Animal and Plant Stem Cells Concepts

Propagation and Engineering 1st

Edition Mirjana Pavlovi■
Visit to download the full and correct content document:
https://textbookfull.com/product/animal-and-plant-stem-cells-concepts-propagation-an
d-engineering-1st-edition-mirjana-pavlovic/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...

Stem Cells Biology and Engineering Phuc Van Pham

https://textbookfull.com/product/stem-cells-biology-and-
engineering-phuc-van-pham/

Studies of Pluripotency in Embryonic Stem Cells and

Induced Pluripotent Stem Cells 1st Edition Xiaoyang
Zhao (Auth.)

https://textbookfull.com/product/studies-of-pluripotency-in-
embryonic-stem-cells-and-induced-pluripotent-stem-cells-1st-
edition-xiaoyang-zhao-auth/

Hepatic Stem Cells: Methods and Protocols Naoki

Tanimizu

https://textbookfull.com/product/hepatic-stem-cells-methods-and-
protocols-naoki-tanimizu/

Skin Stem Cells: Methods and Protocols Kursad Turksen

https://textbookfull.com/product/skin-stem-cells-methods-and-
protocols-kursad-turksen/
Cell Biology and Translational Medicine Volume 3 Stem
Cells Bio materials and Tissue Engineering Kursad
Turksen

https://textbookfull.com/product/cell-biology-and-translational-
medicine-volume-3-stem-cells-bio-materials-and-tissue-
engineering-kursad-turksen/

Perinatal Stem Cells Biology Manufacturing and

Translational Medicine Zhong Chao Han

https://textbookfull.com/product/perinatal-stem-cells-biology-
manufacturing-and-translational-medicine-zhong-chao-han/

Stem Cells and Good Manufacturing Practices Methods

Protocols and Regulations 1st Edition Kursad Turksen
(Eds.)

https://textbookfull.com/product/stem-cells-and-good-
manufacturing-practices-methods-protocols-and-regulations-1st-
edition-kursad-turksen-eds/

Stem Cells in Urology Seyed Mohammad Kazem Aghamir

https://textbookfull.com/product/stem-cells-in-urology-seyed-
mohammad-kazem-aghamir/

Science and Engineering Projects Using the Arduino and

Raspberry Pi: Explore STEM Concepts with Microcomputers
1st Edition Paul Bradt

https://textbookfull.com/product/science-and-engineering-
projects-using-the-arduino-and-raspberry-pi-explore-stem-
concepts-with-microcomputers-1st-edition-paul-bradt/
Mirjana Pavlović · Ksenija Radotić

Animal and
Plant Stem
Cells
Concepts, Propagation and Engineering
Animal and Plant Stem Cells
Mirjana Pavlović Ksenija Radotić
•

Animal and Plant Stem Cells

Concepts, Propagation and Engineering

123
Mirjana Pavlović Ksenija Radotić
Department of Computer and Electrical Department of Life Sciences
Engineering Institute for Multidisciplinary Research,
Florida Atlantic University University of Belgrade
Boca Raton, FL Belgrade
USA Serbia

ISBN 978-3-319-47761-9 ISBN 978-3-319-47763-3 (eBook)

DOI 10.1007/978-3-319-47763-3
Library of Congress Control Number: 2016963320

© Springer International Publishing AG 2017

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt from
the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the
authors or the editors give a warranty, express or implied, with respect to the material contained herein or
for any errors or omissions that may have been made. The publisher remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

Printed on acid-free paper

This Springer imprint is published by Springer Nature

The registered company is Springer International Publishing AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface

This book provides a unique parallel view on animal and plant stem cells from
various aspects. The book maximizes reader’s insight into research and application
in its endeavor to understand the nature of these cells; their sources and categories;
engineering of these cells, reprogramming of their functions, and their role as novel
cellular therapeutic approach. Written by the author who has already published
books and articles in this field, this new one focuses on all aspects of stem cells that
were omitted in previous (such as expansion, propagation in culture, metabolic
aspects) and gives the specific, multifaceted insight into the world of stem cells this
time enriched with contribution of the second author who is the expert in plant cell
domain. For plants, it is characteristic indeterminate growth pattern, requiring
specific features of their stem cells. In contrast, the animal body plan is mostly
defined during embryonic development, and adults generally lack pluripotent stem
cells. These features make certain crucial differences between plant and animal stem
cells, although there are many similarities in their structure and mechanisms of
functioning. This course enhances reader’s understanding of plant and human
ordinary stem cells, their similarities and differences. It introduces the concepts of
emergence of cancer stem cells and different modalities in targeted cancer stem cell
therapies. The book treats both theoretical and practical aspects of stem cell
research and application and covers many different applications with their advan-
tages and limitations. It is a valuable source of fresh information for academics and
researchers, giving an intriguing insight into molecular mechanisms of animal and
plant stem cell regulation and their usage for therapeutic applications. It will be a
great source of information for students at different level of their education in the
fields that require medical and bioengineering background, since it includes cases

v
vi Preface

that illustrate and explain mechanisms, interactions, targeted effects, and multi-
modal therapeutic approaches. This work explores the intersection between animals
and plants and explains their co-operative role in life. Academics, researchers, and
those who want to expand their knowledge in this field will find this to be an
exceptional source of references.

Boca Raton, FL, USA Mirjana Pavlović

Belgrade, Serbia Ksenija Radotić
Acknowledgements

The authors would like to express their gratitude to all of those people who were
supportive and understanding for their ideas in life and work on this book.
Ksenija Radotić wants to thank Acad. Prof. Dr. Radoslav Andjus who was
himself the example of a man emerged from science but always understanding
for those whom it was the way of leaving as well. She is also grateful to Republic
of Serbia Council for grant research support, grant no ON173017 and
Prof. Dr. Vladimir Pavlović for his assistance with technicalities that followed the
book editing.
Mirjana Pavlović has a thank you note for most of her colleagues from
Department of CEECS, Sharmistha Chatterjee, Ph.D., for her help in revising
figures and tables, and her brother Prof. Dr. Vladimir Pavlović for selfless and
enthusiastic technical help with manuscript.
Both authors appreciate the work of book illustrators Aleksandar Hadži Manić
and Jordan Brennan who helped with adoption or original design of illustrations.

Mirjana Pavlović
Ksenija Radotić

vii
Contents

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

Part I Bioengineering and Animal Stem Cells

2 Current Status and Perspectives in Stem Cell Research:
The Concept of Normal Stem (NSC) and Cancer
Stem Cell (CSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 Stemness, Migration, Circulation, and Seeding . . . . . . . . . . . . . 8
2.3 Mobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.4 Similarities and Differences Between Embryonic
and Adult Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.5 Similarities and Differences Between Normal and Cancer
Stem Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.6 Resume Based upon Similarities and Differences Between
NSCs and CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.7 What and Which Molecular Markers of CSCs We Know
Today? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.8 Isolation of CSCs Using Magnetic Beads . . . . . . . . . . . . . . . . . 11
2.9 Theories of Origin of CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.10 Current Tests for Determination of the Presence of CSCs . . . . 12
2.11 Sorting and Isolation of CTC Using the Method
of Magnetic Levitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.12 The Emergence/Origin and Development of CSC:
Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.13 Aberrant Methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.14 What Do We Need to Do with the Sum of Information? . . . . . 14
2.15 How Was the Concept of CSC Therapy Designed? . . . . . . . . . 14
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

ix
x Contents

3 Essential Characteristics of Stem Cells: Self-renewal,

and Plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 17
3.1 The Nature of Stem Cells . . . . . . . . . . . . . . . . . . . . ......... 17
3.2 Self-renewal and Its Significance for Stem Cells. . . ......... 18
3.3 Plasticity/Pluripotency of Animal Stem Cells
and Its Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . ......... 19
3.4 Other Features of Significance . . . . . . . . . . . . . . . . ......... 19
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 20
4 Stem Cell Sources and Types of Animal Stem Cells . . . . . . . . . . . . . 23
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4.2 Embryonic SCs (ES or ESC) . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4.3 Fetal Stem Cells (FSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.4 Bone Marrow and Other Adult Stem Cells Sources . . . . . . . . . 24
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
5 Stemness and Stem Cell Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.2 Human Embryonic Stem Cell Markers and Pluripotency . . . . . 28
5.3 Fetal Stem Cell Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
5.4 Cord Blood Stem Cell Markers. . . . . . . . . . . . . . . . . . . . . . . . . 30
5.5 Placental Stem Cell Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5.6 Adult Stem Cell Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
6 Stem Cell Signaling Molecules and Pathways . . . . . . . . . . . . . . . . . . 33
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
6.2 ESC-Wnt Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
6.2.1 Significant Discoveries in Wnt . . . . . . . . . . . . . . . . . . 35
6.3 Notch Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
6.4 The Human Hedgehog Signaling . . . . . . . . . . . . . . . . . . . . . . . 37
6.5 TGF-Beta Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
6.6 Signaling Mechanisms in Adult Human Cells
(HSC and MSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
6.7 Pathways in Adult HSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
6.8 Pathways in Adult Mesenchymal Stem Cells . . . . . . . . . . . . . . 39
6.9 Pathways in CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
6.10 The JAK (Janus Kinase)/STAT3 (Signal Transducer
and Activator of Transcription) Signal Mechanism . . . . . . .... 41
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 42
Contents xi

7 Expansion of Stem Cells: Propagation of Animal

Stem Cells Ex Vivo (In Culture) . . . . . . . . . . . . . . . . . . . . . . . . .... 45
7.1 What Is Expansion and What Do We Expect
from Expansion? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 45
7.2 Basic Concept—Ex Vivo Expansion of Hematopoietic
Cells Today . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
7.2.1 Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
7.2.2 Distinct Stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
7.2.3 Media for Expansion . . . . . . . . . . . . . . . . . . . . . . . . . . 47
7.2.4 Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8 Stem Cell Pool: What Are the Best Patterns
for Cellular Therapy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
8.2 Organogenesis from Adult Stem Cells and Problems
with Different Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
8.3 Therapeutic Implications for TCSCs as a New Concept . . . . . . 55
8.4 The Concept of VSEL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
8.5 The Concept of Mesenchymal Stem Cell (MSC)
with Dental Pulp Cells (DPSCs) as an Example . . . . . . . . . . . . 60
8.6 Mobilization as a New Noninvasive Therapeutic
Concept. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
8.7 New Concepts in Adult Stem Cell Research
with Development of New Strategies: Personal Experience
in the Light of Significance of Growing Information . . . . . . . . 63
8.7.1 Background and Significance . . . . . . . . . . . . . . . . . . . . 63
8.8 Directions and Relevant Studies: We and Others . . . . . . . . . . . 64
8.9 Optimization of Primitive Stem Cell Patterns
for Regeneration and Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
9 Induced Pluripotent Stem Cells (iPSCs) and Nuclear
Reprogramming. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
9.1 Breakthrough: Induced Pluripotent Stem Cells (iPSC) . . . . . . . 72
9.1.1 Reprogramming as a Therapeutic Event . . . . . . . . . . . 72
9.2 Induced Pluripotent Stem Cells (iPSCs)
and Nanotechnology: State of the Art . . . . . . . . . . . . . . . . . . . . 74
9.3 Nanoparticles for Genetic Reprogramming of Somatic
Cells to iPSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
9.3.1 Poly(Beta-Amino Ester) Nanoparticle-Based
Non-viral Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
9.3.2 Calcium Phosphate Nanoparticle Based Non-viral
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
xii Contents

9.3.3 Polyketal Nanoparticle-Mediated Non-viral,

Non-genetic Approach for Delivery
of Mature MicroRNAs . . . . . . . . . . . . . . . . . . . . . . . . . 77
9.3.4 Non-viral Magnetic Nanoparticle Based
Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
9.3.5 The Impact of iPSC upon the Research
and Practical Application . . . . . . . . . . . . . . . . . . . . . . . 79
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
10 Cancer Stem Cell Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
11 Metabolic Reprogramming in Cancer and Metabolic
Theory of CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 99
11.1 Metabolism of CSC: New Explanations
and the Old Fundament . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
11.2 Terminology of Energy Metabolism . . . . . . . . . . . . . . . . . . . . . 100
11.3 Coupling of the Respiration with OXPHOX . . . . . . . . . . . . . . . 102
11.4 Mechanisms of ATP Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . 102
11.5 Metabolic Abnormalities in Cancer Cells
and Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 102
11.6 The Hallmarks of Cancer Cell Energy Metabolism:
The Warburg Effect and the Crabtree Effect . . . . . . ......... 105
11.7 Resume: Energy Metabolism in Cancer Cell
Compromised? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
11.8 The Overview on CSC Metabolic Activity . . . . . . . . . . . . . . . . 107
11.9 New Cancer Therapy Concept . . . . . . . . . . . . . . . . . . . . . . . . . 109
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
12 Concept of Targeted Cancer Stem Cell Therapy
and New Versions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 113
12.1 The Idea of Targeted Therapy: Before, Now,
and in the Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
12.2 Can We Use NSCs in the Therapy of Cancer? . . . . . . . . . . . . . 114
12.3 Mitochondria as the Target . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
12.4 Other Targeting Possibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
12.5 New Modalities of Targeted Therapy and Idea
of Remote Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
12.6 Principles of Targeted Therapy Based on Nanoparticles . . . . . . 115
12.7 What is the Best Way to Kill CSC: New Methods? . . . . . . . . . 115
12.8 Different Combinations of Targeted Therapy . . . . . . . . . . . . . . 115
12.9 The Use of Electromagnetic Fields for Growth
Inhibition of CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 116
12.10 Cancer and Electromagnetism . . . . . . . . . . . . . . . . . . . . . . .... 116
12.11 Different Principles: Drug Delivery Based upon
Nanoparticles of Ferrous Oxide . . . . . . . . . . . . . . . . . . . . .... 117
Contents xiii

12.12 3D–Printed Tumor Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

12.13 Computerized 3D Model for Analysis of Aggregation
of Tumorigenic Cells Is Revealing Specialized Behavior
and Unique Cell Types Which Accelerate the Aggregation
Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
12.14 Possible Mechanisms of Tumor Cellular Heterogeneity:
Mediated Coalescence? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
12.15 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
12.16 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
12.17 A Look Ahead; What Does the Future Say? . . . . . . . . . . . . . . . 120
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
13 HLA Typization Choice of Donors: Match or Match Me Not . .... 125
13.1 Distinction Between HLA and MSH Genes Molecules
and Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
13.1.1 HLA (H-Histocompatibility Locus Antigen) . . . . . . . . 125
13.1.2 MHC (Major Histocompatibility Complex) . . . . . . . . . 125
13.1.3 HLA Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
13.1.4 MHC Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
13.1.5 Polygeny and Polymorhysm of MHC . . . . . . . . . . . . . 126
13.1.6 The Mechanism of Gene Rearrangement Is
Contributing to and Allowing for Antigen
Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 128
13.1.7 Transplantation Needs Matching to Support
Engraftment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 128
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 129
14 Engraftment: Homing and Use of Genetic Markers . . . . . . . . . . . . . 131
14.1 What is Engraftment/Homing? . . . . . . . . . . . . . . . . . . . . . . . . . 131
14.2 Genetic Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
15 Nanotechnology in Stem Cell Research . . . . . . . . . . . . . . . . . . . . . . . 133
15.1 Nanotechnology/Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . 133
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
16 Stem Cell Therapy: Optimization, Regeneration,
Reprogramming, Expansion, Tissue Engineering . . . . . . . . . . . .... 137
16.1 Optimization of the Pattern for Stem Cell Treatment . . . . .... 137
16.2 Regeneration in Animals and Humans . . . . . . . . . . . . . . . .... 137
16.3 Nuclear Reprogramming as One of the Methodological
Modalities to Get iPSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
16.4 Significance of Expansion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
16.5 Stem Cell as a Part of TE Triangle . . . . . . . . . . . . . . . . . . . . . . 138
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
xiv Contents

17 What Are Positive Results of Stem Cell Therapies? . . . . . . . . . .... 141

17.1 An Overview of Some Disease States in Which Stem
Cells Could Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 141
17.2 Heart Attack . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 142
17.2.1 Optimization of the Stem Cell Source
for Intracoronary Grafting . . . . . . . . . . . . . . . . . . . . . . 142
17.3 Physically Induced Injury of Neural Tissues . . . . . . . . . . . . . . . 143
17.4 Degenerative Injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
17.5 Persistent Disease States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
17.6 Concerns with Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
17.7 The Recovery of the Five Senses with Stem Cells:
Are the Concepts Realistic? . . . . . . . . . . . . . . . . . . . . . . . .... 148
17.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 151
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 152
18 Topic Novelties in Animal Stem Cell Research . . . . . . . . . . . . . . . . . 163
18.1 Biomagnetism, Electromagnetism, and Biophotonics . . . . . . . . 163
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
19 Resume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. 165
19.1 The Directions in Animal Stem Cell Research
Development Require Further Work . . . . . . . . ............. 165
19.2 Quo Vadis? . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. 167
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. 167

Part II Plant Stem Cells and Bioengineering

20 Stem Cells in Plants. Meristems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
21 Shoot and Root Apical Meristems . . . . . . . . . . . . . . . . . . . . . . . .... 175
21.1 Shoot Apical Meristems . . . . . . . . . . . . . . . . . . . . . . . . . . .... 175
21.1.1 Functional Zones and Layers of Shoot Apical
Meristem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 176
21.2 Root Meristems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 177
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 178
22 Lateral Meristems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
23 External Control of the Plant Stem Cells . . . . . . . . . . . . . . . . . . . . . 181
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
24 Signaling and Genetic Regulation of the Plant Stem Cells . . . . . . . . 183
24.1 Signaling and Regulation in Shoot Apical Meristem . . . . . . . . . 183
24.1.1 Regulation by WUS Transcription Factor . . . . . . . . . . . 184
24.1.2 Regulation by KNOX Genes . . . . . . . . . . . . . . . . . . . . 186
Contents xv

24.1.3 Signaling Molecules: Auxin. . . . . . . . . . . . . . . . . .... 186

24.1.4 Signaling Molecules: Cytokinines . . . . . . . . . . . . .... 190
24.2 Signaling and Regulation in the Root Apical Meristem . . .... 191
24.2.1 Control of the Root Stem Cells via Auxin
and PLT Genes . . . . . . . . . . . . . . . . . . . . . . . . . . .... 192
24.2.2 Control of the Root Stem Cells by Transcription
Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
24.3 Regulation in Vascular Meristems . . . . . . . . . . . . . . . . . . . . . . 194
24.4 Regulation in Grass Meristems . . . . . . . . . . . . . . . . . . . . . . . . . 196
24.4.1 Phyllotaxy and Plastochron Regulation . . . . . . . . . . . . 197
24.4.2 The Floral Transition . . . . . . . . . . . . . . . . . . . . . . . . . . 198
24.4.3 Inflorescence Meristem Identity . . . . . . . . . . . . . . . . . . 198
24.5 Similarities of Plant Stem Cells at the Molecular Level . . . . . . 199
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
25 Meristems and Primary and Secondary Growth in a Plant . . . . . . . 207
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
26 Propagation of Plant Stem Cells in Culture . . . . . . . . . . . . . . . . . . . 209
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
27 Cultured Plant Stem Cells as a Source of Plant Natural
Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
27.1 Plant Stem Cell Extracts as a Source for Regenerative
Therapy of Human Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
28 Mitochondria, a Vital Organelle in Stem Cell Maintenance . . . .... 217
28.1 Mitochondria: Energy Metabolism in Animal Normal
and Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 217
28.2 Mitochondria in Plant Stem Cells . . . . . . . . . . . . . . . . . . . .... 219
28.2.1 Specific Architecture of Mitochondria in Shoot
Apical and Leaf Primordial Meristems . . . . . . . . .... 219
28.2.2 Role of Dysfunctional Mitochondria in Meristem
Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 221
28.3 Molecular Difference in Mitochondria Between Plants
and Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 221
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 223
29 Molecular Similarities Between Plant and Animal Stem Cells . . . . . 225
29.1 Stemness Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
29.2 Stem Cells in Plants and the Method of Clonal Analysis . . . . . 228
29.3 Homologs of Breast Cancer Genes in Plants . . . . . . . . . . . . . . . 230
29.4 Chromatin States in Animal and Plant Stem Cells . . . . . . . . . . 231
29.5 Just Coincidence? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Abstract

Stem cells are a noncoherent group of cells that have little in common. Despite the
fact that these diverse cell types are regarded as belonging to the same category,
they do not share molecular markers. The definition of stemness is therefore
descriptive, relating to potentials of the cells rather than to the actual properties that
they harbor. This situation is confusing and causes unnecessary debates in this field
of research. It is therefore of paramount importance to find a new molecular defi-
nition of stemness that would consist of the cellular machineries, which constitute
the stem cell state. Going through this broad area, the authors are showing inter-
esting aspects of the life and behavior of two kingdoms: animal and plant and their
stem cells.

xvii
Chapter 1
Introduction

Stem cell concept in animals and plants: what is stem cell,

and what is stemness?

Stem cells are nowadays topical research issue due to their potential medical use
and due to the ethical debate around human embryonic stem cells. However, from
biological point of view, stem cells have even wider significance as central players
in the development of complex animal and plant organisms. There are surprising
similarities and striking differences between stem cells found in plants and in
animals. How were they discovered, and what are their general implications for
stem cell biology?
Stem cells function as the source of new cells to build tissues and organs giving
the specific features to the development of diversity of complex biological organ-
isms. They are building blocks for the other cells and tissues in animal and plant
kingdoms. Through their three fundamental features (self-renewal,
proliferation/differentiation, and plasticity) animal stem cells play a crucial role in
regeneration, replacement and repair of damaged and lost tissues. The essential
feature of stemness is the existence of unique phenotypic molecular markers and
specific functionality that can be measured and detected after stem cell has reached
the end in its differentiation: maturation into normal, fully functional cell. This
function is also based upon developmental changes of stem cells through the
process of trans-differentiation, as a part of plasticity feature, meaning the plasticity
is a capacity for trans-differentiation [1–5].
It still remains unrevealed whether stem cells have specific stemness factors that
make them pluripotent, or they are simply any kind of cell that divides and is
blocked from the next step of differentiation.
Furthermore, as Dov Zipori mentioned, once, they are rather state than entity if
we are looking for the strict definition, in time and space (Fig. 1.1) [1–3]. This
would fit very well with their heterogeneity within the specific source [4–6]. And
really, it appears to be a heterogeneous population of cells, residing in the niche,
some of which are very primitive and some of which already express lineage
commitment [1–5]. They are maintained in the niche of different animal organs and
tissues, regulated by spectrum of molecules and signals from different mature

© Springer International Publishing AG 2017 1

M. Pavlović and K. Radotić, Animal and Plant Stem Cells,
DOI 10.1007/978-3-319-47763-3_1
2 1 Introduction

Fig. 1.1 The model of stem cell concept according to Dov Zipori

adjacent cells [4–6]. This cross talk between stem cells and their niches is essential
for their self-renewal capacity [6, 7]. Transcriptional profiling experiments are
important in understanding the molecular nature of both animal and plant stem cells
in depth as they can reveal a reason for heterogeneity.
Relatively recently, the “evolutionary stem cell paradigm” based on the rela-
tionship between anaerobiosis and stemness in animal stem cell population was
proposed by Ivanovic’s group [6].
It is possible (by genetically marking stem cells), to show that nearly all cells of
a mature plant, descend from small groups of stem cells located in their growing
apices, in other words, plant stem cells [8]. Experiments with mutant plants and
selective cell killing have shown that plant stem cells are maintained by signals
from other, adjacent, cells. Thus, this feature is shared with animal stem cells and
helps them to adjust stem cell proliferation to the needs of the plant organism. The
functional similarities of stem cells in plants and animals probably have evolved
independently as solutions to the problem of balancing the need to grow with the
need to produce specialized cells, which often cannot divide any further [8].
Probably the most fundamental questions in comparing animal and plant stem cells
is: Do stem cells work the same way in plants as they do in animals? As indicated,
there are similarities and there are differences, as well.
Thus, the mechanisms that control whether a cell continues to function as a stem
cell or starts to differentiate, for instance, show some similarities in plants and
animals [9]. One gene that is conserved between plants and animals and has a
central role in deciding whether a cell continues to divide or differentiate, encodes
the Retinoblastoma (Rb) protein. When activated, Rb represses genes required to
replicate DNA, in addition to other less well-characterized functions that lead to cell
differentiation [10]. In Arabidopsis, if the gene encoding Rb is inactivated in the
root meristem, the descendants of root stem cells cannot differentiate; conversely, if
1 Introduction 3

Rb is artificially activated in the stem cells, they stop dividing and differentiate [11].
Similarly, Rb appears to promote exit from stem cell state in animals [9]. However,
the regenerative mechanisms, are fundamentally different between two kingdoms
[9, 12]. We are hopeful that this book will answer some aspects of those funda-
mental questions and probably induce further consideration in the light of experi-
ence and scientific vision for future studies.

References

1. Zipori D (2004) The nature of stem cells: state rather than entity. Nat Rev Genet 5:873–878
2. Zipori D (2009) Biology of stem cells and the molecular basis of the stem state. Humana Press
(Springer), New York, p 278
3. Zipori D (2011) Towards a molecular rather than a descriptive definition of stemness. Med Sci
(Paris) 27(3):303–307 (Article in French)
4. Pavlovic M, Balint B (2012) Stem cells and tissue engineering. Springer. ISBN-13:
978-1-4614-5505-9, ISBN:1-4614-5505-7
5. Pavlovic M, Balint B (2015) Bioengineering and Cancer Stem Cell Concept. Springer.
ISBN:978-3-319-25668-9
6. Ivanovic Z, Vlaski-Lafarge M (2015) Anaerobiosis and stemness: an evolutionary paradigm
for therapeutic applications, 1st edn. Academic Press. ISBN-10:0128005408,
ISBN-13:978-0128005408
7. Howel JC, Yoder CM (2003) Adult stem cell plasticity defined. NeoReviews 4(7):e181
8. Sablowski R (2010) Stem cells in plants and animals. Nat Educ 3(9):4
9. He ND, Morrison SJ (2009) Mechanisms of stem cell self-renewal. Annu Rev Cell Dev Biol
25:377–406
10. Burkhart DL, Sage J (2008) Cellular mechanisms of tumour suppression by the retinoblas-
toma gene. Nat Rev Cancer 8:671–682
11. Wildwater M et al (2005) The RETINOBLASTOMA-RELATED gene regulates stem cell
maintenance in Arabidopsis roots. Cell 123:1337–1349
12. Heidstra R, Sabatini S (2014) Plant and animal stem cells: similar yet different. Nat Rev Mol
Cell Biol 15201:301–311
 Part I
Bioengineering and Animal Stem Cells

Mirjana Pavlović
Chapter 2
Current Status and Perspectives in Stem
Cell Research: The Concept of Normal
Stem (NSC) and Cancer Stem Cell (CSC)

Science is organized knowledge. Wisdom is organized life.

Immanuel Kant

Abstract This chapter intends to give the audience a basic idea on normal and
cancer stem cells, as two essential types of cells in stem cell phenomenology
connected by the same feature—“stemness.” This is an introductory conceptual
consideration of what these cells are and where are their similarities and differences.
The chapter discusses normal and abnormal mechanisms that are working in both
type of these cells and make them different. General features of both cell types are
given in condensed manner. The development of methodology for their isolation,
purification, and segregation from other cell types in normal and cancerous tissues
is presented. New methods such as magnetic beads separation, magnetic levitation,
detection of microspheres in CSC, the confirmation of CSC entity through injection
of the cells in NOD/SCID mice, are described. The impact of the concepts upon
development of a new movement in cancer therapy, cancer stem cell targeted
therapy is explained.

2.1 Introduction

Today, stem cell research is dedicated to better understanding of the fundamentals

of normal stem cells (NSC) from different sources as well as cancer stem cell
(CSC) concept. In order to understand CSC concept it is necessary to look into
fundamental features of NSC [1–6].
Both types of cells are connected with the feature of “stemness,” which in
essence covers existence of one primitive cell with potential for renewal and
trans-differentiation into advanced progenitors. And, while in a normal stem cell
these progenitors mature into final, morphologically and functionally determined
products, in CSC (due to activity of other mechanisms) we have resulting, immature
cells of the tissue from which the cancer evolves, as a direct consequence of its
tumorigenic functionality.

© Springer International Publishing AG 2017 7

M. Pavlović and K. Radotić, Animal and Plant Stem Cells,
DOI 10.1007/978-3-319-47763-3_2
8 2 Current Status and Perspectives in Stem Cell Research …

2.2 Stemness, Migration, Circulation, and Seeding

In animals, the signaling molecules used within stem cell niches are often the same
used to organize growth and tissue patterning during embryogenesis, such as
homologues of the Notch, Wingless, and Hedgehog proteins from Drosophila
[1, 2]. However, normal stem cell is seen by the eye of stem cell expert as the state
of stemness, which assumes renewal, proliferation, and differentiation into final
mature state [3]. The property of pluripotency is believed to depend at least in part
on the way the chromatin is organized, that is, how the DNA is packed in the
nucleus and how this affects the access of regulatory proteins to genes required for
cell differentiation. As will be mentioned at the other place, polycomb proteins play
an important role in regulating the chromatin to repress differentiation genes and
therefore maintain the pluripotency of animal stem cells [2]. Cancer cell is also
renewing, but does not differentiate completely, and therefore, never reaches mature
state neither according to morphological nor functional criteria. Both types of cells
give heterogeneous populations with different potential of stemness [4–7]. It seems
that phenomenon of stemness is very well conserved through evolution within the
range from planaria across protozoa and ancestral prokaryotes to mammals in
animal world and in the plants between meristems (stem) cells of plants [8]. Thus,
both sorts of humane stem cells: normal and cancerous, express phenomena of
“resistency to drugs and radioactive irradiation” in the context of early evolutionary
origin of stemness. Migration, circulation, and seeding into distant locations are
also the features of both types of these cells.

2.3 Mobilization

Let us take, for example, normal, adult very small embryonic-like stem cell
(VSEL), originated from hypoxic niches of many organs in mammals including
bone marrow (BM). Morphologically, VSEL cell is smaller several times than
hematopoietic stem cell (HSC), has high N/C ratio and embryonic body with the
cells of all three embryonic leaflets within cytoplasm [9]. Specifically stained with
fluorescent dyes, it shows all markers of stemness and phenotypic difference (with
respect to markers) according to HSC, as well as according to other adult stem cells.
These cells can be mobilized from their “niches” through binding with growth
factor (G-CSF/Neupogen/Filgrastim) upon dosage-dependent manner, and thus
dragged into peripheral blood that can be collected by apheresis. Once dragged into
the blood, these cells can be selected and quantified, by Flow Cytometry (FC),
Fluorescence Acquired—sorting, (FACS) which allows for segregation of “good
mobilizers” and bad ones, thus serving as one of the criteria for stem cell therapy [9,
10]. That feature (mobility) have also CSCs which, even without growth factor
added, migrate/metastasize into distant organs carried by blood as circulating tumor
cells (CTC) [11]. The most probably among CTC, there are CSCs as a minor
fraction within entire population, but with very high tumorigenicity.
2.4 Similarities and Differences Between Embryonic and Adult Stem Cells 9

2.4 Similarities and Differences Between Embryonic

and Adult Stem Cells

Both types are non-specialized cells, therapeutically efficient, and they can cure
some diseases if adequately applied. One of the essential features that distinct adult
from embryonic stem cells is the lack of pluripotent stem cells with unlimited
transformation into cells and organs in the repertoire of adult stem cells, which
disables organism to fully regenerate the organs, but only participate in repair and
repopulation with cellular elements in the case when regeneration would be
desirable [11, 12]. The invention of induced pluripotent stem cell (iPSC) from
somatic cells thus has replaced the need for embryonic stem cell in adult diseases
scenarios [13]. The pluripotent stem cells are mostly characteristics of amphibia and
plant meristem, where they can regenerate in first case limb and in the latter, leaf.

2.5 Similarities and Differences Between Normal

and Cancer Stem Cell

Cancer stem cells (CSCs) are tumor cells that have the principal properties of
self-renewal, clonal tumor initiation capacity, and clonal long-term repopulation
potential [12–22]. CSCs reside in niches, which are anatomically distinct regions
within the tumor microenvironment. These niches maintain the principle properties
of CSCs, preserve their phenotypic plasticity, protect them from the immune sys-
tem, and facilitate their metastatic potential [18, 21]. Since CSCs survive many
commonly employed cancer therapies, we examine the prospects of targeting dif-
ferent components of CSC as preferable therapeutic targets. CSC has similarities
with, and it is also fundamentally different than normal stem cell. Thus, Table 2.1,
summarizes similarities and differences between normal and CSCs. It is clear that
beside the similarities which have been described, there are also fundamental dif-
ferences between them, especially with respect to:
• Homeostatic regulation of surrounding tissue regulation (lost in CSC)
• Growth regulation which is controlled by cellular and molecular components of
the niche in normal stem cells and by internal mechanisms in CSC
• Signal responses upon growth factors that exist in normal and do not exist in
CSC
• Apoptotic responses (existing in NSC and non-existing in CSC)
• Limited replication in NSCs and lack of limitations in CSC)
• Angiogenic supportive network—solid Tu (non-existing in NSCs and a present
in CSC)
• Tissue invasion (noncharacteristic for NSCs and typical for CSCs)
• Differentiation of resulting daughter cells (present in NSC and decreased or
nonexisting in CSCs)
• Aberrant methylation (lacking in NSCs and present in CSCs).
10 2 Current Status and Perspectives in Stem Cell Research …

Table 2.1 Normal stem cells versus cancer stem cells

Normal Stem
Endogenous & Exogenous Cues Cancer Stem Cells
Cells
Homeostatic Regulation of Tissue
Maintained Lost
Regeneration Signals
Genetic Plasticity High High
Growth Regulation Niche-driven Self-sustained
Antigrowth Signal Response Yes No
Apoptosis Signal Response Yes No
Limitation to Replication Yes No
Angiogenic Sustainability No Yes
Tissue Invasion & Metastasis No Yes
Differentiation of Resultant
Yes Impaired or None
Daughter Cells
Aberrant DNA Methylation No Yes
Anaerobic Respiration Yes Yes
Heterogeneous population of cells Yes Yes
Different Sets of miRNA involved Yes Yes

Further comparison indicates:

• hypoxic nature (anaerobic respiration) of both types of stem cells,
• heterogenous populations, and
• different sets of miRNA with participation in epigenetic regulation.

2.6 Resume Based upon Similarities and Differences

Between NSCs and CSCs

CSC of mammals including humans, shares a certain number of characteristics with

the adult normal stem cells (NSC). Although CSCs are present with participation of
only 0.1% of the whole tumor, they can regenerate original tumor and migrate
through blood vessels spreading the cancer into secondary locations [20]. It is very
difficult to localize this thin cell fraction within the tumor, since for a long time the
scientists did not know about their existence, and therefore, there were no methods
developed to the work with them. It was looking like a needle in a hay stuck.
2.6 Resume Based upon Similarities and Differences Between NSCs and CSCs 11

However, with the application of magnetic beads today the isolation of purified
CSCs samples especially after specific markers were determined and is a relatively
easy procedure.

2.7 What and Which Molecular Markers of CSCs

We Know Today?

Beside genetic, epigenetic, and biochemical markers, in the past decade almost all
phenotypic markers of protein nature, are identified—mostly of the cluster of
designation type (CD), which do not change during cancerogenesis and perpetuate
through clonal expansion, building up characteristic phenotype and funding the
platform for precise isolation, examination, and targeted treatment (Table 2.1).

2.8 Isolation of CSCs Using Magnetic Beads

Isolation is now possible by magnetic beads coated with antibodies raised against
specific CSC markers and by using the magnet which drags the cell/bead complex
bound to the magnetic beads through antibodies. The cells dragged to the wall of
the tube by the magnet from outside, are washed, and separated from antibodies
with appropriate buffer, being now purified in the solution, ready for further
expansion if needed or are instantly used, if planned so (Quiagen).

2.9 Theories of Origin of CSCs

In the studies related to cancer there are two fundamentally different theoretical
explanations for emergence of the CSCs, none of which can explain all the features
of the cancer: (1) Stochastic theory or the Theory of clonal evolution and hierarchic
theory or the Theory of cancer stem cell [22]. According to the first theory, clonally
expanded cells originate from one clone, are all equally changed/mutated and of the
same tumorigenicity, while according to the Hierarchic Theory, only one cell—
CSC is on the top and it is orchestrating further development of tumorigenicity.
Surrounding cells are of different degrees of differentiation, but they do not have
that tumorigenic strength to renew the cancer. Both theories are equally inspiring
for further understanding of the concept of targeted therapy. However, in order to
develop more efficient treatment of the cancer, it is of critical importance to
determine which of the theories is correct [22–25]. If most of the cells can pro-
liferate and metastasize, then, virtually, all the cells must be eradicated in order for
12 2 Current Status and Perspectives in Stem Cell Research …

the disease to be cured, while the specific elimination of CSC would be enough if
the theory of clonal evolution is only myth.

2.10 Current Tests for Determination of the Presence

of CSCs

Today we have two types of tests for determination of the presence of CSCs:
in vitro and in vivo. In vivo is already described as the repopulation of breast and
pancreas tumor tissue built up from only CSCs in the body of immunodeficient
NOD/SCID mice, while in vitro tests detect occurrence of microspheres during the
growth of CSCs in colonies [23–28].

2.11 Sorting and Isolation of CTC Using the Method

of Magnetic Levitation

Before we continue with CSCs, let us see what are circulating tumor cells
(CTC) and what their significance is. CTCs originate from a primordial tumor mass
and they are entering the peripheral circulation. CTCs are crucial for understanding
the biology of metastasis, and they are also playing a vital role in diagnosis,
prognosis, follow-up of the disease and individual therapy [22]. However, they are
also rare in blood and due to that, problematic for isolation. Besides, viability of
CTC can be easily compromised under high stress while we are deliberating them
from the surface. Their heterogeneity regarding expression of the biomarkers makes
their isolation very challenging; efficacy of isolation and specificity of contempo-
rary applications is in need for improvement. Nanostructured substrates appeared as
promising biosensor platform since they produce better isolation sensitivity with
regards to the price of isolation of CTCs. Method of magnetic levitation has,
however, emerged as one of the newest approaches to isolation of these cells [22].
The immediate question: are among those cells also CSCs, is not yet answered.
Magnetic levitation, or magnetic suspension is the method by which the object is in
suspension without any other support except the support of the magnetic force [22].
Magnetic force is used to contradict the effect of gravitational acceleration and any
kind of acceleration. Cells have components of micro- and nanoscale as well as
material contributing to their fundamental features of density and magneticity. Both
types of cells, eukaryotic and prokaryotic, can levitate and every cell will have its
unique levitation profile [22]. That is how CTCs differ from other cells by density
and magnetic features, which helps their segregation from overall mass. This is
sorting of cancerous cells without labeling and eventual centrifugation since during
centrifugation there is the release of ROS and fragmentation of DNA,
2.11 Sorting and Isolation of CTC Using the Method of Magnetic Levitation 13

consecutively. The costly aspect of labeled antibodies today is not such a problem,
since there are many methods for expansion of normal and cancer stem cells one of
which is natural—hypoxia [1].

2.12 The Emergence/Origin and Development of CSC:

Mechanisms

What mechanisms are leading to establishment of CSC?

a. Genetic
Definitely, it is the first that we would think about, but here we shall not talk about
genetic mechanisms since they are field per se and require another book.
Retinoblastoma gene, genes of Lynch syndrome, the genes changed in the presence
of specific viruses such as Ebstein-Barr and Varicella Virus, are all genetic mecha-
nisms working on the design of the CSC [28–33].
b. Epigenetic
Mammalian embryonic development is tightly regulated process that, from a single
zygote, produces a large number of cell types with widely divergent functions.
Distinct cellular differentiation programmes are facilitated by tight transcriptional
and epigenetic regulation [28]. However, the contribution of epigenetic regulation
to tissue homeostasis after the completion of development is less well understood.
The research on the effects of epigenetic dysregulation on adult stem cell function is
in progress. Current evidence indicates that depending on the tissue type and the
epigenetic regulator affected, the alterations range from minor to stem cell mal-
function and disruption of tissue homeostasis, which may predispose to diseases
such as cancer. Therefore, maybe more intriguing then genetic are these epigenetic
mechanisms in the range from aberrant methylation of DNA to the histone modi-
fications [28–33]. Epigenetic phenomenology is a broad term and these mecha-
nisms broadly contribute to emergence of CSC [33–49].

2.13 Aberrant Methylation

DNA methylation has an important role in epigenetic regulation of genes during

development and during different illnesses. DNA methylation is the process by
which methyl groups are binding to DNA. We know that aberrant methylation is
most frequently described as hyper-or hypomethylation. Methylation is also pos-
sible in the normal non-methylated CpG islands of the genes, which are so
becoming dysfunctional and can influence formation of CSC. Methyl group is on
the wrong site: on cytosine instead of thymine.
14 2 Current Status and Perspectives in Stem Cell Research …

2.14 What Do We Need to Do with the Sum

of Information?

Computer is a great help in organizing information of this kind and here is a

conceptual proposal of the work on sorting of information which would help pre-
diction, and planning of the work on detection and discoveries of cancerous lesions
[49]. Computational modeling is the key factor in the understanding of biological
systems. The interplay of computational modeling and experimental data already
exist for the plants. Some effort is necessary to elevate the knowledge on animal SC
to that extant in order to be able to answer deep questions such as: which signals are
involved in the maintaining of stem cell network organization, how are asymmetric
stem cell division and renewal achieved, which are underlying molecular mecha-
nisms related to stem cell progeny differentiation?

2.15 How Was the Concept of CSC Therapy Designed?

Analyzing research during past few years, it is estimated that situation is mature
enough for establishment of new concept, e.g., targeted cancer stem cell therapy
[28–49]. While many investigators in the field of tumor therapy continue to upgrade
the existing models of chemotherapy and radiation, in an effort to improve their
efficacy by increasing their specificity, a particular cadar of investigators is taking a
new road—directed toward CSC [40–42]. The starting contribution by Dick (1997)
has established evident criteria of the CSC concept, by using NOD/SCID mouse
model). Dick has successfully transplanted stem cells of acute myeloid leukemia
(AML) into NOD/SCID mouse model where the AML human cells were regen-
erated (1994). Classical experiments of Al Hajj (breast carcinoma) and Li (pan-
creatic cancer) have supported concept even more [26–28]. Fluorescent labeling has
detected clonally propagated, stable markers, and high tumorigenicity with resis-
tance to therapy—typical signs of functionality of those, otherwise, rare cells (0.1%
total population). Rarity of CSCs—requires development of therapeutic strategies
different than conventional [49].

References

1. Orford KW, Scadden DT (2008) Deconstructing stem cell self-renewal: genetic insights into
cell-cycle regulation. Nat Rev Genet 2:115–128
2. He S, Nakada D, Morrison SJ (2009) Mechanisms of stem cell self-renewal. Annu Rev Cell
Dev Biol 25:377–406
3. Zipori D (2004) The nature of stem cells: state rather than entity. Nat Rev Genet 5(11):1471
4. Ramalho-Santos M, Yoon S, Matsuzaki et al (2002) Stemness: transcriptional profiling of
embryonic and adult stem cells. Science 298(5593):597
References 15

5. Reya T, Morrison SJ, Clarke MF (2001) Stem cells, cancer, and cancer stem cells. Nature 414
(6859):105
6. Nguyen LV et al (2012) Cancer stem cells: an evolving concept. Nat Rev Cancer 12(2):
133–143
7. Jain M et al (2012) Highlights from Recent Cancer Literature. Cancer Res 72:13
8. Ivanovic Z, Vlaski-Lafarge M (2016) Anaerobiosis and stemness: an evolutionary paradigm
for therapeutic applications, 1st edn. Academic Press, p 2015, ISBN-10:0128005408,
ISBN-13:978-0128005408
9. Ratajczak MZ (2014) Adult stem cell therapies: alternatives to plasticity. Stem Cell Biology
and regenerative medicine. ISBN 978-1-4939-1001-4
10. Ivanovic Z, Kovacevic-Filipovic M, Jeanne M, Ardilouze L, Bertot A, Szyporta M,
Hermitte F, Lafarge X, Duchez P, Vlaski M, Milpied N, Pavlovic M, Praloran V, Boiron JM
(2010) CD34+ cells obtained from “good mobilizers” are more activated and exhibit lower
ex vivo expansion efficiency than their counterparts from “poor mobilizers”. Transfusion 50
(1):120–127
11. Durmusa NGH, Tekinc C, Guvenc S, Demirci U et al (2015) Magnetic levitation of single
cells. PNAS published on line E3661-E3668
12. Gil J, Stembalska A, Pesz KA, Sasiadek MM (2008) Cancer stem cells: the theory and
perspectives in cancer therapy. J Appl Genet 49(2):193
13. Singh A, Settleman J (2010) EMT, cancer stem cells and drug resistance: an emerging axis of
evil in the war on cancer. Oncogene 29(34):4741–4751
14. Welte Y, Adjaye J, Lehrah H, Regenbrecht CRA (2010) Cancer stem cells in solid tumors:
elusive or illusive? Welte Cell Commun Signal 8:6
15. Greaves M (2010) Cancer stem cell: back to Darvin? Semin Cancer Biol 20:65–70
16. William JL (2012) Cancer stem cells. Clin Lab Sci J Am Soc Med Technol 25(1):50
17. Gupta PB et al (2009) Identification of selective inhibitors of cancer stem cells by
high-throughput screening. Cell 4138:645–659
18. Gupta PB, Chaffer CL, Weinberg RA (2009) Cancer stem cells: mirage or reality? Nat Med
15(9):1010–1012
19. Diehn N et al (2009) Association of reactive oxygen species levels and radioresistance in
cancer stem cells. Nature 458(7239):780–783
20. Singh A, Settleman J (2010) EMT, cancer stem cells and drug resistance: an emerging axis of
evil in the war on cancer. Oncogene 29(34):4741–4751
21. Rosen JM, Jordan CT (2009) The increasing complexity of the cancer stem cell paradigm.
Science 324(5935):1670–1673
22. Shackleton M et al (2009) Heterogeneity in cancer: cancer stem cells versus clonal evolution.
Cell 138(5):822–829
23. Hirsch HA et al (2009) Metformin selectively targets cancer stem cells, and acts together with
chemotherapy to block tumor growth and prolong remission. Cancer Res 69(19):7507–7511
24. Jamieson CHM et al (2009) Methods of identifying and isolating stem cells and cancer stem
cells. US Patent No. 7,622,255
25. Lapidot T et al (1994) Cell initiating human acute myeloid leukaemia after transplantation
into SCID mice. Nature 367:645–648
26. Li C, Heidt DG, Dalerba P et al (2007) Identification of pancreatic cancer stem cells. Cancer
Res 67(3):1030
27. Li C, Lee C, Simeone DM (2009) Identification of human pancreatic cancer stem cells.
Cancer Stem Cells. Humana Press, pp 161–173
28. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective
identification of tumorigenic breast cancer cells. Proc Natl Acad Sci 100(7):3983
29. Shimono Y et al (2009) Down regulation of miRNA-200c links breast cancer stem cells with
normal stem cells. Cell 138(3):592–603
30. Charafe-Jauffret E et al (2009) Breast cancer cell lines contain functional cancer stem cells
with metastatic capacity and a distinct molecular signature. Cancer Res 69(4):1302–1313
16 2 Current Status and Perspectives in Stem Cell Research …

31. Christophe G et al (2010) CXCR1 blockade selectively targets human breast cancer stem cells
in vitro and in xenografts. J Clin Investig 120(2):485
32. Neve RM, Chin K, Fridlyand J et al (2006) A collection of breast cancer cell lines for the
study of functionally distinct cancer subtypes. Cancer Cell 10(6):515
33. Zhao C et al (2009) Hedgehog signaling is essential for maintenance of cancer stem cells in
myeloid leukaemia. Nature 458(7239):776–779
34. Collins AT, Berry PA, Hyde C et al (2005) Prospective identification of tumorigenic prostate
cancer stem cells. Cancer Res 65(23):10946
35. Jachetti E, Mazzoleni S, Grioni M et al (2013) Prostate cancer stem cells are targets of both
innate and adaptive immunity and elicit tumor-specific immune responses. OncoImmunology
2(5)
36. Singh SK, Hawkins C, Clarke ID et al (2003) Identification of a cancer stem cell in human
brain tumors. Cancer Res 63(18):5821
37. Vermeulen L et al (2010) Wnt activity defines colon cancer stem cells and is regulated by the
microenvironment. Nat Cell Biol 12(5):468–476
38. Puglisi MA, Tesori V, Lattanzi W, Gasbarrini GB, Gasbarrini A (2001) Colon cancer stem
cells: controversies and perspectives. World J Gastroenterol 19(20):2997
39. Shukrun R, Shakked NP, Dekel B (2013) Targeted therapy aimed at cancer stem cells: Wilm’s
tumor as an example. Pediatr Nephrol 29(5):815–23
40. Fisher B, Wolmark N, Rockette H, Redmond C et al (1988). Postoperative adjuvant
chemotherapy or radiation therapy for rectal cancer: results from NSABP Protocol R-011.
J Natl Cancer I.8(1):21
41. Brannon-Peppas L, Blanchette JO (2004) Nanoparticle and targeted systems for cancer
therapy. Adv Drug Deliver Rev 56(11):1649
42. Davis ME, Chen Z, Shin DM (2008) Nanoparticle therapeutics: an emerging treatment
modality for cancer. Nat Rev Drug Discov 7(9):771
43. Gaitanis A, Staal S (2010) Liposomal doxorubicin and nab-paclitaxel: nanoparticle cancer
chemotherapy in current clinical use. Cancer Nanotechnol 624:385
44. Gradishar WJ, Rjulandin S, Davidson N et al (2005) Phase III trial of nanoparticle
albumin-bound paclitaxel compared with polyethylated castor oil–based paclitaxel in women
with breast cancer. J Clin Oncol 23(31):7794
45. Farokhzad OC, Jon S, Khademhosseini A et al (2004) Nanoparticle-aptamer bioconjugates a
new approach for targeting prostate cancer cells. Cancer Res 64(21):7668
46. Kirson ED, Gurvich Z, Schneiderman R et al (2004) Disruption of cancer cell replication by
alternating electric fields. Cancer Res 64(9):3288
47. Dylla SJ, Park I-K, Gurney AL (2009) Cancer stem cells. Emerging technology platforms for
stem cells, vol 129, no. 09, p 129
48. Pavlovic M, Balint B (2012) Stem cells and tissue engineering. Springer.
ISBN-13:978-1-4614-5505-9, ISBN:1-4614-5505-7
49. Pavlovic M, Balint B (2015) Bioengineering and cancer stem cell concept. Springer.
ISBN:978-3-319-25668-9
Another random document with
no related content on Scribd:
offered him entertainment, which he scrupulously refused. ‘Then I
went to my lord and said, I was sent by God to cast the devil out of
his son, by faith in Christ. He seemed to be like that lord who had the
charge of the gate in Samaria. Then I said to him: “My lord, do you
not believe me?” Then he bade me go and 1720.
speak to many ministers that was near by
him; but I said I was not sent to them. Then he went to them himself,
and spoke to them what I said; but they would not hear of it; so I
went to three witches and a warlock, to examine them, in sundry
places. Two of them denied, and two of them confessed. I have no
time to relate here all that I said to them, and what they said; but I
asked them, “When they took on in that service?” The wife said:
“Many years;” and the man said: “It was ten years to him.” Then I
asked the wife: “What was her reason for taking on with the devil?”
And she said: “He promised her riches, and she believed him.” Then
she called him many a cheat and liar in my hearing. Then I went to
the man, because he was a great professor, and could talk of religion
with any of the parish, as they that was his neighbours said, and he
was at Bothwell Bridge fighting against the king; and because of that,
I desired to ask questions at him; but my lord’s officer said: “His lord
would not allow me.” I said I would not be hindered neither by my
lord nor by the devil, before many there present. Then I asked:
“What iniquity he found in God, that he left his service?” He got up
and said: “Oh, sir, are ye a minister?” So ye see the devil knows me to
be a minister better than the magistrates. He said: “He found no fault
in God; but his wife beguiled him;” and he said: “Wo be to the
woman his wife!” and blamed her only, as Adam did his wife, and the
woman blamed the devil; so ye see it is from the beginning. This is a
caution to us all never to hearken to our wives except they have
Scripture on their side. Then I asked at him: “Did he expect heaven?”
“Yes,” said he. Then I asked at him if he could command the devil to
come and speak to me? He said: “No.” Then I said again: “Call for
him, that I may speak with him.” He said again: “It was not in his
power.” Then my lord sent more servants, that hindered me to ask
any more questions, otherwise I might have seen the devil, and I
would have spoken about his son.’[539]
On this fast-day, a sermon was preached in Calder kirk by the Rev.
Mr John Wilkie, minister of Uphall, the alleged sorcerers being all
present. Lord Torphichen subsequently caused the discourse to be
printed. His boy in time recovered, and going to sea, rose by merit to
the command of an East Indiaman, but perished in a storm. It brings
us strangely near to this wild-looking affair, 1720.
that the present tenth Lord Torphichen
(1860) is only nephew to the witch-boy of Calder.

The exportation of some corn from Feb. 5.

Dundee being connected unfavourably in
the minds of the populace with a rise of the markets, a tumult took
place, with a view to keeping the grain within the country. The mob
not only took possession of two vessels loaded with bear lying in the
harbour, the property of Mr George Dempster, merchant, but
attacked and gutted the house, shops, cellars, and lofts of that
gentleman, carrying off everything of value they contained, including
twelve silver spoons, a silver salver, and two silver boxes, one of
them containing a gold chain and twelve gold rings, some hair ones,
and others set with diamonds. Dempster advertised that whoever
shall discover to him ‘the havers of his goods,’ should have ‘a
sufficient reward and the owner’s kindness, and no questions
asked.’[540]
A similar affair took place at Dundee nine years later. The country-
people in and about the town then ‘carried their resentment so high
against the merchants for transporting of victual, that they furiously
mobbed them, carried it out of lofts, and cut the sacks of those that
were bringing it to the barks.’[541]

Died, Alexander Rose, who had been Mar. 20.

appointed Bishop of Edinburgh just before
the Revolution. He was the last survivor of the unfortunate
episcopate of Scotland, and also outlived all the English bishops who
forfeited their sees at the Revolution. Though strenuous during all
these thirty-one years as a nonjuror—for which in 1716 he was
deprived of a pension assigned him by Queen Anne—he is testified to
by his presbyter Robert Keith as ‘a sweet-natured man.’[542] His
aspect, latterly, was venerable, and the gentleness of his life secured
him the respect of laymen of all parties. Descended of the old House
of Kilravock, he had married a daughter of Sir Patrick Threipland of
Fingask, a family which maintained fidelity to the House of Stuart
with a persistency beyond all parallel, never once swerving in
affection from the days of the Commonwealth down to recent times.
‘Mr John Rose, son of the Bishop of Edinburgh,’ is in the list of rebels
who pled guilty at Carlisle in December 1716. The good bishop,
having come to his sister’s house in the Canongate, to see a brother
who was there lying sick, had a sudden fainting-fit, and calmly
breathed his last. He was buried in the 1720.
romantic churchyard of Restalrig, which
has ever since been a favourite resting-place of the members of the
Scottish Episcopal communion.

There was a jubilation in Edinburgh on Apr. 28.

what appears to us an extraordinary
occasion. The standing dryness between the king and Prince of Wales
had come to a temporary end. The latter had gone formally to the
palace, and been received by his father ‘with great marks of
tenderness’ [the king was sixty, and the prince thirty-seven]. At a
court held on the occasion, ‘the officers and servants on both sides,
from the highest to the lowest, caressed one another with mutual
civilities,’ and there were great acclamations from the crowd outside.
The agreeable news having been received in the northern metropolis,
the magistrates set the bells a-ringing, and held an entertainment for
all persons of note then in town, at which loyal toasts were drunk,
with feux de joie from the City Guard. Demonstrations of a like
nature took place at Glasgow—the music-bells rung—the stairs of the
town-house covered with carpets—toast-drinking—and discharges of
firearms from the Earl of Stair’s regiment. Nor was there a similar
expression of joy wanting even in the Cavalier city of Aberdeen—
where, however, such expressions were certainly more desirable.[543]

One is startled at finding in the May 2.

Edinburgh Evening Courant of this date
the following advertisement: ‘Taken up a stolen negro: whoever owns
him, and gives sufficient marks of his being theirs, before the end of
two weeks from the date hereof, may have him again upon payment
of expenses laid out upon him; otherwise the present possessor must
dispose of him at his pleasure.’
Yet true it is that colonial negro slaves who had accompanied their
masters to the British shores, were, till fifty-five years after this
period, regarded as chattels. One named Joseph Knight came with
his master, John Wedderburn, Esq., to Glasgow in 1771, and
remained with him as his bound slave for two years. A love-affair
then set the man upon the idea of attempting to recover his liberty,
which a recent decision by Lord Mansfield in England seemed to
make by no means hopeless. With the help of friends, he carried his
claim through a succession of courts, till a 1720.
decision of the Court of Session in 1775
finally established that, however he might be a slave while in the
West Indies, he, being now in Scotland, was a free man.

Horse-racing had for many years been considerably in vogue in

Scotland. There were advertised in the course of this year—a race at
Cupar in Fife; one at Gala-rig, near Selkirk, for a piece of plate given
by the burgh, of £12 value; a race at Hamilton Moor for £10; a race
on Lanark Moor for a plate of £12, given by the burgh; a race on the
sands of Leith for a gold cup of about a hundred guineas value, and
another, for a plate of £50 value, given by the city of Edinburgh;
finally, another race at Leith for a silver punch-bowl and ladle, of £25
value, given by the captains of the Trained Bands of Edinburgh—the
bowl bearing an inscription which smacks wonderfully like the
produce of the brain of Allan Ramsay:
‘Charge me with Nantz and limpid spring,
Let sour and sweet be mixt;
Bend round a health syne to the king,
To Edinburgh captains next,
Wha formed me in sae blithe a shape,
And gave me lasting honours;
Take up my ladle, fill and lap,
And say: “Fair fa’ the donors.”’

The genius of Scott has lent an Oct. 17.

extraordinary interest to a murder
perpetrated at this date. Nicol Mushet appears to have been a young
man of some fortune, being described as ‘of Boghall,’ and he had
studied for the profession of a surgeon; but for some time he had led
an irregular and dissipated life in Edinburgh, where he had for one of
his chief friends a noted profligate named Campbell of Burnbank,
ordnance store-keeper in the Castle. The unhappy young man was
drawn into a marriage with a woman named Hall, for whom he soon
discovered that he had neither affection nor respect; and he then
became so eager to be free from the connection, as to listen to a
project by Burnbank for obtaining a divorce by dishonourable
means. An obligation passed between the parties in November 1719,
whereby a claim of Burnbank for an old debt of nine hundred merks
(about £50) was to be discharged by Mushet, as soon as Burnbank
should be able to furnish evidence calculated to criminate the
woman. Burnbank then deliberately hired a wretch like himself, one
Macgregor, a teacher of languages, to enter into a plot for placing
Mrs Mushet in criminative circumstances; 1720.
and some progress was made in this plan,
which, however, ultimately misgave. It was then suggested by
Burnbank that they should go a step further, and remove the woman
by poison. One James Mushet and his wife—a couple in poor
circumstances—readily undertook to administer it. Several doses
were actually given, but the stomach of the victim always rejected
them. Then the project for debauching her was revived, and Mushet
undertook to effect it; but it was not carried out. Dosing with poison
was resumed, without effect; other plans of murder were considered.
James Mushet undertook to knock his sister-in-law on the head for
twenty guineas, and got one or two in hand by anticipation, part of
which he employed in burying a child of his own. These diabolically
wicked projects occupied Mushet, his brother, his brother’s wife, and
Burnbank, in the Christian city of Edinburgh, during a course of
many months, without any one, to appearance, ever feeling the
slightest compunction towards the poor woman, though it is
admitted she loved her husband, and no real fault on her side has
ever been insinuated.
At length, the infatuated Nicol himself borrowed a knife one day,
hardly knowing what he wanted it for, and, taking his wife with him
that night, as on a walk to Duddingston, he embraced the
opportunity of killing her at a solitary place in the King’s Park. He
went immediately after to his brother’s, to tell him what he had done,
but in a state of mind which made all afterwards seem a blank to
him. Next morning, the poor victim was found lying on the ground,
with her throat cut to the bone, and many other wounds, which she
had probably received in struggling with her brutal murderer.
Mushet was seized and examined, when he readily related the
whole circumstances of the murder and those which had led to it. He
was adjudged to be hanged in the Grassmarket on the ensuing 6th of
January. His associate Burnbank was declared infamous, and
sentenced to banishment. The common people, thrilled with horror
by the details of the murder, marked their feelings in the old national
mode by raising a cairn on the spot where it took place; and Mushet’s
Cairn has ever since been a recognised locality.[544]

There was published this year in Edinburgh a small treatise at the

price of a shilling, under the title of Rules of 1720.
Good Deportment and Good Breeding. The
author was Adam Petrie, who is understood as having commenced
life as domestic tutor in the family of Sinclair of Stevenston, and to
have ended it in the situation of a parish schoolmaster in East
Lothian. He dedicated his treatise to the magistrates of Edinburgh,
acknowledging them to be ‘so thoroughly acquainted with all the
steps of civility and good breeding, that it is impossible for the least
misrepresentation of them to escape your notice.’
Adam sets out with the thesis, that ‘a courteous way gilds a denial,
sweetens the sharpness of truth ... sets off the defects of reason,
varnishes slights, paints deformities ... in a word, disguises
everything that is unsavoury.’ Everything, however, required to have
some reference to religion in that age, and Adam takes care to
remind us that civility has a divine basis, in the injunctions, ‘Be
courteous to all men,’ and ‘Give honour to whom honour is due.’
As to ordinary demeanour, Mr Petrie was of opinion that ‘a
gentleman ought not to run or walk too fast in the streets, lest he be
suspected to be going a message.’ ‘When you walk with a superior,
give him the right hand; but if it be near a wall, let him be next to it.’
The latter rule, he tells us, was not yet followed in Scotland, though
established in England and Ireland. ‘When you give or receive
anything from a superior, be sure to pull off your glove, and make a
show of kissing your hand, with a low bow after you have done.’ In
this and some other instances, it strikes us that a too ceremonious
manner is counselled; but such was the tendency of the time. There
was, however, no want of rude persons. ‘I have,’ says Adam, ‘seen
some noblemen treat gentlemen that have not been their
dependents, and men of ancienter families than they could pretend
to, like their dependents, and carry to the ambassadors of Jesus
Christ as if they had been their footmen.’
Mr Petrie deemed it proper not to come amongst women abruptly,
‘without giving them time to appear to advantage: they do not love to
be surprised.’ He also thought it was well ‘to carry somewhat
reserved from the fair sex.’ One should not enter the house or
chamber of a great person with a great-coat and boots, or without
gloves—though ‘it is usual in many courts that they deliver up their
gloves with their sword before they enter the court, because some
have carried in poison on their gloves, and have conveyed the same
to the sovereign that way.’ Women, on their 1720.
part, are equally advised against
approaching superiors of their own sex with their gown tucked up.
‘Nor,’ says he, ‘is it civil to wear a mask anywhere in company of
superiors, unless they be travelling together on a journey.’ In that
case, ‘when a superior makes his honours to her, she is to pull off her
mask, and return him his salute, if it be not tied on.’
There is a good deal about the management of the handkerchief,
with one general recommendation to ‘beware of offering it to any,
except they desire it.’ We also are presented with a rule which one
could wish to see more universally observed than it is, against
making any kind of gesticulations or noises in company.
There were customs of salutation then, which it is now difficult to
imagine as having ever been practised. ‘In France,’ says Adam, ‘they
salute ladies on the cheek; but in Britain and Ireland they salute
them on the lips.’ Our Scottish Chesterfield seems to have felt that
the custom should be abated somewhat; or perhaps it was going out.
‘If,’ says he, ‘a lady of quality advance to you, and tender her cheek,
you are only to pretend to salute her by putting your head to her
hoods: when she advances, give her a low bow, and when you retreat,
give her another.’ He adds: ‘It is undecent to salute ladies but in
civility.’
Formulæ of address and for the superscription of letters are fully
explained; but Adam could not allow ‘the Right Reverend Father in
God the Lord Bishop of London’ to pass as an example of Episcopal
style, without remarking that many have not ‘clearness’ to use such
titles. Adam is everywhere inclined to an infusion of piety. He
denounces ‘an irreligious tippling’ of coffee, tea, and chocolate,
which he observed to be continually going on in coffee-houses,
‘because not one in a hundred asks a blessing to it.’ He is very much
disposed, too, to launch out into commonplace morals. Rather
unexpectedly in a lover of the politenesses, he sets his face wholly
against cards and dice, stage-plays, and promiscuous dancing,
adducing a great number of learned references in support of his
views.
The editor of a very scarce reprint of this curious volume,[545]
remarks that, from the manifest sincerity of the author’s delineations
of good breeding, and the graphic character of many of his scenes, it
may fairly be presumed that they were painted from nature. We are
told by the same writer, that ‘Helen Countess of Haddington, who
died in 1768, at the advanced age of ninety-one, and to whom Petrie
was well known, used to describe his own 1720.
deportment and breeding as in strict
accordance with his rules.’

James Dougal, writing the news of 1721. Jan. 4.

Edinburgh to his friend Wodrow at
Eastwood, has a sad catalogue to detail. ‘There was four pirates
hanged at Leith this day ... very hardened. They were a melancholy
sight, and there is three to be hanged next Wednesday. Nicol Mushet
is to be hanged on Friday ... for murdering his wife: he appears to be
more concerned than he was before. Ane woman brought from Leith
is to die the first Wednesday of February for putting down
[destroying] a child. Another man is laid up in prison, that is thought
to have murdered his wife. The things falling out now are very
humbling.’
 He goes on to tell that several persons ‘in trouble of mind’ are
frequently prayed for in Edinburgh churches. ‘But they do not name
them but after such a manner—A man there is in such trouble (or a
woman), and desires the congregation to praise God with them for
signal deliverance that the Lord hath given them from great troubles
that they have been in.’
The end of the letter is terrible: ‘There is some of the Lord’s people
that lives here, that are feared for melancholy days, iniquity doth so
abound, and profanity; and if there were not a goodly remnant in this
town, it would sink.’[546]

A sperm whale, ‘the richest that has ever Jan. 30.

been seen in this country,’ was advertised in
the Courant as having come ashore in the Firth of Forth near
Culross, and to be sold by public roup.
At the end of June 1730, three wounded whales ran ashore at
Kilrenny in Fife, on the property of Mr Bethune of Balfour. The
produce, consisting of a hundred and forty-six barrels of speck, or
blubber, and twenty-three barrels of spermaceti speck, was
afterwards advertised for sale.

With regard to several of the forfeited Oct.

estates which lay in inaccessible situations
in the Highlands, the Commissioners had been up to this time
entirely baffled, having never been able even to get surveys of them
effected. In this predicament in a special manner lay the immense
territory of the Earl of Seaforth, extending from Brahan Castle in
Easter Ross across the island to Kintail, and 1721.
including the large though unfertile island
of Lewis. The districts of Lochalsh and Kintail, on the west coast, the
scene of the Spanish invasion of 1719, were peculiarly difficult of
access, there being no approach from the south, east, or north,
except by narrow and difficult paths, while the western access was
only assailable to a naval force. To appearance, this tract of ground,
the seat of many comparatively opulent ‘tacksmen’ and cattle-
farmers, was as much beyond the control of the six Commissioners
assembled at their office in Edinburgh, as if it had been amongst the
mountains of Tibet or upon the shores of Madagascar.
During several years after the insurrection, the rents of this district
were collected, without the slightest difficulty, for the benefit of the
exiled earl, and regularly transmitted to him. At one time, a
considerable sum was sent to him in Spain, and the descendants of
the man who carried it continued for generations to bear ‘the
Spanyard’ as an addition to their name.[547] The chief agent in the
business was Donald Murchison, descendant of a line of faithful
adherents of the ‘high chief of Kintail’—the first of whom, named
Murcho, had come from Ireland with Colin the son of Kenneth, the
founder of the clan Mackenzie in the thirteenth century. The later
generations of the family had been intrusted in succession with the
keeping of Ellan Donan Castle, a stronghold dear to the modern
artist as a picturesque ruin, but formerly of serious importance as
commanding a central point from which radiate Loch Alsh and Loch
Duich, in the midst of the best part of the Mackenzie country. Donald
was a man worthy of a more prominent place in his country’s annals
than he has yet attained; he acted under a sense of right which,
though unfortunately defiant of acts of parliament, was still a very
pure sense of right; and in the remarkable actions which he
performed, he looked solely to the good of those towards whom he
had a feeling of duty. A more disinterested hero—and he was one—
never lived.
When Lord Seaforth brought his clan to fight for King James in
1715, Donald Murchison and a senior brother, John, went as field-
officers of the regiment—Donald as lieutenant-colonel, and John as
major. Sir Roderick I. Murchison, the distinguished geologist, great-
grandson of John, possesses a large ivory and silver ‘mill,’ which
once contained the commission sent from France to Donald, as
colonel, bearing the inscription: ‘James 1721.
Rex: Forward and Spare not.’ John fell at
Sheriffmuir, in the prime of life; Donald, returning with the remains
of the clan, was intrusted by the banished earl with the management
of estates no longer legally his, but still virtually so, through the
effect of Highland feelings in connection with very peculiar local
circumstances. And for this task Donald was in various respects well
qualified, for, strange to say, the son of the castellan of Ellan Donan
—the Sheriffmuir colonel—had been ‘bred a writer’ in Edinburgh,
and was as expert at the business of a factor or estate-agent as in
wielding the claymore.[548]
In bold and avowed insubordination to the government of George
the First, the Mackenzie tenants continued for ten years to pay their
rents to Donald Murchison, on account of their forfeited and exiled
lord, setting at nought all fear of ever being compelled to repeat the
payment to the commissioners.
In 1720, these gentlemen made a movement for asserting their
claims upon the property. In William Ross of Easterfearn, and
Robert Ross, a bailie of Tain, they found two men bold enough to
undertake the duty of stewardship in their behalf over the Seaforth
property, and also the estates of Grant of Glenmorriston and
Chisholm of Strathglass. Little, however, was done that year beyond
sending out notices to the tenants, and preparing for strenuous
measures to be entered upon next year. The stir they made only
produced excitement, not dismay. Some of the duine-wassels from
about Loch Carron, coming down with their cattle to the south-
country fairs, were heard to declare that the two factors would never
get anything but leaden coin from the Seaforth tenantry. Donald was
going over the whole country, shewing a letter he had got from the
earl, encouraging his people to stand out; at the same time telling
them that the old countess was about to come north with a factory
for the estate, when she would allow as paid any rents which they
might now hand to him. The very first use to be made of this money
was, indeed, to bring both the old and the young countesses home
immediately to Brahan Castle, where they would live as they used to
do. Part of the funds thus acquired, he used in keeping on foot a
party of about sixty armed Highlanders, whom, in virtue of his
commission as colonel, he proposed to employ in resisting any
troops of George the First which might be sent to Kintail. Nor did he
wait to be attacked, but, in June 1720, 1721.
hearing of a party of excisemen passing
near Dingwall with a large quantity of aqua-vitæ, he fell upon them,
and rescued their prize. The collector of the district reported this
transaction to the Board of Excise; but no notice was taken of it.
In February 1721, the two factors sent officers of their own into the
western districts, to assure the tenants of good usage, if they would
make a peaceable submission; but the men were seized, robbed of
their papers, money, and arms, and quietly remanded over the Firth
of Attadale, though only after giving solemn assurance that they
would never attempt to renew their mission. Resenting this
procedure, the two factors caused a constable to take a military party
from Bernera barracks into Lochalsh, and, if possible, capture those
who had been guilty. They made a stealthy night-march, and took
two men; but the alarm was given, the two men escaped, and began
to fire down upon their captors from a hillside; then they set fire to
the bothy as a signal, and such a coronach went over all Kintail and
Lochalsh, as made the soldiers glad to beat a quick retreat.
After some further proceedings, all of them ineffectual, the two
factors were enabled, on the 13th of September, to set forth from
Inverness with a party of thirty soldiers and some armed servants of
their own, with the design of enforcing submission to their legal
claims. Let it be remembered there were then no roads in the
Highlands, nothing but a few horse-tracks along the principal lines in
the country, where not the slightest effort had ever been made to
smooth away the natural difficulties of the ground. In two days, the
factors had got to Invermorriston; but here they were stopped for
three days, waiting for their heavy baggage, which was storm-stayed
in Castle Urquhart, and there nearly taken in a night-attack by a
partisan warrior bearing the name of Evan Roy Macgillivray. The
tenantry of Glenmorriston at first fled with their bestial; but
afterwards a number of them came in and made at least the
appearance of submission. The party then moved on towards
Strathglass, while Evan Roy respectfully followed, to pick up any
man or piece of baggage that might be left behind. At Erchless Castle,
and at Invercannich, seats of the Chisholm, they held courts, and
received the submission of a number of the tenants, whom, however,
they subsequently found to be ‘very deceitful.’
There were now forty or fifty miles of the wildest Highland country
before them, where they had reason to believe they should meet
groups of murderous Camerons and 1721.
Glengarry Macdonalds, and also encounter
the redoubted Donald Murchison, with his guard of Mackenzies,
unless their military force should be of an amount to render all such
opposition hopeless. An appointment having been made that they
should receive an addition of fifty soldiers from Bernera, with whom
to pass through the most difficult part of their journey, it seemed
likely that they would appear too strong for resistance; and, indeed,
intelligence was already coming to them, that ‘the people of Kintail,
being a judicious opulent people, would not expose themselves to the
punishments of law,’ and that the Camerons were absolutely
determined to give no further provocation to the government. Thus
assured, they set out in cheerful mood along the valley of Strathglass,
and, soon after passing a place called Knockfin, were reinforced by
Lieutenant Brymer, with the expected fifty men from Bernera. There
must have now been about a hundred well armed men in the invasive
body. They spent the next day (Sunday) together in rest, to gather
strength for the ensuing day’s march of about thirty arduous miles,
by which they hoped to reach Kintail.
At four in the morning of Monday the 2d October, the party set
forward, the Bernera men first, and the factors in the rear. They were
as yet far from the height of the country, and from its more difficult
passes; but they soon found that all the flattering tales of non-
resistance were groundless, and that the Kintail men had come a
good way out from their country in order to defend it. The truth was,
that Donald Murchison had assembled not only his stated band of
Mackenzies, but a levy of the Lewis men under Seaforth’s cousin,
Mackenzie of Kildun; also an auxiliary corps of Camerons, Glengarry
and Glenmorriston men, and some of those very Strathglass men
who had been making appearances of submission. Altogether, he
had, if the factors were rightly informed, three hundred and fifty men
with long Spanish firelocks, under his command, and all posted in
the way most likely to give them an advantage over the invading
force.
The rear-guard, with the factors, had scarcely gone a mile, when
they received a platoon of seven shots from a rising ground near
them to the right, with, however, only the effect of piercing a soldier’s
hat. The Bernera company, as we are informed, left the party at eight
o’clock, as they were passing Lochanachlee, and from this time is
heard of no more: how it made its way out of the country does not
appear. The remainder still advancing, Easterfearn, as he rode a little
before his men, had eight shots levelled at 1721.
him from a rude breastwork near by, and
was wounded in two places, but was able to appear as if he had not
been touched. Then calling out some Highlanders in his service, he
desired them to go before the soldiers, and do their best, according to
their own mode of warfare, to clear the ground of such lurking
parties, so that the troops might advance in safety. They performed
this service pretty effectually, skirmishing as they went on, and the
main body advanced safely about six miles. They were here arrived at
a place called Aa-na-Mullich (Ford of the Mull People), where the
waters, descending from the Cralich and the lofty mountains of
Kintail, issue eastwards through a narrow gorge into Loch Affaric. It
was a place remarkably well adapted for the purposes of a resisting
party. A rocky boss, called Tor-an-Beatich, then densely covered with
birch, closes up the glen as with a gate. The black mountain stream,
‘spear-deep,’ sweeps round it. A narrow path wound up the rock,
admitting only of passengers in single file. Here lay Donald with the
best of his people, while inferior adherents were ready to make
demonstrations at a little distance. As the invasive party approached,
they received a platoon from a wood on the left, but nevertheless
went on. When, however, they were all engaged in toiling up the
pass, forty men concealed in the heather close by fired with deadly
effect, inflicting a mortal wound on Walter Ross, Easterfearn’s son,
while Bailie Ross’s son was also hurt by a bullet which swept across
his breast. The bailie called to his son to retire, and the order was
obeyed; but the two wounded youths and Bailie Ross’s servant were
taken prisoners, and carried up the hill, where they were quickly
divested of clothes, arms, money, and papers. Young Easterfearn
died next morning. The troops faced the ambuscade manfully, and
are said to have given their fire thrice, and to have beat the
Highlanders from the bushes near by; but, observing at this juncture
several parties of the enemy on the neighbouring heights, and being
informed of a party of sixty in their rear, Easterfearn deemed it best
to temporise.
He sent forward a messenger to ask who they were that opposed
the king’s troops, and what they wanted. The answer was that, in the
first place, they required to have Ross of Easterfearn delivered up to
them. This was pointedly refused; but it was at length arranged that
Easterfearn should go forward, and converse with the leader of the
opposing party. The meeting took place at Bal-aa-na-Mullich (the
Town of the Mull Men’s Ford), and Easterfearn found himself
confronted with Donald Murchison. It 1721.
ended with Easterfearn giving up his
papers, and covenanting, under a penalty of five hundred pounds,
not to officiate in his factory any more; after which he gladly
departed homewards with his associates, under favour of a guard of
Donald’s men, to conduct them safely past the sixty men lurking in
the rear. It was alleged afterwards that the commander was much
blamed by his own people for letting the factors off with their lives
and baggage, particularly by the Camerons, who had been five days
at their post with hardly anything to eat; and Murchison only
pacified them by sending them a good supply of meat and drink. He
had in reality given a very effectual check to the two gentlemen-
factors, to one of whom he imparted in conversation that any scheme
of a government stewardship in Kintail was hopeless, for he and
sixteen others had sworn that, if any person calling himself a factor
came there, they would take his life, whether at kirk or at market,
and deem it a meritorious action, though they should be cut to pieces
for it next minute.[549]
A bloody grave for young Easterfearn in Beauly Cathedral
concluded this abortive attempt to take the Seaforth estates within
the scope of a law sanctioned by statesmen, but against which the
natural feelings of nearly a whole people revolted.[550]
A newspaper advertisement informed the 1721. Dec.
world that ‘There is a certain gentleman
living at Glasgow, who has put forth a problem to the learned—
proposing, if no man answer it, to do it himself in a few weeks—viz.,
Whether or not it is possible so to dispose a ship, either great or
small, that, although she, or it, be rent in the bottom, and filled full of
water, or however tossed with tempest, she, or it, shall never sink
below the water; and also that the same may be reduced to
practice.’[551]

An election of a member of parliament for 1722. Apr. 27.

a Highland county was apt to bring forth
somewhat strenuous sentiments, and the scene sometimes partook a
good deal of the nature of a local civil war.
 A representative of Ross-shire being to be chosen, there came, the
night before, to Fortrose, the greatest man of the north, the Earl of
Sutherland, heading a large body of armed and mounted retainers,
who made a procession round the streets, while an English sloop-of-
war, in friendly alliance with him, came up to the town and fired its
guns. Hundreds of Highlanders, his lordship’s retainers, at the same
time lounged about. The reason of all this was, that the opposition
interest was in a decided majority, and a defeat to the Whig
candidate seemed impending. When the election came on, there
were thirty-one barons present, of whom eighteen gave their votes
for General Charles Ross of Balnagowan, the remainder being for
Captain Alexander Urquhart of Newhall. Hereupon, Lord
Sutherland’s relative and friend, Sir William Gordon of Invergordon,
sheriff of the county, retired with the minority, and went through the
form of electing their own man, notwithstanding a protest from the
other candidate. ‘Immediately after this separation, Colin Graham of
Drynie, one of the deputy-lieutenants of the county, came into the
court-house, with his sword in his hand, accompanied by Robert
Gordon of Haughs and Major John Mackintosh, with some of the
armed Highlanders whom they had posted at the door, with drawn
swords and cocked firelocks, and did require the majority (who
remained to finish the election), in the name of the Earl of
Sutherland, to remove out of the house, otherwise they must expect
worse treatment. Major Mackintosh said they would be dragged out
by the heels. Upon which the barons protested against those violent
proceedings, declaring their resolution to 1722.
remain in the court-house till the election
was finished, though at the hazard of their lives; which they
accordingly did.’[552]

The Catholics had of late been getting up Apr. 29.

their heads in the north, especially in
districts over which the Gordon family held sway; and the open
practice of the Romish rites before large congregations in the
Banffshire valleys, was become a standing subject of complaint and
alarm in the church-courts. When at length the government obtained
scent of the Jacobite plot in which Bishop Atterbury was concerned,
it sympathised with these groans of the laden spirits in Scotland, and
permitted some decided measures of repression to be taken.
Accordingly, this day, being Sunday, as the Duchess-Dowager of
Gordon—Elizabeth Howard, daughter of the Duke of Norfolk—was
having mass performed at her house in the Canongate, Edinburgh, in
the presence of about fifty professors like herself of the Catholic
religion, Bailie Hawthorn, a magistrate of the Canongate, broke open
the doors, and seized the whole party. The ladies were bailed, and
allowed to depart; but the priest, Mr John Wallace, was marched to
prison. We are informed by Wodrow that Wallace had been ordained
a Protestant minister thirty-five years before.[553] The Lord Advocate
would not at first listen to any proposal for his liberation, though
several persons of distinction came to plead for it; but at length bail
was taken for him to the extent of a thousand merks Scots. Being
indicted under the statute of 1700, he failed to stand his trial, and
was outlawed.[554]
Before the upbreak of this plot, considerable numbers of
gentlemen under attainder daily presented themselves on the streets
of Edinburgh, emboldened of course by the mildness of the
government; but, one or two of them having been seized and put up
in the Castle, it came to pass, 15th May, that not one was any longer
to be seen. Mr Wodrow, who records these circumstances, expresses
the feeling of the hour. ‘It’s certain we are in a most divided and
defenceless state; divisions on the one hand, rancour and malice on
the other, and a wretched indolence among too many. But the Lord
liveth!’[555]

The Canongate, which had so often, in the Aug. 3.

sixteenth century, 1722.
been reddened
with the best blood in Scotland, was still occasionally the scene of
wild transactions, though arising amongst a different class of persons
and from different causes. A local journalist chronicles a dreadful
tragedy as occurring on its pavé at this date.
‘In the afternoon, Captain Chiesley and Lieutenant Moodie, both of
Cholmly’s Regiment, which lies encamped at Bruntsfield Links,
having quarrelled some time before in the camp, meeting on the
street of the Canongate, the captain, as we are told, asked Mr Moodie
whether he had in a certain company called him a coward? And he
owning he had, the captain beat him first with his fist, and then with
a cane; whereupon Mr Moodie drew his sword, and, shortening it,
run the captain into the great artery. The captain, having his sword
drawn at the same time, pushed at Mr Moodie, who was rushing on
him with his sword shortened, and thus run him into the lower belly,
of which in a few minutes he died, without speaking one word,
having had no more strength or life left him than to cross the street,
and reach the foot of the stair of his lodgings, where he dropped
down dead. The captain lived only to step into a house near by, and
to pray shortly that God might have mercy on his soul, without
speaking a word more. ’Tis said Mr Moodie’s lady was looking over
the window all the while this bloody tragedy was acting.’[556]
A duel which happened about the same time between Captains
Marriot and Scroggs proved fatal to both.

‘Four of those poor deluded people called Aug. 7.

Quakers, two men and two women, came
about noon to the Cross [of Edinburgh], when one of the women,
who by her accent seemed to be of Yorkshire, after several violent
agitations, said, that she was appointed by God to preach repentance
to this sinful city; that a voice of mortality, as she called it, had
sounded in her ears, and that desolation and all kinds of miseries
would befall the inhabitants if they did not repent. After she had
spoke about a quarter of an hour, a party of the city-guard carried
her and the other three prisoners to the main guard.’
Some years after, one Thomas Erskine, a brewer, made himself
conspicuous as a Quaker preacher in Edinburgh. One Saturday,
January 17, 1736, he ‘made a religious peregrination through this
city. He made his first station at the Bow-head [reputed as the head-
quarters of the saints in Edinburgh], where 1722.
he pronounced woes and judgments on the
inhabitants of the Good Town, if they did not speedily repent. Thence
he walked to the Cross, where he recapitulated what he had
evangelised by the way, and concluded with desiring his auditory to
remember well what he had told them. However, he gave them forty
days to think on’t.’
 One day, in the ensuing July, Erskine sent a notice to the quiet
little country town of Musselburgh, to the effect that the Spirit had
appointed him to hold forth to them in the marketplace at five in the
afternoon; and, accordingly, at the appointed hour, he mounted the
Cross, and discoursed to a large audience.[557]

A second attempt was now made to Aug. 29.

obtain possession of the forfeited Seaforth
estates for the government. It was calculated that what the two
factors and their attendants, with a small military force, had failed to
accomplish in the preceding October, when they were beat back with
a fatal loss at Aa-na-Mullich, might now be effected by means of a
good military party alone, if they should make their approach
through a less critical passage. A hundred and sixty of Colonel Kirk’s
regiment left Inverness under Captain M‘Neil, who had at one time
been commander of the Highland Watch. They proceeded by
Dingwall, Strath Garve, and Loch Carron, a route to the north of that
adopted by the factors, and an easier, though a longer way. Donald
Murchison, nothing daunted, got together his followers, and
advanced to the top of Maam Attadale, a high pass from Loch Carron
to the head of Loch Long, separating Lochalsh from Kintail. Here a
gallant relative named Kenneth Murchison, and a few others,
volunteered to go forward and plant themselves in ambush in the
defiles of the Choille Van [White Wood], while the bulk of the party
should remain where they were. It would appear that this ambush
party consisted of thirteen men, all peculiarly well armed.
On approaching this dangerous place, the captain went forward
with a sergeant and eighteen men to clear the wood, while the main
body came on slowly in the rear. At a place called Altanbadn, in the
Choille Van, he encountered Kenneth and his associates, whose fire
wounded himself severely, killed one of his grenadiers, and wounded
several others of the party. He persisted in advancing, and attacked
the handful of natives with sufficient 1722.
resolution. They slowly withdrew, as unable
to resist; but the captain now obtained intelligence that a large body
of Mackenzies was posted in the mountain-pass of Attadale. It
seemed as if there was a design to draw him into a fatal ambuscade.
His own wounded condition probably warned him that a better
opportunity might occur afterwards. He turned his forces about, and
made the best of his way back to Inverness. Kenneth Murchison
quickly rejoined Colonel Donald on Maam Attadale, with the
cheering intelligence that one salvo of thirteen guns had repelled the
hundred and sixty sidier roy.[558] After this, we hear of no renewed
attempt to comprise the Seaforth property.
Strange as it may seem, Donald Murchison, two years after thus a
second time resisting the government troops, came down to
Edinburgh with eight hundred pounds of the earl’s rents, that he
might get the money sent abroad for his lordship’s use. He remained
a fortnight in the city unmolested. He would on this occasion appear
in the garb of a Lowland gentleman; he would mingle with old
acquaintances, ‘doers’ and writers; and appear at the Cross amongst
the crowd of gentlemen who assembled there every day at noon.
Scores would know all about his doings at Aa-na-Mullich and the
Choille Van; but thousands might have known, without the chance of
one of them betraying him to government.
General Wade, writing a report to the king in 1725, states that the
Seaforth tenants, formerly reputed the richest of any in the
Highlands, are now become poor, by neglecting their business, and
applying themselves to the use of arms. ‘The rents,’ he says, ‘continue
to be collected by one Donald Murchison, a servant of the late earl’s,
who annually remits or carries the same to his master into France.
The tenants, when in a condition, are said to have sent him free gifts
in proportion to their several circumstances, but are now a year and
a half in arrear of rent. The receipts he gives to the tenants are as
deputy-factor to the Commissioners of the Forfeited Estates, which
pretended power he extorted from the factor (appointed by the said
commissioners to collect those rents for the use of the public), whom
he attacked with above four hundred armed men, as he was going to
enter upon the said estate, having with him a party of thirty of your
majesty’s troops. The last year this Murchison marched in a public
manner to Edinburgh, to remit eight hundred pounds to France for
his master’s use, and remained fourteen days there unmolested. I
cannot omit observing to your majesty, that 1722.
this national tenderness the subjects of
North Britain have one for the other, is a great encouragement for