . . . . . . 321
Akihito Yamamoto, Kohki Matsubara, Fumiya Kano,
and Kiyoshi Sakai

PART VI IMMUNEMODULATION BY STEM CELLS

27 Therapeutic Application of Mesenchymal Stromal Cells in Murine
Models of Inflammatory Bowel Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Elena Gonzalez-Rey and Mario Delgado
28 Mesenchymal Stem Cells Attenuate Rat Graft-Versus-Host Disease . . . . . . . . . 341
Masayuki Fujino, Ping Zhu, Yusuke Kitazawa, Ji-Mei Chen,
Jian Zhuang, and Xiao-Kang Li
29 Assessment of Anti-donor T Cell Proliferation and Cytotoxic
T Lymphocyte-Mediated Lympholysis in Living Donor Kidney
Transplant Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Aruna Rakha, Marta Todeschini, and Federica Casiraghi
30 Modulation of Autoimmune Diseases by iPS Cells. . . . . . . . . . . . . . . . . . . . . . 365
Fengyang Lei, Rizwanul Haque, Xiaofang Xiong, and Jianxun Song
31 A Chimeric Mouse Model to Study Immunopathogenesis
of HCV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Moses T. Bility, Anthony Curtis, and Lishan Su

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Contributors

REEVA AGGARWAL • Cardiovascular Stem Cell Laboratories, The Dorothy M. Davis Heart
and Lung Research Institute, Wexner Medical Center at The Ohio State University,
Columbus, OH, USA
ANDREAS ANDROUTSELLIS-THEOTOKIS • Department of Medicine, University of Dresden,
Dresden, Germany; Center for Regenerative Therapies Dresden, Dresden, Germany
GREG ASATRIAN • Department of Pathology and Laboratory Medicine, David Geffen School
of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
AGNIESZKA BANAS • Division of Molecular and Cellular Medicine, National Cancer Center
Research Institute, Tokyo, Japan
ZWI BERNEMAN • Laboratory of Experimental Hematology, University of Antwerp,
Antwerp, Belgium; Vaccine and Infectious Disease Institute (Vaxinfectio),
University of Antwerp, Antwerp, Belgium
GAIL E. BESNER • Department of Pediatric Surgery, Center for Perinatal Research,
The Research Institute at Nationwide Children’s Hospital, Nationwide Children’s
Hospital, Ohio State University College of Medicine, Columbus, OH, USA
MOSES T. BILITY • Lineberger Comprehensive Cancer Center, University of North Carolina
at Chapel Hill, Chapel Hill, NC, USA
DEBBIE LE BLON • Laboratory of Experimental Hematology, University of Antwerp,
Antwerp, Belgium; Vaccine and Infectious Disease Institute (Vaxinfectio),
University of Antwerp, Antwerp, Belgium
ANNIE C. BOWLES • Department of Cell and Molecular Biology, Tulane University School
of Medicine, New Orleans, LA, USA; Center for Stem Cell Research and Regenerative
Medicine, Tulane University School of Medicine, New Orleans, LA, USA
THOMAS BRAUN • Department of Cardiac Development and Remodelling, Max Planck
Institute for Heart and Lung Research, Bad Nauheim, Germany
WALTER BREHM • Large Animal Clinic for Surgery, Faculty of Veterinary Medicine,
University of Leipzig, Leipzig, Germany; Translational Centre for Regenerative
Medicine, University of Leipzig, Leipzig, Germany
STEFANIA BRUNO • Department of Molecular Biotechnology and Healthy Science,
Molecular Biotechnology Center, University of Torino, Torino, Italy
NICOLAS BUCHON • Department of Entomology, Cornell University, Ithaca, NY, USA
BRUCE A. BUNNELL • Department of Pharmacology, Tulane University School of Medicine,
New Orleans, LA, USA; Center for Stem Cell Research and Regenerative Medicine,
Tulane University School of Medicine, New Orleans, LA, USA
JANINA BURK • Large Animal Clinic for Surgery, Faculty of Veterinary Medicine,
University of Leipzig, Leipzig, Germany; Translational Centre for Regenerative
Medicine, University of Leipzig, Leipzig, Germany
GIOVANNI CAMUSSI • Department of Medical Sciences; Department of Molecular
Biotechnology and Healthy Science, University of Torino, Torino, Italy
FEDERICA CASIRAGHI • Department of Clinical Immunology and Transplantation,
Centro Ricerche Trapianti “Chiara Cucchi de Alessandri e Gilberto Crespi”,
Azienda Ospedaliera Papa Giovanni XXIII, IRCCS-Istituto di Ricerche
Farmacologiche Mario Negri, Ranica-Bergamo, Italy

xi
xii Contributors

CHAWNSHANG CHANG • George Whipple Lab for Cancer Research, Departments of Pathology,
Urology, Radiation Oncology, The Wilmot Cancer Center, University of Rochester
Medical Center, Rochester, NY, USA; Sex Hormone Research Center, China Medical
University/Hospital, Taiwan, China
LE CHANG • Department of Pathology and Laboratory Medicine, David Geffen School
of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
JI-MEI CHEN • Guangdong Cardiovascular Institute, Guangdong General Hospital,
Guangdong Academic of Medical Sciences, Guangzhou, China
BRUNO CHRIST • Department of Surgery, University of Leipzig, Leipzig, Germany
HANS CHRISTIANSEN • Department of Radiotherapy and Radiation Oncology,
University Medical Centre, Georg-August-University Goettingen, Goettingen, Germany
SANGMI CHUNG • Harvard Medical School, Harvard Stem Cell Institute,
McLean Hospital/Harvard Medical School, Belmont, MA, USA
EMMANUELLE CORNALI • Aurigon Life Science GmbH, Tutzing, Germany
ANTHONY CURTIS • Lineberger Comprehensive Cancer Center, University of North Carolina
at Chapel Hill, Chapel Hill, NC, USA
JASMIJN DAANS • Laboratory of Experimental Hematology, University of Antwerp, Antwerp,
Belgium; Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp,
Antwerp, Belgium
QIANG DANG • George Whipple Lab for Cancer Research, Departments of Pathology,
Urology, Radiation Oncology, The Wilmot Cancer Center, University of Rochester
Medical Center, Rochester, NY, USA
SYLVIE DARCHE • Innate Immunity Unit, Institut Pasteur, Inserm U668, Paris, France
HIRANMOY DAS • Cardiovascular Stem Cell Laboratories, The Dorothy M. Davis Heart
and Lung Research Institute, Wexner Medical Center at The Ohio State University,
Columbus, OH, USA
MARIO DELGADO • Institute of Parasitology and Biomedicine “Lopez-Neyra”, CSIC,
Granada, Spain
UTA DELLING • Large Animal Clinic for Surgery, Faculty of Veterinary Medicine,
University of Leipzig, Leipzig, Germany
PETER TEN DIJKE • Department of Molecular Cell Biology, Cancer Genomics Centre,
Leiden University Medical Center, Leiden, The Netherlands
YVETTE DRABSCH • Department of Molecular Cell Biology, Cancer Genomics Centre,
Leiden University Medical Center, Leiden, The Netherlands
MATHILDE DUSSÉAUX • Innate Immunity Unit, Institut Pasteur, Inserm U668, Paris,
France
SUOMI M.G. FOURASCHEN • Department of Surgery, Laboratory of Experimental
Transplantation and Intestinal Surgery (LETIS), Erasmus MC-University Medical
Center, Rotterdam, The Netherlands
RACHELLE FRANZEN • Groupe Interdisciplinaire de Génoprotéomique appliquée (GIGA),
Neurosciences Unit, University of Liège, Liège, Belgium
MASAYUKI FUJINO • Division of Transplantation Immunology, National Research Institute
for Child Health and Development, Tokyo, Japan; AIDS Research Center,
National Institute of Infectious Diseases, Tokyo, Japan
ELENA GONZALEZ-REY • Institute of Parasitology and Biomedicine “Lopez-Neyra”, CSIC,
Granada, Spain
HERMAN GOOSSENS • Vaccine and Infectious Disease Institute (Vaxinfectio), University of
Antwerp, Antwerp, Belgium
 Contributors xiii

SEAN R.R. HALL • Division of Thoracic Surgery, University Hospital Bern, Bern, Switzerland
SANG WON HAN • Department of Biophysics, Universidade Federal de Sao Paulo, Sao Paulo,
SP, Brazil
RIZWANUL HAQUE • Department of Microbiology and Immunology, The Pennsylvania State
University College of Medicine, Hershey, PA, USA
NIEL HENS • Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp,
Antwerp, Belgium; Center for Statistics, I-Biostat, Hasselt University, Hasselt, Belgium;
Centre for Health Economic Research and Modeling Infectious Diseases (Chermid),
University of Antwerp, Antwerp, Belgium
CHLOÉ HOORNAERT • Laboratory of Experimental Hematology, University of Antwerp,
Antwerp, Belgium, Germany; Vaccine and Infectious Disease Institute (Vaxinfectio),
University of Antwerp, Antwerp, Belgium
PHILIP L. HOUTZ • Department of Entomology, Cornell University, Comstock Hall,
Ithaca, NY, USA
CHIUNG-KUEI HUANG • George Whipple Lab for Cancer Research, Departments of Pathology,
Urology, Radiation Oncology, The Wilmot Cancer Center, University of Rochester
Medical Center, Rochester, NY, USA
TSUYOSHI ISHII • Regenerative Medicine Research and Planning Division,
Rohto Pharmaceutical Co. Ltd., Minato-ku, Tokyo, Japan
SHUMPEI ISHIKAWA • Department of Genomic Pathology, Tokyo Medical and Dental
University, Bunkyo-ku, Tokyo, Japan
AARON W. JAMES • Department of Pathology and Laboratory Medicine,
University of California, Los Angeles, Los Angeles, CA, USA; David Geffen School
of Medicine, Los Angeles, CA, USA
JEROEN DE JONGE • Department of Surgery, Laboratory of Experimental Transplantation
and Intestinal Surgery (LETIS), Erasmus MC-University Medical Center, Rotterdam,
The Netherlands
FUMIYA KANO • Department of Oral and Maxillofacial Surgery, Nagoya University
Graduate School of Medicine, Showa-ku, Nagoya, Japan
TAKESHI KATSUDA • Division of Molecular and Cellular Medicine, National Cancer Center
Research Institute, Chuo-ku, Tokyo, Japan
KWANG-SOO KIM • Department of Cardiac Development and Remodelling,
McLean Hospital/Harvard Medical School, Belmont, MA, USA
JOHNNY KIM • Department of Cardiac Development and Remodelling,
Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
YUSUKE KITAZAWA • Division of Transplantation Immunology, National Research Institute
for Child Health and Development, Tokyo, Japan
SARAH KOENIG • Department of General, Visceral, and Pediatric Surgery,
University Medical Centre, Georg-August-University Goettingen, Goettingen, Germany
PETRA KRAUSE • Department of General, Visceral, and Pediatric Surgery,
University Medical Centre, Georg-August-University Goettingen, Goettingen, Germany
SANJAY KUMAR • Center for Stem Cell Research, Christian Medical College, Vellore,
Tamilnadu, India
HAYATO KURATA • Division of Molecular and Cellular Medicine, National Cancer Center
Research, Chuo-ku, Tokyo, Japan; Regenerative Medicine Research and Planning
Division, Rohto Pharmaceutical Co. Ltd., Minato-ku, Tokyo, Japan
xiv Contributors

LUC J.W. VAN DER LAAN • Department of Surgery, Laboratory of Experimental

Transplantation and Intestinal Surgery (LETIS), Erasmus MC-University Medical
Center, Rotterdam, The Netherlands
ANNE-MARIE LAU • Research Group Adipocyte Development, German Institute of Human
Nutrition, Nuthetal, Germany
SOO OK LEE • George Whipple Lab for Cancer Research, Departments of Pathology,
Urology, Radiation Oncology, The Wilmot Cancer Center, University of Rochester
Medical Center, Rochester, NY, USA
FENGYANG LEI • Department of Microbiology and Immunology, The Pennsylvania State
University College of Medicine, Hershey, PA, USA
XIAO-KANG LI • Division of Transplantation Immunology, National Research Institute
for Child Health and Development, Setagaya-ku, Tokyo, Japan
ANNEMIE VAN DER LINDEN • Bio-Imaging Laboratory, University of Antwerp, Antwerp,
Belgium
JIE LUO • George Whipple Lab for Cancer Research, Departments of Pathology,
Urology, Radiation Oncology; The Wilmot Cancer Center, University of Rochester
Medical Center, Rochester, NY, USA
PRISCILA KEIKO MATSUMOTO MARTIN • Department of Biophysics, Universidade Federal de
São Paulo, São Paulo, Brazil; Research Center for Gene Therapy, Universidade Federal
de São Paulo, São Paulo, Brazil
LEONARDO MARTINS • Department of Biophysics, Universidade Federal de São Paulo,
São Paulo, Brazil; Research Center for Gene Therapy, Universidade Federal de São Paulo,
São Paulo, Brazil
KOHKI MATSUBARA • Department of Oral and Maxillofacial Surgery, Nagoya University
Graduate School of Medicine, Showa-ku, Nagoya, Japan
JISOOK MOON • Molecular Neurobiology Laboratory, Department of Psychiatry
and Program in Neuroscience, CHA University/CHA Stem Cell Institute, Seoul,
South Korea; Department of Bioengineering, CHA University/CHA Stem Cell Institute,
Seoul, South Korea
CHRISTINE MUMMERY • Department of Anatomy and Embryology, Leiden University
Medical Center, Leiden, The Netherlands
VIRGINIE NEIRINCKX • Groupe Interdisciplinaire de Génoprotéomique appliquée (GIGA),
Neurosciences Unit, University of Liège, Liège, Belgium
TAKAHIRO OCHIYA • Division of Molecular and Cellular Medicine, National Cancer Center
Research Institute, Chuo-ku, Tokyo, Japan
VALERIA V. ORLOVA • Department of Anatomy and Embryology, Leiden University Medical
Center, Leiden, The Netherlands
VINCENT J. POMPILI • Cardiovascular Stem Cell Laboratories, The Dorothy M. Davis Heart
and Lung Research Institute, Wexner Medical Center at The Ohio State University,
Columbus, OH, USA
PETER PONSAERTS • Laboratory of Experimental Hematology, University of Antwerp,
Antwerp, Belgium; Vaccine and Infectious Disease Institute (Vaxinfectio),
University of Antwerp, Antwerp, Belgium
STEVEN W. POSER • Department of Medicine, University of Dresden, Dresden, Germany
JELLE PRAET • Laboratory of Experimental Hematology, University of Antwerp, Antwerp,
Belgium; Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp,
Antwerp, Belgium; Bio-Imaging Laboratory, University of Antwerp, Antwerp, Belgium
 Contributors xv

ARUNA RAKHA • Department of Translational and Regenerative Medicine, Postgraduate

Institute of Medical Education and Research, Chandigarh, India
MARGRET RAVE-FRANK • Department of Radiotherapy and Radiation Oncology,
University Medical Centre, Georg-August-University Goettingen, Goettingen, Germany
KRISTIEN REEKMANS • Laboratory of Experimental Hematology, University of Antwerp,
Antwerp, Belgium; Vaccine and Infectious Disease Institute (Vaxinfectio), University of
Antwerp, Antwerp, Belgium
BERNARD ROGISTER • Groupe Interdisciplinaire de Génoprotéomique appliquée (GIGA),
Neurosciences Unit, University of Liège, Liège, Belgium; GIGA, Development,
Stem Cells and Regenerative Medicine Unit, University of Liège, Liège, Belgium;
Neurology Department, Centre Hospitalier Universitaire de Liège, Liège, Belgium
MARIA ADELE RUEGER • Department of Neurology, University of Cologne, Cologne, Germany
KIYOSHI SAKAI • Department of Oral and Maxillofacial Surgery, Nagoya University
Graduate School of Medicine, Showa-ku, Nagoya, Japan
EVA SANTERMANS • Vaccine and Infectious Disease Institute (Vaxinfectio),
University of Antwerp, Antwerp, Belgium; Center for Statistics, I-Biostat,
Hasselt University, Hasselt, Belgium
INSA S. SCHROEDER • Department of Biophysics, GSI Helmholtz Center for Heavy Ion
Research, Department of Biophysics, Darmstadt, Germany
TIM J. SCHULZ • Research Group Adipocyte Development, German Institute of Human
Nutrition, Nuthetal, Germany
BRITTNI A. SCRUGGS • Center for Stem Cell Research and Regenerative Medicine and
Department of Pharmacology, Tulane University School of Medicine, New Orleans,
LA, USA
JIANXUN SONG • Department of Microbiology and Immunology, The Pennsylvania State
University College of Medicine, Hershey, PA, USA
PEGGY STOCK • Department of Surgery, University of Leipzig, Leipzig, Germany
HELENE STRICK-MARCHAND • Innate Immunity Unit, Institut Pasteur, Inserm U668,
Paris, France
LISHAN SU • Lineberger Comprehensive Cancer Center, University of North Carolina
at Chapel Hill, Chapel Hill, NC, USA; Center for Infection and Immunity, Institute
of Biophysics, Chinese Academy of Sciences, Beijing, China
YANWEI SU • Department of Cardiovascular and Respiratory Medicine, Tongji Medical
College, Wuhan Puai Hospital, Huazhong University of Science and Technology, Wuhan,
Hubei, China
RIE TAMAI • Division of Molecular and Cellular Medicine, National Cancer Center
Research Institute, Chuo-ku, Tokyo, Japan; Regenerative Medicine Research and
Planning Division, Rohto Pharmaceutical Co. Ltd., Minato-ku, Tokyo, Japan
MARTA TODESCHINI • Department of Clinical Immunology and Transplantation,
Centro Ricerche Trapianti “Chiara Cucchi de Alessandri e Gilberto Crespi,”
Azienda Ospedaliera Papa Giovanni XXIII, IRCCS- Istituto di Ricerche
Farmacologiche Mario Negri, Ranica-Bergamo, Italy
YU-HUA TSENG • Integrative Physiology and Metabolism, Joslin Diabetes Center,
Harvard Medical School, Boston, MA, USA
NATHALIE DE VOCHT • Laboratory of Experimental Hematology, University of Antwerp,
Antwerp, Belgium; Vaccine and Infectious Disease Institute (Vaxinfectio),
University of Antwerp, Antwerp, Belgium; Bio-Imaging Laboratory,
University of Antwerp, Antwerp, Belgium
xvi Contributors

RONGHAO WANG • George Whipple Lab for Cancer Research, Departments of Pathology,
Urology, Radiation Oncology; The Wilmot Cancer Center, University of Rochester
Medical Center, Rochester, NY, USA
SANDRA WINKLER • Department of Surgery, University of Leipzig, Leipzig, Germany
SABINE WISLET-GENDEBIEN • Groupe Interdisciplinaire de Génoprotéomique appliquée
(GIGA), Neurosciences Unit, University of Liège, Liège, Belgium
XIAOFANG XIONG • Department of Microbiology and Immunology, The Pennsylvania State
University College of Medicine, Hershey, PA, USA
AKIHITO YAMAMOTO • Department of Oral and Maxillofacial Surgery, Nagoya University
Graduate School of Medicine, Showa-ku, Nagoya, Japan
JIXIN YANG • Department of Pediatric Surgery, Tongji Medical College, Tongji Hospital,
Huazhong University of Science and Technology, Wuhan, China
PING ZHU • Division of Transplantation Immunology, National Research Institute for Child
Health and Development, Tokyo, Japan; Guangdong Cardiovascular Institute,
Guangdong General Hospital, Guangdong Academy of Medical Sciences,
Guangzhou, China
JIAN ZHUANG • Guangdong Cardiovascular Institute, Guangdong General Hospital,
Guangdong Academic of Medical Sciences, Guangzhou, China
 Part I

Introduction
 Chapter 1

Stem Cells: Are We Ready for Therapy?

Insa S. Schroeder

Abstract
Cell therapy as a replacement for diseased or destroyed endogenous cells is a major component of regenerative
medicine. Various types of stem cells are or will be used in clinical settings as autologous or allogeneic
products. In this chapter, the progress that has been made to translate basic stem cell research into phar-
maceutical manufacturing processes will be reviewed. Even if in public perception, embryonic stem (ES)
cells and more recently induced pluripotent stem (iPS) cells dominate the field of regenerative medicine
and will be discussed in great detail, it is the adult stem cells that are used for decades as therapeutics.
Hence, these cells will be compared to ES and iPS cells. Finally, special emphasis will be placed on the
scientific, technical, and economic challenges of developing stem cell-based in vitro model systems and cell
therapies that can be commercialized.

Key words Multipotent stem cells, Pluripotent stem cells, Disease modeling, Stem cell therapy,
Translation

1 Introduction

Over the last century, healthcare has advanced significantly enhancing

life expectancy and/or quality of life of many patients. Yet there are
still a number of degenerative diseases that cannot be treated ade-
quately leading to organ dysfunction and tissue degeneration.
Moreover, extended life expectancy comes at a price of higher
incidence of diseases such as diabetes mellitus, myocardial infarc-
tion, and stroke that eventually may require intervention by
cell-based therapies as well. However, due to the shortage of trans-
plantable organs, tissues, or cells, sourcing for replacement thera-
pies is difficult. Therefore, cells of ample supply that can satisfy the
medical demands regarding functionality, long-term survival, and
safety are badly needed. Embryonic, induced as well as adult stem
cells may serve as such sources for the alleviation or cure of a given
disease. To date, hematopoietic stem cell transplantation is the most
successful stem cell therapy used in genetic blood disorders, immune
deficiencies, or malignancies like leukemia. Other stem cells are

Bruno Christ et al. (eds.), Animal Models for Stem Cell Therapy, Methods in Molecular Biology, vol. 1213,
DOI 10.1007/978-1-4939-1453-1_1, © Springer Science+Business Media New York 2014

3
4 Insa S. Schroeder

tested in clinical trials or are on the cusp of reaching this translational

gate. Outside such peer-reviewed, well-controlled clinical trials,
unproven stem cell therapies are widely performed without any
legitimate investigation of efficacy or safety, misleading patients
and their families and trivializing possible adverse effects. Therefore,
blinded randomized trials are the minimum requirement for stem
cell therapies to reach clinical translation. However, before reach-
ing preclinical or clinical levels, protocols for the isolation, expan-
sion, or differentiation of stem cells that are established in a research
laboratory have to be adapted to mass production. Not all labora-
tory protocols may be easily translated and scaled up one to one
into a pharmaceutical manufacturing procedure under GMP con-
ditions. Therefore, already early on in the process of developing
cell-based proof-of-concept studies, which are supposed to be used
in a clinical setting later on, one should consider designs that allow
the implementation of GMP guidelines. Also, thorough cell
characterization and functional assays that will be applicable in a
high-throughput setting need to be designed as soon as possible.
Thus, this review is intended to report about the progress that has
been made to translate basic stem cell research into pharmaceutical
manufacturing processes and the challenges that still need to be
considered.

2 Adult Stem Cells in Regenerative Medicine

2.1 Hematopoietic Hematopoietic stem cells were first postulated by Alexander

Stem Cells (HSCs) A. Maximow [1] and eventually identified in the 1960s [2]. Since
then, they have been used excessively in stem cell therapy. As
progenitor cells residing in the bone marrow, they will form all
blood and immune cells (myeloid and lymphoid cells). Therefore,
they are successfully used to cure blood disorders like thalassemia,
Fanconi’s anemia, immune deficiency, and blood cancers such as
leukemia and lymphomas [3]. HSCs are positive for the cell surface
markers CD34/CD90/CD59/Thy1 and negative for CD38/
CD45Ra or mature blood lineage markers (lin−). In the bone mar-
row, 1 out of 10,000–15,000 cells is thought to be a bona fide
HSC. In the peripheral blood, this number drops to 1 in 100,000
cells. However, HSCs can also be found in umbilical cord blood
that gains increasing importance as a HSC cell source [4, 5]. In a
quiescent state, HSCs reside in a stem cell niche consisting of
spindle-shaped N-cadherin+ osteoblasts embedded in stromal
fibroblasts [6], but they are easily mobilized and migrate into the
blood stream when patients are treated with granulocyte colony-
stimulating factor. HSCs are a very heterogenic population
that can be divided into subpopulations according to their repop-
ulating or differentiation behavior: short-term repopulating
HSCs versus long-term repopulating HSCs or lymphoid-biased,
 Stem Cells for Therapy 5

myeloid-biased, and balanced HSCs. As HSCs age, the myeloid-

biased HSC subpopulation accumulates at the expense of the
lymphoid one [7]. Propagation of HSCs is challenging as they
readily undergo differentiation, but factors such as stem cell factor,
Flk2/Flt3 ligand, thrombopoietin, and IL-6/sIL-6R can be used
for ex vivo expansion of human transplantable HSCs [8]. Currently,
there are 1,795 open clinical studies using hematopoietic stem cells
(www.clinicaltrials.gov, accessed Oct. 28, 2013) demonstrating the
importance of this stem cell source. Based on single HSC
transplantation, HSCs show certain plasticity as cell types such as
fibroblasts, myofibroblasts, adipocytes, and osteo-chondrocytes
were derived from HSCs (reviewed in [9]). However, whether
they are able to generate cell types outside the mesodermal lineage
is still debated.

2.2 Mesenchymal A second stem cell population that can be found in the bone marrow
Stem or Stromal Cells consists of mesenchymal stem cells, originally discovered by
(MSCs) Friedenstein [10] that since then have been found in a number of
other tissues [11]. These cells were initially identified by their
ability to adhere to plastic surfaces, and for many years findings
regarding MSCs were difficult to compare as laboratories used
different standards to characterize the MSCs. Finally, in 2006, the
International Society for Cellular Therapy defined the minimal
criteria MSCs have to fulfill: (1) adherence to plastic under stan-
dard tissue culture conditions; (2) the expression of the surface
markers CD73, CD90, and CD105 and lack of expression of
CD45, CD34, CD14, CD11b, CD79a, and CD19 and HLA-DR
surface molecules; and (3) the capacity to differentiate into osteo-
blasts, adipocytes, and chondrocytes in vitro [12]. Later, CD271
was found to be a unique marker of MSCs [13]. Like HSCs, MSCs
are used in a vast number (n = 221, www.clinicaltrials.gov, accessed
Oct 28, 2013) of clinical studies that are currently pursued for a
variety of diseases, among them 19 phase III trials (studies that
gather more information about safety and effectiveness by studying
different populations and different dosages) related to stroke,
myocardial infarction, type 1 and 2 diabetes, chronic graft-versus-
host disease, articular cartilage defects, Crohn’s disease, and others.
However, currently no phase IV trial (studies occurring after FDA
has approved a drug/procedure for marketing. These include
postmarket requirement and commitment studies that are required
of or agreed to by the sponsor. These studies gather additional
information about a drug’s/procedure’s safety, efficacy, or optimal
use) using MSCs is performed. As MSCs are able to home to sites
of inflammation, may differentiate into other cell types, secrete
bioactive substances that may initiate, improve, or accelerate
regenerative processes, and are immunomodulatory, they are ideal
candidates of clinical applications. The most acknowledged feature
of MSCs is their immunomodulatory ability that makes them
6 Insa S. Schroeder

useful in diseases such as acute graft-versus-host disease, which

may occur after allogeneic HSC transplantation. A recent study
though indicates that this ability varies based on MSC isolation
techniques [14]. In a small Dutch trial applying MSCs after myo-
cardial infarction (www.trialregister.nl, no. NTR1553), MSC treat-
ment was shown to be feasible and safe at short term and up to
5 years of follow-up. However, the 5-year event-free survival was
not different from the control group, and even though global left
ventricular function assessed by echocardiography showed contin-
uous improvements in left ventricular systolic function after MSC
injection during the first 12 months, these results were comparable
to the control group [15]. Therefore, even though beneficial
effects have been seen in animal models after MSC transplantation
and so far no adverse side effects have been correlated to MSCs,
evidence for their usefulness is questioned [16], and more insight
has to be gained regarding their efficacy in humans.
MSCs have also been reported to differentiate into other cell
types like hepatocytes [17, 18], cardiomyocytes [19], or neuronal
cells [20] showing some plasticity even though the degree of dif-
ferentiation may not reach that of fully functional, mature cell
types [16, 21].
In general, even though the underlying molecular mechanism
of HSC- or MSC-mediated effects may not always be fully under-
stood, novel stem cell-based therapies will benefit from the experi-
ences acquired during adult stem cell-based transplantations.

3 Embryonic Stem Cells in Regenerative Medicine: Possibilities

Unlike multipotent adult stem cells, pluripotent human embryonic

stem (hES) cells can be propagated indefinitely in vitro and can
differentiate into any cell type of the human body if given the right
stimulus. These characteristics make them suitable for regenerative
medicine, drug discovery, but more importantly for disease model-
ing, and developmental studies, which can only be examined in a
limited way using adult stem cells. Ever since embryonic stem cells
have been generated first from the inner cell mass of mice in 1981
[22] and a few years later from human blastocysts [23], hundreds
of lines were generated including disease-specific hES lines, and
every endeavor has been made to establish in vitro protocols lead-
ing to functional cells of various organs via the stepwise recapitula-
tion of embryonic development. This has led to the establishment
of a number of hES cell-based toxicology assays [24], the screen-
ing of teratogenous compounds [25], and neuro- and cardiotoxic-
ity tests [26].
Clinical applications, however, require animal-free isolation,
culture, and differentiation of hES cells. Yet early methods of isolating
and maintaining hES cells required xenogeneic components such
 Stem Cells for Therapy 7

as enzymes, the coculture with irradiated mouse embryonic fibro-

blasts (MEF), and enriched culture media containing fetal bovine
serum. MEF support the self-renewal of hES cells by secreting
essential growth factors, cytokines, and extracellular matrices
(e.g., transforming growth factor beta, activin A, laminin 511, or
vitronectin). Over the last decade, these procedures have been
adapted to xenogeneic-free GMP-compliant isolation and culture
methods that now allow using hES cells in clinical applications
[27]. The group of O. Hovatta greatly contributed to the develop-
ment of animal-free substrates and culture media to isolate, main-
tain, and cryopreserve hES cells [28]. This included the
development of a method to mechanically isolate the inner cell
mass replacing the use of immunosurgery, which involves animal
components and enzymes [29], and the substitution of MEF by
human recombinant laminin 511 allowing a feeder-free culture of
hES cells [30]. However, the most common approach still is the
culture of hES cells on culture plates coated with Matrigel, which
is composed of laminin, collagen IV, heparan sulfate proteogly-
cans, entactin, and growth factors. Yet Matrigel is derived from
Engelbreth-Holm-Swarm mouse sarcomas and exhibits lot-to-lot
variability and also may induce xenogeneic contaminants [31]. It is
increasingly replaced by vitronectin coating, which supports undif-
ferentiated proliferation of pluripotent stem cells [32]. Of great
concern is the establishment of culture conditions that will allow
large-scale expansion of hES cells for clinical applications. To date,
hES cells are commonly grown as adherent colonies requiring
elimination of differentiated clones every now and then by manual
passage. Upon detachment, hES cells form embryoid bodies and
differentiate. However, mass production of hES cells will require
suspension culture in bioreactors. Indeed, such suspension culture
of hES cells is possible using proper culture media [33, 34],
which has helped on bringing hES cell-based cell technology from
bench to bedside.
With the progress made in the development of GMP proce-
dures regarding the culture and differentiation of hES cells and
extensive preclinical testing, three hES cell-based clinical trials
have been approved: Geron launched a study using oligodendro-
cytes (GRNOPC1) derived from hES cells to treat patients with
spinal cord injuries. Even though no adverse effects were reported,
Geron surprisingly stopped recruiting patients in November 2011
due to financial reasons. This leaves two phase I/II clinical trials
for the treatment of dry age-related macular degeneration and
Stargardt’s macular dystrophy, which are currently carried out by
Advanced Cell Technology. In these studies, hES cell-derived retinal
pigment epithelial cells are injected subretinally. Four months
after implantation, survival and engraftment of cells as well as
visual improvement could be shown, whereas no tumorigenicity
was observed [35].
8 Insa S. Schroeder

4 Embryonic Stem Cells in Regenerative Medicine: Challenges

One of the most challenging issues regarding hES cell-based therapies

is the possible tumorigenicity of residual ES cells that have escaped
differentiation. Therefore, thorough characterization and purifica-
tion of the hES cell derivatives are mandatory. However, such homo-
geneous, well-defined cell populations can be obtained by double
selection as reported, e.g., by Kahan et al. [36]. In a mouse ES cell
model based on embryoid body formation, they generated pancre-
atic cells. Negative selection of pluripotent stem cells using an SSEA1
antibody followed by positive selection of EpCAM-positive pancre-
atic progenitors resulted in no tumor formation after transplantation
(n = 31), while transplantation of the unsorted progeny led to tumor-
igenicity in seven out of eight cases. This proves the efficiency of
double selection and may be used also in clinical settings.
Another issue raised is the genomic instability of ES cells that
may prevent the use of their progeny due to the risk of oncogenic
transformation. Lund et al. [37] found that in karyotypically
abnormal hES cells, the histone deacetylase proteins, HDAC1 and
HDAC2, are increased. Inhibition of these markers resulted in
reduced proliferation, induction of the proliferation inhibitor
cyclin-dependent kinase inhibitor 1A (CDKN1A), and altered reg-
ulation of the tumor suppressor protein retinoblastoma 1 (RB1).
Genome-wide analyses revealed altered expression of genes linked
to severe developmental and neurological diseases and cancers.
Thus, genetic instability in hES cells and their progeny is a param-
eter that has to be closely monitored.
A third safety issue is the possible immunogenicity of ES cells
and their derivatives [38]. This issue has been circumvented in the
abovementioned clinical studies by transplanting cells into immune-
privileged sites, which was shown to require only mild transient
immunosuppression [35]. However, more insight into the immune
response of hES cells and their derivatives need to be gained to
completely abolish the danger of cell rejection and subsequent fail-
ure of cell therapy strategies.
A still remaining technical challenge is the development of
simple, scalable differentiation protocols that give rise to mature,
functional cell types or to progenitors that are able to mature
in vivo in a reasonable time. This goal has not been achieved for
several cell types including insulin-producing beta cells [39, 40]
requiring more basic research.

5 Induced Pluripotent Stem Cells in Regenerative Medicine

The idea of reprogramming adult somatic cells has been pursued

for several decades. Already in 1952, Briggs and King showed that
transferring nuclei of blastocysts into an enucleated frog egg
 Stem Cells for Therapy 9

resulted in tadpole clones [41]. However, as the experiment was

difficult to reproduce, the authors concluded that cell plasticity
declines with differentiation. Ten years later, Sir John Gurdon
repeated these experiments succeeding in generating frogs even
though such reprogramming process through somatic cell nuclear
transfer (SCNT) was inefficient [42]. In contrast to Briggs and
King, he concluded that differentiation of cells is not accompanied
by irreversible nuclear changes, but that instead cell specialization
is reversible. However, apart from cloning the sheep Dolly by Ian
Wilmut some 30 years later [43], little public attention was given
to the potential of reprogramming cells. This changed dramatically
in 2006, when Takahashi and Yamanaka reported the successful
reprogramming of mouse embryonic and adult fibroblasts to a
pluripotent ES cell-like stage generating the so-called induced
pluripotent stem (iPS) cells by only four factors: Oct3/4, Sox2,
c-Myc, and Klf4 [44]. In honor of these hallmark discoveries,
Gurdon and Yamanaka were awarded the Nobel Prize in medicine
in 2012. Indeed, the generation of iPS cells opens up completely
new avenues in regenerative medicine. As acquisition of patient
samples generally is challenging, analyzing the cause and pro-
gression of many diseases was exacerbated in the past. However, as
now fibroblasts or other cells from patients suffering from any
given disease theoretically can be reprogrammed and differentiated
in vitro into almost any cell type desired, it is possible to investigate
the underlying mechanisms leading to the disease. This is especially
interesting with respect to those diseases, which are correlated with
genetic alterations or genetic predispositions. Thus, the iPS
technology not only allows establishing in vitro test systems and
drug screening methods but also offers the possibility of autolo-
gous cell therapy.

5.1 Reprogramming There are several viral and nonviral methods of iPS generation,
Methods each harboring its own advantages and disadvantages in respect to
their use in a clinical setting. Viral methods use inducible or non-
inducible integrating retro- or (polycistronic) lentiviruses and non-
integrative adenoviruses or more recently Sendai viruses. Excisable
integrating vectors such as loxP (polycistronic) lentiviruses or
(inducible) PiggyBac or Sleeping Beauty transposons are successfully
used as well. Nonviral methods employ nonintegrative vectors
such as polycistronic plasmids, Epstein-Barr nuclear antigen-1-
based vectors or minicircles, proteins, small molecules, mRNA,
and microRNA.
In the original method, pluripotency markers were introduced
in somatic cells by a replication-defective gammaretroviral Moloney
murine leukemia virus (Mo-MLV)-based vector [45]. This
approach is very efficient in actively dividing cells but fails to trans-
duce slow- or nondividing cells [46]. In contrast, lentiviral vectors
such as HIV or SIV derivatives also transduce nondividing cells as
10 Insa S. Schroeder

long as they are metabolically active. However, while retrovirally

introduced transgenes are silenced through methylation and epi-
genetic modifications, lentiviral vectors may lead to prolonged and
thus unwanted transgene expression and the generation of only
partially reprogrammed progeny [47]. Yet the lentiviral approach
is the most robust and most commonly used reprogramming
method. Of the nonintegrating systems, the Sendai virus system is
becoming increasingly important in the generation of clinically
relevant iPS cells. In contrast to other nonintegrating vectors that
are still based on DNA intermediates, which in theory may be
introduced accidentally into the host genome by DNA repair
mechanisms, the Sendai virus possesses an RNA genome, which
will never be incorporated into host chromosomes. It efficiently
transduces a plethora of host cells, shows no human pathogenicity
[48], and is diluted out of the host cells by 5–8 passages [49].
Reprogramming strategies using proteins have been explored using
mouse somatic cells [50], and recombinant proteins can be used in
a combination with viral systems [51], but to date no stable human
iPS line has been generated using proteins, even if combined with
epigenetic modifiers [52]. Nonetheless, small molecules that
maintain pluripotency/self-renewal or modulate the epigenetic
status of the host genome such as the DNA methyltransferase
inhibitor 5-azacytidine or the histone deacetylase inhibitors
trichostatin A or valproic acid induce chromatin remodeling and
may complement other reprogramming methods improving their
efficiency (reviewed in [53]). Usage of RNA for reprogramming
purposes has been shown to be successful, too [54, 55], but it
requires multiple transfections. As pluripotency is regulated and
fine-tuned by microRNAs such as the miR-302, miR294, and
miR-181 family [56], these noncoding RNAs were also used to
reprogram somatic cells [57, 58]. They may also be used to dis-
tinguish human ES cells from iPS cells and to judge about repro-
gramming efficacy [59].
Finally, because reprogramming efficiency largely depends on
cell cycle progression, synchronizing cells in GO/G1 by serum
starvation increased reprogramming efficiency, since the cells when
released went through mitosis when the transgenes expressed the
reprogramming factors [60]. Modulating cell cycle regulators such
as mitogen-activated protein kinase kinase (MEKK) [61] or using
such regulators in combination with other small molecules [62]
increased reprogramming efficiency up to 200-fold.

5.2 Reprogramming The original factors used by the Yamanaka group comprise of
Factors Oct4, Sox2, Klf4, and c-Myc (OSKM) and are not only efficient in
combination with viral delivery systems but also with nonintegrat-
ing approaches such as adenoviruses, Sendai viruses, mRNA, epi-
somal vectors, or proteins albeit with low efficiency (for review see
[63]). The Thomson group used a slightly different cocktail
 Stem Cells for Therapy 11

substituting KLF4 and c-MYC by NANOG and LIN28 (OSNL)

[64]. Besides using cocktails with up to six pluripotency markers to
enhance or facilitate reprogramming [65], many more factors that
substitute the abovementioned pluripotency factors and are known
to be lineage specifiers (reviewed in [66]) have been established
such as Nr5a2, which replaces Oct4 [67], or Esrrb that can substi-
tute Klf4 and c-Myc [68].

5.3 Donor Cells Choosing the right donor material starts with the question of
young versus old cells. As cellular aging may be correlated with
irreversible cell cycle arrest, accumulation of mutations, and DNA
damage or altered signaling pathways and epigenetic alterations,
cell reprogramming could be hampered. Whether embryonic cells
are more easily reprogrammable than adult tissue cells that would
be the most interesting for disease modeling is still debated.
Similarly, it also remains to be seen whether the differentiation
capacity of iPS cells generated from elderly equals that of younger
donors. In a recent work, Wen et al. neither found difference in the
reprogramming efficiency of vaginal fibroblast obtained from
young and old women nor differences in the expression of senes-
cence or apoptosis markers [69]. There was also no difference in
the differentiation capacity of iPS cells produced from young and
old donors [69]. The same was shown by Ohmine et al. [70], who
successfully differentiated iPS cells from patients into islet-like
cells. However, aging cells express higher levels of Ink4/Arf [71],
which is a barrier to reprogramming [72]. Nonetheless, the limited
capacity to be reprogrammed may be overcome by factors such as
sirtuin 6, which was shown to improve reprogramming efficiency
in human dermal fibroblast of elderly subjects [73]. As important
as the age of donor cells is the right choice of the cell type. Even
though most iPS protocols use fibroblasts as a starting material,
other cells such as keratinocytes [74] or, more attractive for regen-
erative medicine, blood cells, which are easily accessible from rou-
tine sampling, have been proven to be suitable for iPS cell
generation (reviewed in [75]).

6 Human iPS Cells as Models of Human Diseases: Potentials

So far, animal models have been used to investigate the onset and
progression of human diseases. Yet, because of genetic and devel-
opmental differences between human and animals, it is question-
able whether human diseases can be properly simulated using
animal models alone. Having the iPS technology at our disposal,
especially those diseases that are caused by genetic mutations can
be analyzed by producing iPS from patients with genetic disorders
and their healthy relatives. This way, the impact of genetic muta-
tions or predispositions in disease initiation and progression can be
12 Insa S. Schroeder

investigated, even though this requires not only the bona fide
production of iPS cells but also the establishment of differentiation
protocols that closely mimic the in vivo development.
Numerous efforts have been undertaken to establish patient-
specific pluripotent cells from almost all organs affected by heredi-
tary diseases. Using dermal fibroblasts, Rashid et al. generated iPS
lines from patients with inherited metabolic disorders (IMD) of
the liver such as alpha-1-antitrypsin deficiency, familial hypercho-
lesterolemia, and glycogen storage disease type 1a [76]. In this
study, dermal fibroblasts from seven individuals suffering from five
hepatic metabolic disorders were reprogrammed using the
Yamanaka factors and compared to iPS lines derived from three
healthy individuals. All iPS lines showed endogenous expression of
pluripotency markers and were able to differentiate into the three
germ layers. However, as these lines showed karyotypic abnormali-
ties after prolonged culture (passages ≥40), differentiation
experiments, using among others activin A, oncostatin M, and
hepatocyte growth factor [77], were performed only up to passage
30. Differentiated cells showed functional maturity based on albu-
min secretion and cytochrome P450 metabolic activity. More
importantly, iPS cells derived from patients with IMD recapitu-
lated key pathological features of the diseases affecting the patients
from which they were derived such as aggregation of misfolded
alpha-1-antitrypsin in the endoplasmic reticulum, deficient LDL
receptor-mediated cholesterol uptake, and elevated lipid and gly-
cogen accumulation. Advancing the idea of examining and modu-
lating disease-causing genes further, Eggenschwiler et al. [78] used
disease-specific iPS cells to target severe α-1-antitrypsin (A1AT)
deficiency by overexpressing a human microRNA 30 (miR30)-
styled shRNA directed against the PiZ variant of A1AT, which is
known to cause chronic liver damage in affected patients. In this
study, a functional relevant reduction (-66 %) of intracellular PiZ
protein in hepatic cells after differentiation of patient-specific iPS
cells could be shown, demonstrating that not only mimicking the
disease but also modeling it is possible using patient-specific iPS
cells. Likewise, mutation-based congenital heart diseases such as
the arrhythmogenic long QT syndrome (LQTS) can be recapitu-
lated using iPS cells from patients affected [79]. When differentiated
into cardiomyocytes, these cells revealed significant prolongation
of the action potential duration when compared to healthy control
cells. Voltage-clamp studies confirmed that QT prolongation origi-
nated from a significant reduction of the cardiac potassium current
I(Kr). Importantly, LQTS-derived cells also showed marked
arrhythmia [79]. Patient-specific iPS cells have also been estab-
lished from patients suffering from diseases such as type 2 diabetes
mellitus that are not correlated with a monogenetic modification
[80, 70]. However, genome-wide association studies robustly
revealed a number of single nucleotide polymorphisms (SNPs) that
 Stem Cells for Therapy 13

contribute or predispose to diabetes mellitus type 2 (reviewed in

[81]). Analyzing these SNPs may broaden our insight into disease
pathogenesis leading to new pathways for therapeutic intervention,
strategies for patient stratification, and biomarkers for identifying
those at greatest risk of developing diabetes. Predicting the risk of
type 2 diabetes mellitus with genetic risk models on the basis of
established genome-wide association markers even when combined
with conventional risk models has been limited [82]. Here, inte-
gration-free iPS cell technology could greatly facilitate such risk
assessment as it is possible to create isogenic stem cell lines that
differ only in the mutation under study followed by differentiation
into relevant cell types. This approach is possible thanks to genome
editing technologies using zinc finger nucleases (ZFNs) or tran-
scription activator-like effector nuclease (TALEN) [83, 84]. These
techniques can also be used for gene correction of autologous
human iPS cells intended for cell therapy. Even if currently such
therapies are prohibitive due to the enormous costs, it has been
suggested to generate human iPS cell banks created from a limited
number of homozygous HLA-haplotyped donors that would cover
a large proportion of the population [85, 86]. For example, even
in an ethnically very heterogeneous population like the United
States, it has been calculated that only 100 iPS lines would cover
the majority of ethnics [87]. Recently, it has been shown that not
only monogenetic disorders but even more complex genetic abnor-
malities may be targeted in the future as well: Li et al. reported the
ablation of the extra chromosome 21 in iPS cells derived from
Down syndrome patients [88].

7 Human iPS Cells as Models of Human Diseases: Challenges

Even if looking at the abovementioned possibilities, the iPS cells

appear to offer several advantages over ES cells or adult stem cells
and the first iPS-based clinical study started recruiting patients with
exudative (wet-type) age-related macular degeneration in August 2013
(http://www.riken.jp/en/pr/press/2013/20130730_1/, accessed
Oct. 27, 2013), several caveats of the iPS technology need to be
addressed before iPS cell-derived progeny can be broadly applied in
disease modeling or clinical applications. As discussed for hES cell-
based strategies, the genetic stability of iPS cells has to be proven,
tumor formation must be prevented without doubts, and it has to be
ensured that iPS cell differentiation produces only the cell type of
interest with the required functionality. Unique for pluripotent iPS
cells, however, are hurdles regarding the reprogramming process
itself: (1) genetic and epigenetic differences such as copy number
variations and (insertional) mutations, (2) incomplete demethyl-
ation and remethylation, or (3) aberrant X-chromosome or imprint-
ing and repeat instability (reviewed in [89]), all of which may be
14 Insa S. Schroeder

caused by the reprogramming technique alone or by prolonged

culture. Besides raising safety concerns, these changes may lead to
partially reprogrammed cells, alter the differentiation potential of
fully reprogrammed iPS cells, or mask the impact of genetic varia-
tions observed in many diseases rendering them even unsuitable for
in vitro disease modeling.
A major concern regarding therapeutic applications is the
possible immunogenicity of iPS cells. Even though patient-derived
iPS cells for autologous therapeutic reasons have the same genetic
background and express the same major histocompatibility com-
plex molecules as the recipient, therefore not requiring immune
suppression, a study by Zhao et al. [90] raised concerns as it
revealed that autologous iPS cells, but not autologous, strain-
matched ES cells triggered a T cell-mediated immunogenicity after
transplantation. However, in another study by Guha et al., no evi-
dence of increased T cell proliferation in vitro, rejection of synge-
neic iPS cell-derived EBs/tissue-specific cells after transplantation,
or an antigen-specific secondary immune response was found [91].
In addition, differentiated cells derived from syngeneic iPS cells
were not rejected after transplantation. The authors also found
little evidence of an immune response to undifferentiated, synge-
neic iPS cells. It has to be seen whether immunogenicity may be
correlated with certain reprogramming methods or donor cells.

8 Direct Lineage Reprogramming

Reprogramming cells to the pluripotent “ground state” newly

sparked the interest in transforming mature cells from one lineage to
another without going through pluripotent intermediates via over-
expression of key transcription factors required for the development
of the cell type of interest. Meanwhile a range of cells were generated
that way, e.g., neurons [92], cardiomyocytes [93], blood cell
progenitors [94], hepatic stem cells [95], retinal pigment epithelium-
like cells [96], or insulin-producing cells [97]. However, as the
mature starting material may be as scarce as the cells they are sup-
posed to be turned into, it needs to be seen if such strategies will
enter translation into clinical strategies that would require large
quantities of donor cells. Direct reprogramming has also been
achieved in vivo converting pancreatic exocrine acinar cells to endo-
crine insulin-producing cells [98]. Likewise, cardiac fibroblasts were
reprogrammed in vivo to cardiomyocytes with an efficiency of about
6 % [99]. However, if such a procedure after myocardial infarction
would be beneficial is debatable. Due to massive infiltration of
cardiac fibroblasts that may prevent proper alignment of cardiomyo-
cytes, there is the danger of incomplete coupling of cardiomyocytes
leading to the induction of arrhythmias.
 Stem Cells for Therapy 15

9 General Criteria and Challenges for Stem Cell-Based Therapies

Regardless of the cell type used, all stem cell-based therapies have
to fulfill four major criteria that will rule over clinical or commer-
cial outcome: safety, identity, purity, and potency. Thus, the
International Society for Stem Cell Research (ISSCR) composed
“The ISSCR Guidelines for the Clinical Translation of Stem Cells”
(http://www.isscr.org/home/publications/ClinTransGuide, accessed
Oct 27th, 2013). Currently, many approaches that have been
published as a proof of principle by basic researchers do not fulfill
one or more of the abovementioned requirements. However, only
if cell therapy products comprise solely of the intended cell type
that shows the desired function, without adverse residual cells or
adventitious components or adverse side effects such as tumorige-
nicity, all requirements for successful approval by regulatory agen-
cies and clinical application will be met. Conversely, some diseases
will require the replacement of complex tissues involving the
assembly of several cell types within a three-dimensional matrix.
Whether such complex tissue replacements will ever reach market
authorization is questionable. However, such systems may still be
beneficial in drug screening, toxicity testing, or disease modeling
and should be pursued regardless of their marketability. The benefits
of stem cell therapies and standard pharmaceutical approaches will
have to be weighed up against each other taking into account the
efficiency, feasibility, and cost-effectiveness. One will also have to
take into account whether autologous or allogeneic therapies will
be more favorable. Allogeneic products derived from one donor
may be administered to a large number of recipients that share the
expenditures for research and development and quality control
[100], while this is not the case for autologous therapies possibly
making this strategy too expensive even though additional lifelong
immunosuppression will not be required. A “concise review on
guidance in developing commercializable autologous/patient-
specific cell therapy manufacturing” has recently been published by
Eaker et al. and may serve as an advice for basic researchers [101].

10 Conclusions

Stem cell-based disease modeling or cell therapies may be challenging

and cost intensive. However, if combined with meaningful animal
models, they can give invaluable insights into the onset and
progression of various diseases and alleviate or cure a number of
ailments. None of the stem cell sources whether adult or embryonic/
pluripotent are dispensable; rather all of them advance our knowl-
edge in terms of disease development and cure. Even if the estab-
lishment of such stem cell-based tools and therapeutics will take
16 Insa S. Schroeder

decades and be accompanied by many failures, the knowledge

gained will vindicate the endeavor. Nevertheless, it has to be
ensured that stem cell-based interventions are not applied prema-
turely or outside of well-controlled clinical trials as otherwise
legitimate studies and the public trust in regenerative medicine
could be jeopardized. Such unproven interventions already led to
the death of a child that received intracranial injections of cord
blood and to the formation of a brain tumor following neural stem
cell transplantation in an ataxia telangiectasia patient [102].
Nevertheless, taken the progress into account that has been made
in the last years, stem cell-based disease modeling or cell therapies
may be feasible when preceded with caution.

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 Chapter 2

Aurigon’s Point of View on the Safety Assessment

of Cell-Based Therapies: An Experience Based
on the Participation in 15 ATMPs Projects
Emmanuelle Cornali

Abstract
Cell-based medicinal products (CBMP) require the use of a case-by-case, risk-based, and scientifically
justified nonclinical safety testing approach. The challenge is to translate regulatory requirements into
concrete testing designs while overcoming technical limitations inherent to animal models and the CBMP
and assaying combination strategies to reduce animal use and save time and budget.

Key words Cell-based medicinal products, CBMP, Safety testing

1 Introduction

Although cell-based medicinal products (CBMPs) have been

clinically used for more than a decade, robust nonclinical scientific
and regulatory provisions for these products have only recently
been adopted. Safety assessment of CBMPs has not been per-
formed systematically. Either health benefits were greater com-
pared to any risk (e.g., bone marrow cell transplantation) or side
effects were socially accepted (e.g., plastic surgery).
Since the first description of human pluripotent stem cells
(1998), a lot of development has been made in new innovative
cell-based therapies for diseases and tissue defects, for which tradi-
tional therapies and medicinal products have not provided satisfac-
tory outcomes. Developments include induction of adult somatic
pluripotent stem cells (iPSCs), 2D or 3D tissue engineering,
genetic manipulation of cells, and combination of cells with medical
device products.
This wide variety and high complexity of Advanced Therapy
Medicinal Products (ATMPs), their foreseen technical limitations
(human origin, limited amount, and viability), and their particular
risks (identity/variability, plasticity, immunogenicity, tumorigenicity,

Bruno Christ et al. (eds.), Animal Models for Stem Cell Therapy, Methods in Molecular Biology, vol. 1213,
DOI 10.1007/978-1-4939-1453-1_2, © Springer Science+Business Media New York 2014

23
Exploring the Variety of Random
Documents with Different Content
the other side, there were murmurs among the well-to-do, who were
deprived of their favourite beverages unless they could obtain a
doctor’s certificate of ill-health, which did not, however, seem difficult
to arrange. I was asked more than once whether King George was
about to follow the lead given by the Tsar, Russians not being very
clear as to the limitations of a constitutional monarchy.
Soldiers were to be seen everywhere, sometimes drilling near the
great red Winter Palace, sometimes as reservists, with numbers
chalked upon their backs, or again as small parties of wounded in
charge of kind-faced hospital nurses. I heard pathetic accounts of
the extreme poverty of the men who were being nursed back to
health in the English Hospital directed by Lady Georgina Buchanan,
who had had the kindly thought of fitting them out when they were
dismissed to their peasant homes; the totally disabled being trained
in basket-making. Both at Petrograd and at Moscow, our next
halting-place, those actively engaged in nursing spoke highly of the
courage and gratitude of their patients. In the latter city an English
girl of only nineteen and a Russian lady of the same age, neither of
whom had had any training in nursing, were in charge of a hospital
containing forty-five wounded soldiers. They did all the bandaging
themselves, assisted at every operation, and supervised the peasant
women who performed the more menial share of the work. My
devoted compatriot told me that the men called her “Little Sister,”
and were marvellously brave when operated upon, saying that her
presence gave them courage. Owing to the absence of the great
majority of the trained nurses at the front, these capable amateurs
were of the utmost service. We heard that the Russian medical
faculty disapproved of inoculation for typhoid, giving the somewhat
inadequate reason that “there were so many worse diseases,” and
consequently the soldiers suffered terribly from this scourge.
My brother and I did the sights of Petrograd, with its many gold-
covered domes, cupolas and spires, but I will refrain from describing
the gorgeous interior of St. Isaak, the pictures of the Hermitage, or
even the deeply interesting house in which Peter the Great lived
while building his “window opening to the West.”
Moscow, with its hundreds of gilt-domed or purple or blue or green
cupolas, that bizarre orgy of colour and fantastic design called the
Church of Ivan the Terrible, and the ancient Kremlin built to resist
Tartar inroads, gave me, as indeed it does to most travellers, the
impression of a semi-Oriental city.
We were in the very heart of Russia, and no one could fail to be
struck by the intense devotion—I refrain from calling it superstition—
of the people. In the dim magnificence of the small but lofty
Coronation Chapel, which has its walls literally encrusted with
jewelled icons, crowds were kissing the hands and feet of the sacred
pictures all day long, in defiance of every hygienic principle. Long-
haired priests in embroidered copes were chanting services, and as
the body of a saint, dead centuries ago, had just been exhumed, it
was confidently expected that many miracles of healing would be
wrought by the remains. Gilded and jewelled banners to be carried in
procession stood in the ornate chapels, which had gorgeous doors
through which no woman might pass. On the great day of his
coronation the Tsar passed through these portals, anointed and
crowned himself, then issued forth, the Father of his people, to
perform the same ceremony on the Tsaritsa.
The monarch, in common with the humblest of his subjects,
uncovers himself as he passes under one of the entrances to the
Kremlin, above which stands a particularly holy icon. Indeed in every
room of every Russian house, even in the hotels, hangs some
pictured saint with a little lamp in front of him, while the railway
stations and waiting-rooms are all provided with sacred guardians.
To these people the War was then a holy one. The chambermaid of
our hotel, who spoke German—a language it is forbidden to use in
public—told me with tears that her only son had been killed at the
front, that his father had died of grief when the news reached them,
and that her daughter, working at a hospital, had had no news of her
soldier-husband for three months and naturally feared the worst. “But
we must not grumble,” she ended bravely; “it is terrible for all of us,
but with God’s help our Tsar will conquer his enemies and we shall
have peace once more.”
Russians struck us as being somewhat silent in the streets, and we
never heard any one whistle. It was explained that they have the
same superstition about whistling as have the Persians, and look
upon it as “devilish speech.” In connection with this we were told that
on one occasion an American bishop and his chaplain were visiting a
monastery in Moscow, and to the horror of the monks the chaplain
kept on bursting into snatches of whistling. But one of the holy men
was equal to the occasion and, walking close behind the
unconscious offender, made the sign of the cross repeatedly in order
to avert any evil consequences!
The lack of efficiency in Russia was very noticeable. For example, to
cash our letters of credit in a bank was a tedious business, the
money being slowly counted with the aid of an abacus. The
shopkeepers also depend greatly on these aids to arithmetic. It was
moreover a land of tips. In every private house the servant who
helped you on and off with your fur coat and galoshes expected a
pourboire, and on leaving a hotel we were surrounded by a throng of
waiters, porters of different grades, and a bevy of small boys, all
intent on fees.
During the next section of our journey to Tashkent the trains were by
no means as comfortable as before. Our only light was a guttering
candle in a lantern placed high above the carriage door, and, what
was worse, the double windows were screwed up for the winter, all
the air we breathed passing through most inadequate ventilators in
the roof. After some thirty hours of semi-suffocation it was a relief
when the train stopped at Samara, and its great bridge over the
Volga. Before we crossed, soldiers with fixed bayonets filed into the
corridors and lined the train, and henceforward sentries stood with
fixed bayonets on all the platforms. Instead of going through to
Tashkent, our train stopped for eighteen hours, so we drove perforce
to the best hotel in the place. There I was ushered into a bedroom
which had only a mattress on the bedstead; but a cheery maid soon
produced sheets, pillows and towels, these articles from now onward
being charged separately in the bill: she also filled up the water-tank
which discharged itself into the basin by a kind of squirt, liable to
drench the unwary. A hot bath is an expensive luxury in Russia,
costing from three to five shillings; but I never appreciated it at its
proper value. The bath, filled with water too hot for me to plunge my
hand into, was invariably taken in a tiny room without ventilation in
which a stove was fiercely burning, and the attendant, armed with a
thermometer, was always greatly astonished when I demanded a
copious admixture of cold water. Half the room would be occupied by
a divan covered with a sheet on which to repose after the bath, and
once or twice I had some difficulty in getting rid of the maid, so
anxious was she to wrap me in a second sheet, with which Russians
drape themselves before they step into the water.
Samara is an important provincial town, but the whole place looked
poor and shabby, partly because the coloured plaster coating of the
houses was dropping off in unsightly patches. The wide streets
radiated from a small public garden in which stood a statue of
Alexander II., the Liberator, and, as it was Sunday, all the world was
promenading in its best clothes along the slush-covered pavements,
the thaw having set in. The peasants looked picturesque in short
sheepskin coats, worn with the wool inside, fur caps with lappets to
protect the ears, long leather riding-boots, putties tied up with string
and thick leather gloves. The shaggy hats of black or white
sheepskin made their wearers look like brigands in opera, and
beside them the women, in long black coats much kilted at the waist,
with their heads tied up in woollen shawls, appeared decidedly tame.
We made our way down to the Volga and walked on the frozen river,
which was a mile wide, watching the drinking-water of the town being
drawn from various holes in the ice.
At the railway station that evening we found a large crowd on the
platform assembled to give a hearty send-off to a trainload of
soldiers evidently hailing from the neighbourhood. The men were
travelling to the front in horse-boxes, and leant over the wooden
barriers wildly cheering and waving their caps, full of health and
spirits, and one could hardly bear to think that many would never
return, or, sadder still, would come home incapacitated for the rest of
their days.
Owing to the War there were no restaurant-cars attached to the
trains, and as the time-tables were unaltered we had halts of only ten
or twelve minutes three or four times a day, when the passengers
made a frenzied rush to get what they could at the inferior station
buffets. We usually bought something in the way of meat, cheese
and bread, and carried it back with us to our carriage, after we had
gulped down plates of the excellent cabbage soups called stchee or
borsch. The only long halt we made—one of forty minutes—was at a
station with no buffet whatever. The farther east we went the less
food could we procure: sometimes packets of inferior Russian
biscuits were the only stock-in-trade of the buffet, and if it had not
been for our soup-packets we should have been half-starved. As it
was, we were often unpleasantly hungry, hot water being the only
thing that we could be sure of obtaining.
In spite of this the journey was full of interest. We were travelling
across limitless steppes, and the melting of the snow in patches
showed that spring was at hand, when the sun would break forth
from the grey, lowering skies. Near Orenburg we noticed many tons
of hay ready to be despatched to the front, and as we halted at
Alexis I suddenly saw the ungainly forms of camels. Nearer and
nearer they came, padding across the snow, drawing sleighs laden
with hay, and with a leap of the heart I realized that we were once
again in the East, that Europe was left behind, and that we had
entered that vast mysterious continent of Asia, cradle of the human
race and birthplace of its great religions.
The following day we passed the Sea of Aral, with masted ships
riding at anchor in its port; and by now all traces of snow had gone,
and the sandy steppe was scantily dotted with coarse grasses.
Sometimes we traversed stretches of salt-encrusted ground, and in
places the rolling sand-dunes were planted and bound together with
rushes in order to prevent them from encroaching upon the railway,
or long lines of fencing answered the same purpose for the
snowdrifts.
We saw few signs of life, and the loneliness of the steppe made me
realize something of those vast empty spaces of Asia which from
lack of water will for ever be dreary wastes forsaken by mankind. Yet
a picturesque crowd was usually assembled at the stations. Hairless-
faced men with high cheek-bones were clad in long padded coats
reaching to their heels, or wore sheepskins, their rope or straw-soled
shoes being tied with leather thongs criss-cross from knee to ankle
over thick woollen stockings. Among a variety of headgear the
quaintest resembled early Victorian coal-scuttle bonnets tied under
the chin. They were made of brightly coloured velvet, with broad fur-
lined brims, a fur-lined flap behind and lappets over the ears, and
looked most comical when worn by brawny Kirghiz, who strode up
and down the platforms trailing long whips in their hands.
The warm weather was now beginning, and the Russian women who
sold tea and hot water from big brass samovars had discarded their
winter clothes and appeared in flowered cotton dresses with gaily
coloured handkerchiefs over their heads. Their children were running
about barefoot, and I was amused at watching an encounter
between a lightly clad urchin and a smart little boy who was travelling
in our overheated train. This latter, who had a long fur-lined coat, a
fur cap and galoshes over his boots, held up his foot for the
admiration of the platform youngster, who laughed good-humouredly,
and stretched out his dusty toes in response.
In spite of the warm sunshine, ours were the only windows open in
the whole train, and when, after leaving Samara, my brother had
obtained fresh air by freely tipping a most reluctant conductor, an
official higher in rank came to enquire whether it was not a mistake
and whether after all we did not wish to be screwed up again! I could
not imagine why our fellow-passengers did not follow our example,
because, before we reached Tashkent, the sun flamed down from a
cloudless blue sky; the hoopoe, harbinger of spring, chased its mate;
the crested larks sang, and the children offered big bunches of the
little mauve iris. Ploughing was visible in places, and a faint green
flush was spreading over the vast plain, which near Tashkent gave
way to grassy downs on which cattle grazed.
At the imposing-looking station of Turkestan we made enquiries
respecting the flags that we noticed hanging out on all the platforms,
and to our joy were told that they were in honour of the taking of
Przemyzl. An officer of military police with whom my brother talked,
said that this victory had come at an opportune moment, as there
was considerable unrest among the native population.
We were sorry not to see the tomb erected by Tamerlane in the old
city of Turkestan to the memory of a Kirghiz saint, for M. Romanoff,
an authority on Mohamedan art, who has visited a large proportion of
the mosques and shrines of Central Asia, considers this splendid
building to be a masterpiece.
 CHAPTER II
BEYOND THE TIAN SHAN TO KASHGAR
Farghana is a country of small extent, but abounding in grain and fruits;
and it is surrounded with hills on all sides except on the west.... Andijan is
the capital. The district abounds in birds and beasts of game. Its
pheasants are so fat that the report goes that four persons may dine on
the broth of one of them and not be able to finish it.—Memoirs of Baber.

After three days and nights in the train it was pleasant to make a
halt at Tashkent, the capital of Russian Turkestan, though the
sudden change of climate was somewhat exhausting. It was towards
the end of March, and the whole town, famous for its fruit trees, was
embowered in pink and white blossom, and the avenues of
magnificent poplars, willows and beautiful Turkestan elms were
shaking out their fresh green leaves.
The Russians, under General Kaufmann, took Tashkent about fifty
years ago, and have laid out the new town with broad roads planted
with fine trees that are watered by irrigation. There are churches,
public parks, tram-lines and imposing-looking shops, the
considerable Russian population appearing to mix freely with the
Sarts, as the inhabitants are termed by the dominant race. In India a
white woman of whatever class has a position with the natives, but
here the ordinary Russian woman is seemingly on an equality with
them, and not infrequently marries them. In the best confectioner’s
shop, served by Russian girls, natives came in and bought and ate
cakes and sweets on the premises, side by side with smart officers
or elegant ladies evidently belonging to the upper circles of Tashkent
society.
Even in this remote part of the Russian Empire the War was brought
home to the inhabitants by the presence of fifteen thousand
prisoners, Germans and Austrians. The latter, who were mostly
Slavs, had the privilege of shopping in the town, and we heard that
they were on excellent terms with their captors, whereas the
Germans were permitted no such relaxation of their captivity.
A long narrow street led from the Russian city straight into the native
town with its mud-built houses, its little stalls of food and clothing, its
mosques and shrines, and above all its gaily clad populace. But for
the people I could have imagined myself to be in a Persian city; but
here, instead of men in dingily coloured frock-coats and tall
astrakhan hats, and women shrouded in black from head to foot, the
inhabitants of both sexes revelled in colour. All wore smart velvet or
embroidered caps, round which the greybeards swathed snowy
turbans. The men had striped coats of many colours, the brighter the
better, the little girls rivalling them with bold contrasts, such as a
short, gold-laced magenta velvet jacket worn above a flowered,
scarlet cotton skirt, or a coat of emerald green with a vivid blue
under-garment. For the most part they were pretty, rosy-cheeked,
velvet-eyed maidens, with their hair hanging down their backs in a
dozen plaits, and I felt sorry to think that all their charm would shortly
have to disappear behind the long cloak, beautifully embroidered
though it might be, and the hideous black horsehair veil affected by
their mothers.
One fascinating little figure adorned with big earrings and bracelets
came dancing down an alley into the street, holding out the ends of a
scarlet veil which she had thrown over her head, her cotton dress
and trousers being in two shades of rose. She pirouetted up to a tall
man in a rainbow-coloured silk coat who was carrying a tin can, and
had paused at the steps of the mosque to let the children gather
round him. To my surprise he began to dole out ice-cream in little
glasses, and boys and girls had delicious “licks” in exchange for
small coins. I remembered how envious I had felt in early youth
when I saw English street urchins partaking of what seemed to me to
be food fit for the gods, although my nurse allowed me no chance of
sampling it, and in a moment the East and the West seemed to
come very near, the ice-cream man acting as the bridge across the
gulf.
After leaving Tashkent we travelled through a rich alluvial country
watered by the Sir Daria, the classical Jaxartes, and halted on our
way to Andijan at the ancient city of Khokand. As at Tashkent, the
Russian and native towns are separate, and we hired a moon-faced,
beardless Sart, attired in a long red and blue striped coat and with an
embroidered skull-cap perched on his shaven head, to drive us
round.
He raced his wiry little ponies at a great pace along a wide tree-
planted avenue ending in a church of preternatural ugliness set in a
public garden. Near by were Russian houses and shops, while small
victorias containing grey-uniformed officers or turbaned Sarts
dashed past, and native carts laden with bales of cotton creaked
slowly by. Many of these carts had big tilts, the wooden framework
inside being gaudily painted, and the horses themselves were
decked with handsome brass trappings.
The old town, with its high mud walls, flat-roofed squalid dwellings, a
bazar closely resembling those to be found in any Asiatic city, and
comparatively modern mosques, had little of interest, though a well-
known traveller speaks of its thirty-five theological colleges: its roads,
as usual, were bad and narrow, and must be rivers of mud in wet
weather.
Many women were unveiled, others wore the ghoul-like horsehair
face coverings, and some of their embroidered coats were so
charming in design and colouring that I longed to do a “deal” with the
wearers. Many of the people were squatting, eating melons which
they store during the winter, or drinking tea, a Russian woman being
evidently a member of one family group. We had one or two narrow
shaves of colliding with other carriages, as our coachman threaded
his way far too fast for safety and exchanged abusive epithets with
his brother Jehus, among whom were Russians in black, sleeveless,
cassock-like garments worn over scarlet cotton blouses. The
harness of the little horses was adorned with many tufts of coloured
wools, giving a pretty effect as these tassels nearly swept the ground
or waved in the air. The life on the roads, the spring sunshine, the
fresh green leaves, the white and pink of the blossom, and the orgy
of colour furnished by the inhabitants, made the drive an
unforgettable experience.
A few hours later we reached Andijan, where the railway ended, and
here we had our last clean resting-place until we arrived at Kashgar.
I noticed that the native women wore long grey burnouses with black
borders ending in two tails that were always trailing in the dust, and
all hid their faces in the mask-like horsehair veils. It was the day
before Palm Sunday, and as we strolled in the evening up the
cobbled street of the town a large congregation was issuing from the
church, every one carrying a small branch and a little candle, which
each had lit in the sanctuary. In the darkness the scores of tiny lights
looked like fire-flies, and I observed how carefully the sacred flame
was sheltered from any draught, as it is considered most important
to convey it home unextinguished. Our hotel was fairly good, but I
was not pleased on retiring to find that my door did not lock, and that
my window, opening on to a public balcony, had no fastening. To
supplement these casual arrangements I made various “booby-
traps” by which I should be awakened if any robber entered my
room, but luckily slept undisturbed.
It may give some idea of the vast extent of the plains of Russia
which we had crossed by train, when I mention that there was not a
single tunnel on the hundreds of miles of rail between Petrograd and
Andijan.
It was the end of March when we set out to drive the thirty miles from
Andijan to Osh. We packed ourselves, our suit-cases and the lunch-
basket into a little victoria, while Achmet, the Russian Tartar cook we
had engaged at Tashkent, accompanied our heavy baggage in the
diligence. The sky was overcast with heavy clouds, so there was no
glare from the sun, and the rain of the previous night had laid the
dust on the broad road full of ruts and holes. Ploughing was in full
swing, barley some inches high in the fields, fruit blossom
everywhere, and the poplars and willows planted along the countless
irrigation channels made a delicate veil of pale green. Beyond the
cultivation lay bare rolling hills, behind which rose the lofty mountain
ranges which we must cross before we could reach our destination.
The whole country seemed thickly populated, and we passed
through village after village teeming with life, the source of which is
the river, which ran at this time of year in a surprisingly narrow
stream in its broad pebbled bed, and was so shallow that men on
foot or on donkey-back were perpetually crossing it. Tortoises were
emerging from their winter seclusion, the croak of the frog filled the
land, hoopoes and the pretty doves which are semi-sacred and
never molested flew about, and the ringing cry of quail and partridge
sounded from cages in which the birds were kept as pets.
The men, if not busied with agriculture, were usually fast asleep or
drinking tea on the mud platforms in front of their dwellings, and the
gaily clad women slipped furtively from house to house, or, if riding,
sat on a pillion behind the men. In fine contrast to her veiled sisters
was a handsome Kirghiz lady following her husband on horseback
through the Osh bazar, and making a striking figure in a long green
coat, her head and chin wrapped in folds of white that left her
massive earrings exposed to view. She rode astride every whit as
well as the man did, exchanged remarks freely with him, and was
moreover holding her child before her on the saddle. Other women
were carrying cradles which must have made riding difficult, and
often a child stood behind, clinging to its mother’s shoulders. On
entering the native town of Osh, mentioned in Baber’s Memoirs as
being unsurpassed for healthiness and beauty of situation, we
passed a mosque with such a badly constructed mud dome that it
looked like a turnip, and made our way along a broad tree-planted
Russian road to the nomera. This was a house with “furnished
apartments to let,” and the small rooms, by no means overclean,
were supplied with beds, tables and chairs. We set to work to
unpack our camp things, and sent Achmet out to buy bread, butter,
meat, eggs, etc., for our two hundred and sixty mile ride to Kashgar.
Our host made no pretensions to supply food, but exactly opposite
our lodgings was the officers’ mess; with true Russian hospitality its
members invited us to take our meals there, and next day at lunch
we met a dozen officers, with their jovial, long-haired chaplain in
black cassock with a broad silver chain and crucifix round his neck.
Luckily for me there were a couple of officers who spoke German,
though the others threatened them with heavy fines for daring to
converse in the language of the Huns. In spite of the Tsar’s edict,
vodka and wine flowed freely (the doctor had evidently given medical
certificates liberally to the mess) and numerous toasts were drunk,
every one clinking his glass with my brother’s and mine as the health
of King George, the Tsar, our journey, and so on were given. All were
most kind, though I could have wished Russian entertainments were
not so long—that luncheon lasted over three hours—and we left in a
chorus of good wishes for our ride to Kashgar.
We were roused early next morning by the arrival of our caravan of
small ponies, and with much quarrelling on the part of their drivers
the loads were at last adjusted. We had our saddles put on a couple
of ill-fed animals and started off beside the rushing river on our first
stage of twenty miles. The ponies were very inferior to the fine mules
with which we had travelled in Persia, and our particular steeds
would certainly have broken down long before we reached Kashgar
if we had not dismounted and walked at frequent intervals
throughout the whole journey.
At first the road was excellent as we left pretty little Osh nestling
under Baber’s “mountain of a beautiful figure,” and made our way up
a highly cultivated valley towards the distant snowy peaks. We were
escorted by a fine-looking Ming Bashi or “Commander of a
Thousand,” who had a broad velvet belt set with bosses and clasps
of handsome Bokhara silver-work. He wore the characteristic Kirghiz
headgear, a conical white felt with a turned-up black brim, and four
black stripes, from the back to the front and from side to side of the
brim, meeting at the top and finishing off with a black tassel. We
were to see this headgear constantly during the next eight months,
as it is worn throughout Chinese Turkestan and the Pamirs. Owing to
the presence of these Ming Bashis we met with extreme
consideration, village Begs and their servants escorting us at every
stage and securing the right of way for us with caravans. This was a
privilege that for my part I keenly appreciated, as the track, when it
skirted the flanks of the mountains, was hardly ever wide enough for
one animal to pass another, and I had no wish to be pushed out of
my saddle over the precipice by the great bales of cotton that formed
the load of most of the ponies we met. These officials usually
secured some garden or field, a place of trees and running water,
where we could lunch and rest at mid-day, and often they brought a
silken cushion which they offered to my brother. They were surprised
when he handed it on to me, for in Mohamedan countries the woman
is considered last—if at all.
In the Osh district horses, camels, donkeys, cows, goats and sheep
were in abundance, the sheep having the dumba or big bunch of fat
as a tail, which nourishes the animal when grass runs short during
the winter months. They had long hair like goats and rabbit-like ears,
were coloured black, white, brown, grey or buff, and looked far larger
in proportion than the undersized cattle and ponies. On the road we
saw many of the characteristic carts that had immensely high wheels
with prominent hubs. The driver sat on a saddle on the horse’s back,
supporting his feet on the shafts, thereby depriving the animal of half
its strength for pulling the load and proving that this nation of born
riders has not grasped the elementary principles of driving. These
carts had no sides, but carried their loads in a curious receptacle of
trellis-work, as shown in the illustration.

CART USED IN THE OSH DISTRICT.

Page 26.
We reached our first night’s lodging about four o’clock, and I was
glad to dismount, as riding at a foot pace on an animal that is a slow
walker is a tedious business. All these halting-places in Russian
territory were much alike—a couple of small plastered rooms, often
with bedsteads, table and stools, sometimes looking into a courtyard
where the ponies were tied for the night, but often with no shelter for
the animals and their drivers. Jafar Bai, the chuprassi from the
Kashgar Consulate sent to escort us, was of the utmost service to us
on the road. I noticed that many of the men we passed saluted him
by throwing their whips from right to left across their chests, and their
deference made me realize the high esteem in which he was held.
He put up our camp beds, tables and chairs, and found water for our
folding baths. It was usually cold at night, and besides warm
underclothing I had a sleeping sack, rugs and my fur-lined coat. We
always got up at 5.30 a.m., and I did a hasty toilette in the dark with
the aid of my torchlight, Achmet producing coffee, eggs, bread,
butter and jam for our early breakfast, while Jafar Bai packed our
bedding.
Once or twice we were accommodated in the house of a village Beg,
and found the floors covered with felts and carpets, and a table
spread with bread, sweets, raisins, almonds and pistachios. One of
our hosts kept his treasures in a wonderful gilt, red and black chest,
from which he produced a handsome watch given him by the
Russians. This chest emitted a loud musical note when opened or
shut, in order, I presume, to warn the owner if thieves attempted to
rifle it. At night his servants removed his bedding of Bokhara silken
quilts, but with touching confidence left the box in our charge!
Our second day’s march found us approaching the mountains, and
we rode to the top of a low pass where hills slashed with scarlet,
crimson and yellow rose one behind another, to be dominated by the
glorious snow-covered Tian Shan peaks clear cut against a superb
blue sky. Walking down the passes was certainly preferable to sitting
on a stumbling pony, but I found it rather hard work, as the track was
usually very steep and littered with loose stones, on which one could
easily twist an ankle or tumble headlong. Every now and again it
looked as if we had reached the bottom, when lo, after turning a
corner, the track zigzagged down beneath our feet seemingly longer
and steeper than ever.
During this march we passed a party of Chinese bound for Kashgar,
consisting of an official and a rich merchant with their retinues. The
ladies of the party travelled in four mat-covered palanquins, each
drawn by two ponies, one leading and one behind, and I pitied them
having to descend these steep places in such swaying conveyances.
They were attended by a crowd of servants in short black coats, tight
trousers and black caps with hanging lappets lined with fur, the
leaders being old men clad in brocades and wearing velvet shoes
and quaint straw hats. As seems usual with upper-class Chinese,
they were very indifferent horsemen, and sat on bundles of silk
quilts, not attempting to guide their ponies in any way, but letting the
burly Kirghiz lead them by the halters. In striking contrast to them
was a fine-looking man in a long green and purple striped coat, from
the handsome girdle of which hung a silver-sheathed knife. His
boldly cut aquiline features were surmounted by a black fur cap, and
as he rode down the pass on a beautiful Badakshani horse the pair
made a delightful picture.
Caravans laden with bales of cotton toiled uphill towards us, and
sometimes we met a string of camels; but ponies did most of the
work here, their small heads peering out from between their bulky
loads. They had bells hung round their necks, enabling the approach
of a pack-train to be heard at a considerable distance, and specially
favoured animals wore collars of blue beads to avert the evil eye.
Besides caravans we met gangs of Kashgaris going to work at Osh
or Andijan during the summer, in order to earn the money on which
they live throughout the winter. They were sturdy men, their white
teeth flashing in faces tanned almost black by the sun, and they
wore long padded cotton coats of all colours, the most usual being
scarlet, faded to delicious tints. As these coats were turned back to
enable them to walk more freely, we had the contrast of a bright
turquoise blue, or an emerald green or a purple lining. Some walked
barefoot, others in long leather riding-boots or felt leggings, and all
had leather caps edged with fur. Each man carried a bundle of his
belongings, out of which cooking-pots often peeped, and some one
in the gang was certain to have a tar, a kind of mandoline, with which
to amuse the party, or perhaps a bagpipe or a small native drum; it
was pleasant to come across a group of these wayfarers beguiling
their long march by listening to the music that has so strong a
fascination for Orientals.
The farther we left Osh behind us the more barren became the
country, until we marvelled how the flocks and herds could support
life on the scanty vegetation. At one point the hills were a bright
scarlet and it was strange to see a red mud-built village with sheep
grazing in this brilliantly coloured setting. We crossed rivers and
streams many times, but they were not deep, for the mountain snow
had not yet melted, and we found the bridges formed of rough poplar
stems, with big holes into which boulders were stuck, far more
dangerous than the water. It was during this march that my pony
nearly ended our joint careers by backing with me to the edge of a
precipice. We were passing a donkey laden with brushwood, an
ordinary sight, of which my brother’s horse on ahead had not taken
the smallest notice, when my animal made a big shy, and if Jafar Bai
had not seized the rein I held out to him and hauled at it manfully
while I urged my mount with whip and voice, we should both have
fallen into the river rushing far below.
The crux of our journey was the crossing of the Terek Dawan or
Pass, 12,000 feet high, and the night before we lodged in akhois, at
its foot, in place of the usual rest-house.
It was my first experience of the bee-hive like homes of the Kirghiz
—“a dome of laths and o’er it felts were spread”—and, as we had
ridden through heavy rain and hail the last part of the way, I was
extremely thankful to pass behind a felt curtain and find myself in a
snug circular room lined with felts and embroideries. A fire was lit on
the ground in the centre, the smoke escaping from a large hole in the
roof, and by squatting on the floor we could more or less avoid the
acrid smoke that made our eyes water.
In the morning we started at seven o’clock, anxious to reach the top
of the pass before the sun, now hot during the day, could melt the
snow. To our intense relief it was a superb day, a few fleecy clouds
sailing across a deep turquoise sky. I was clad in a mixture of arctic
and tropical attire, wearing a leather coat under my thick tweed habit,
woollen putties and fur-lined gloves, along with a pith hat, blue
glasses and gauze veil. We soon came to the snow and zigzagged
upwards on a narrow track moving in single file, any animal trying to
pass another being liable to fall headlong in the soft deep snow on
either side, a fate that befell two of our party early in the day. After a
while, as we advanced, the great peaks towered on all sides, sharply
silhouetted against their blue background—nothing but white as far
as eye could reach; and here and there skeletons sticking out of the
snow bore eloquent witness to the terrible annual toll paid by the
hundreds of horses and donkeys that have to cross this cruel pass. I
could hardly believe that it was possible to ride over these
mountains, so steeply did they rise above us; and at the worst part of
the ascent some sturdy Kashgaris coming down towards us had
much ado to keep their feet, even though they carried long staves,
one man falling headlong and rolling a considerable distance. The
last pull to the crest is almost perpendicular, and is noted for
accidents—here my brother’s pony nearly went over—but finally,
caravan and all, we reached the summit of the pass in safety, and
dismounted to enjoy the fine view. Before us lay the great Alai
Range, peak towering above peak of boldly serrated mountains.
Over us hovered a huge vulture, and as I looked down the track in
front where the snow was partly melted, hideous heaps of bones
were revealed, and I felt that the ill-omened bird knew that it would
never lack food so long as Russia did nothing to improve this
execrable road.
In books of travel the writer frequently “swings down” such places,
but my experience was very different, as we crept down the worst
parts on foot. The snow on the farther side was rotten, and our feet
broke through it to water running underneath and big boulders. It
was the kind of path on which one could easily break a leg, and for a
loaded pony was a cruel ordeal, if not almost impossible. Even
where the snow had entirely melted near the foot of the pass the way
lay through a mass of boulders and slippery mud most trying to any
baggage animal.
For ourselves we had nothing to complain of, and a march of seven
hours found us at the little rest-house enjoying some lunch; but our
caravan fared very differently. The distance was only twelve miles,
but so bad was the going that the ponies, though lightly laden, were
about thirteen hours on the road, and four poor animals stayed out
all night. We had no evening meal till nine o’clock, and our hold-alls
when they arrived were encrusted with ice that had made its way
inside and soaked our bedding. We had no means of drying it in the
serai, and so were obliged to sleep in our clothes. We were too
thankful to be safely over the pass to heed such minor discomforts,
and were indeed most fortunate; for the road was closed for some
days after our journey in order that a fresh track might be trampled
down by driving unloaded animals across it.
On the morrow our caravan had a much-needed rest till mid-day,
while we unpacked our boxes and dried our wet belongings in the
sun. I was concerned about my face, as in spite of all my precautions
I found that my cheeks, nose and lips were terribly swollen, and
besides being burnt a bright scarlet, all my skin was coming off in
patches, making me most unsightly in appearance. On my
mentioning this experience not long ago to an eminent geographer
and traveller, he assured me that, if I had thickly powdered my
unlucky visage before encountering sun and snow, it would have got
off scot-free, and I insert the hint for the benefit of future travellers.
Our next stage was Irkeshtam, situated at the junction of the Osh-
Kashgar and Alai routes. In the time of Ptolemy it was an important
centre on the great trade route which ran from Rome across Asia to
China, the “Stone Tower” mentioned by the Greek geographer being
either here or in the vicinity. To-day it consists of a small fort
garrisoned by Cossacks, with customs and telegraph offices all set
down in hopelessly barren surroundings.
We were hospitably welcomed by the customs official’s wife and
sister, but were sorry to find that our host was ill. After the nine
o’clock supper we retired, my brother sleeping in some outhouse,
and I in a little room which my hostess’s sister had kindly vacated for
me, where I had a queer experience. As the window was
hermetically sealed up for the winter, and the stove was lit, I had
perforce to leave the door open in order to escape partial
suffocation. A large carpet was suspended from the ceiling above
the bedstead, across which it was carried, and hung down to the
floor, and upon the bed were a sheet, a velvet bedspread and a
couple of lace-covered pillows. Slipping into my rugs I put out the
lamp, and as I was composing myself for slumber I became aware of
a stirring under the bed, and a breathing. Thinking it must proceed
from the dog or cat, with both of which I had made friends, I tapped
the carpet and said “Ssh!” reflecting that if I troubled to drive the
animal out it would be sure to return again by the open door, and as
all was quiet I thought no more about the matter and went to sleep.
Some time in the middle of the night I was suddenly roused by
feeling the bed violently jolted and to my horror heard loud and
unmistakably human snores proceeding from under it. Considerably
startled, I sat up in the pitch darkness and listened to heavy
breathing while I summed up the situation. The intruder could not be
a burglar, as there was nothing to steal, and of course I was in no
danger, as I could rouse the house in a moment, my door being
open. I felt it would be wrong to make a disturbance as our host was
so ill; I could not communicate with my brother, for I had no idea
where he was, and it would have been impossible to leave the house
and search for him in the wind and darkness, with savage dogs
roaming about. Another alternative would have been to light the lamp
and turn out the intruder myself; but I feared that my lack of Russian
and Turki would make this difficult, and it would certainly rouse the
establishment. All things considered, I decided to lie and watch for
daylight, my matches being to my hand. After the unknown had
turned over again I heard the regular breathing of deep slumber, and
soon, contrary to my intention, I dropped off to sleep myself.
When I woke about seven o’clock it was quite light. Examining my
bed with some trepidation, I found a space between it and the wall at
each end. Behind my pillows was a heavy red felt, and pulling this up
I came upon a makeshift bed with pillow and bedding underneath
mine. The occupant had gone, and I discovered the place at the end
of the bed where “it” must have crept out noiselessly through the
open door!
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