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Stem Cell Therapy
for Vascular Diseases
State of the Evidence

and Clinical Applications
Tulio Pinho Navarro
Lara Lellis Navarro Minchillo Lopes
Alan Dardik
Editors
123
Stem Cell Therapy for Vascular Diseases
Tulio Pinho Navarro
Lara Lellis Navarro Minchillo Lopes
Alan Dardik
Editors
Stem Cell Therapy for

Vascular Diseases
State of the Evidence and Clinical
Applications
Editors
Tulio Pinho Navarro Lara Lellis Navarro Minchillo Lopes
Department of Surgery Postgraduate Program in Sciences Applied
Vascular Surgery Section to Surgery
Federal University of Minas Gerais Federal University of Minas Gerais
Belo Horizonte Belo Horizonte
Minas Gerais Minas Gerais
Brazil Brazil
Alan Dardik
Vascular Biology and Therapeutics Program
and the Departments of Surgery and
Cellular and Molecular Physiology
Yale University School of Medicine
New Haven, CT
USA
ISBN 978-3-030-56953-2 ISBN 978-3-030-56954-9 (eBook)

https://doi.org/10.1007/978-3-030-56954-9
© Springer Nature Switzerland AG 2021

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
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Preface
Despite major medical and scientific advances in the last 50 years, vascular disease
continues to challenge us. Aneurysms, atherosclerosis, heart failure, stroke, malfor-
mations, fistulae, and vasculitis all continue to provide diagnostic and therapeutic
challenges that keep vascular disease the leading the cause of death and a major
source of morbidity and suffering in modern society. Physicians and surgeons con-
tinue to treat vascular disease with the technology at hand. Early advances with
cautery and suture have been supplemented by stents, wires, and coils to give mod-
ern practitioners a range of therapeutic options. But there is still room to improve.
What is next? It is likely to be exciting.
Understanding the biology of stem cells has been a tremendous advance in mod-
ern medicine and has captured the imagination of many people. The initial require-
ment to derive stem cells from embryonic cells prompted ethical concerns that
limited utility and clinical translation of therapy. However, advances in understand-
ing the biology of stem cells continue to open new possibilities. In 2012, the Nobel
Prize was awarded to Drs. Yamanaka and Gurdon for the discovery that somatic
cells could be reprogrammed into induced pluripotent stem cells. This groundbreak-
ing discovery has completely changed our view of development and cellular spe-
cialization, opening new frontiers in biology; stem cell therapy is not far behind.
Although some roadside clinics claim to offer stem cell therapy, a safe and effective
therapy must be based on solid science and understanding of biology and developed
into consistent products that are tested rigorously through adequately powered clini-
cal trials with reasonable endpoints that are then published in peer-reviewed litera-
ture. You and I want this, and our patients deserve this.
The goal of this book is to describe the current status of stem cell biology and
therapy as related to the cardiovascular system and vascular diseases. The editors
have been involved with stem cell research for over 30 years and are involved with
human clinical trials using stem cells; this endeavor capitalizes on their long-term
friendship and collaboration that has stood the test of time and distance. The authors
are recognized experts in their fields with relevant and real world experience. We
hope you will enjoy this book as much as we have enjoyed putting it together.
v
vi Preface
Belo Horizonte, Brazil Tulio Pinho Navarro, MD, PhD

Belo Horizonte, Brazil Lara Lellis Navarro Minchillo Lopes, MD
New Haven, CT, USA Alan Dardik, MD, PhD
Contents
1 Introduction to Stem Cell Therapy and Its Application

in Vascular Diseases �� 1
Lara Lellis Navarro Minchillo Lopes, Tulio Pinho Navarro,
and Alan Dardik
2 Types and Origin of Stem Cells�� 33
Lucíola da Silva Barcelos, Pollyana Ribeiro Castro, Elisabeth
Tamara Straessler, and Nicolle Kränkel
3 Stem Cell Delivery Techniques for Stroke and Peripheral
Artery Disease�� 69
Shin-Rong Lee, Arash Fereydooni, and Alan Dardik
4 The Ethical Challenges of Stem Cell Therapy
in Vascular Disorders�� 105
Ramesh K. Batra
5 Bone Marrow-Derived Cells: From the Laboratory
to the Clinic�� 115
Justin R. King, Jie Xie, and Michael P. Murphy
6 Angiogenesis: Perspectives from Therapeutic Angiogenesis�� 129
Monique Bethel, Vishal Arora, and Brian H. Annex
7 Stem Cell Therapy for Diabetic Foot Ulcers�� 155
Hallie J. Quiroz, Zhao-Jun Liu, and Omaida C. Velazquez
8 Venous Foot and Leg Ulcers�� 173
Edith Tzeng and Kathy Gonzalez
9 Induced Pluripotent Stem Cell-Derived Vascular
Smooth Muscle Cells for Vascular Regeneration�� 199
Biraja C. Dash
vii
viii Contents
10 Mesenchymal Stem Cell and Hematopoietic Stem

Cell Transplantation for Vasculitis �� 221
Lianming Liao and Yongquan Gu
11 Mesenchymal Stem Cell and Endothelial Progenitor
Cell Transplantation for Buerger’s Disease�� 231
Lianming Liao and Yongquan Gu
12 Changing the Course of Peripheral Arterial Disease
Using Adult Stem Progenitor Cells�� 245
Mark Niven, Galit Sivak, Shlomo Baytner, Roman Liberson,
Shlomo Bulvik, Yael Porat, Michael Frogel, Louis Shenkman,
Martin Grajower, Frank Veith, and Michael Belkin
13 Stem Cell Delivery for the Treatment of Arteriovenous
Fistula Failure�� 281
Akshaar N. Brahmbhatt and Sanjay Misra
14 Stem Cell Therapy to Improve Acute Myocardial
Infarction Remodeling�� 299
Jolanta Gorecka and Alan Dardik
15 Stem Cell Therapy for Stroke �� 331
S. M. Robert and C. Matouk
16 Use of Stem Cells in the Treatment of Erectile Dysfunction�� 347
Benjamin Press and Stanton C. Honig
17 Stem Cell Therapy for Ophthalmic Vascular Disease�� 367
Caio Vinicius Regatieri, Augusto Vieira,
and Marcio Bittar Nehemy
18 Stem Cell Therapy Delivery in Liver Disease�� 385
John Langford and Gregory T. Tietjen
19 Stem Cell Therapy for Lymphedema �� 407
Dylan McLaughlin, Angela Cheng, and Luke Brewster
Index�� 421
Contributors
Brian H. Annex, MD Division of Cardiology and Department of Medicine,

Medical College of Georgia at Augusta University, Augusta, GA, USA
Vishal Arora, MD Division of Cardiology and Department of Medicine, Medical
College of Georgia at Augusta University, Augusta, GA, USA
Ramesh K. Batra, MBBS Yale School of Medicine, Yale University, Yale New
Haven Transplant Center, New Haven, CT, USA
Shlomo Baytner, MD Sanz Medical Center, Laniado Hospital, Netanya, Israel
Michael Belkin, MD Sanz Medical Center, Laniado Hospital, Netanya, Israel
Tel Aviv University, Tel Aviv, Israel
Monique Bethel, MD Division of Cardiology and Department of Medicine,
Medical College of Georgia at Augusta University, Augusta, GA, USA
Akshaar N. Brahmbhatt, MD Diagnostic Radiology - Department of Imaging
Sciences, University of Rochester, Rochester, NY, USA
Luke Brewster, MD, PhD Emory University, Department of Surgery, Vascular
Surgery and Endovascular Therapy, Atlanta, GA, USA
Atlanta VA Medical Center, Surgical and Research Services, Decatur, GA, USA
Georgia Institute of Technology, Institute for Bioengineering and Bioscience,
Atlanta, GA, USA
Shlomo Bulvik, MD Sanz Medical Center, Laniado Hospital, Netanya, Israel
Pollyana Ribeiro Castro, MD Physiology and Biophysics, Federal University of
Minas Gerais (UFMG), Belo Horizonte, Brazil
Angela Cheng, MD Emory University, Department of Surgery, Division of Plastic
and Reconstructive Surgery, Atlanta, GA, USA
ix
x Contributors
Lucíola da Silva Barcelos, PhD Physiology and Biophysics, Federal University

of Minas Gerais (UFMG), Belo Horizonte, Brazil
Alan Dardik, MD, PhD Vascular Biology and Therapeutics Program and the
Departments of Surgery and Cellular and Molecular Physiology, Yale University
School of Medicine, New Haven, CT, USA
Biraja C. Dash, PhD Department of Surgery, Division of Plastic Surgery, Yale
School of Medicine, Yale University, New Haven, CT, USA
Arash Fereydooni, MD Yale School of Medicine, New Haven, CT, USA
Michael Frogel, MD Cohen’s Children’s Medical Center, New Hyde Park, NY, USA
Kathy Gonzalez, MD Division of Vascular Surgery, University of Pittsburgh
Medical Center, Pittsburgh, PA, USA
Jolanta Gorecka, MD Vascular Biology and Therapeutics Program and the
Department of Surgery, Yale University School of Medicine, New Haven, CT, USA
Martin Grajower, MD Albert Einstein College of Medicine, Bronx, NY, USA
Yongquan Gu, MD Department of Vascular Surgery, Xuan Wu Hospital of Capital
Medical University, Beijing, China
Stanton C. Honig, MD Department of Urology, Yale School of Medicine, New
Haven, CT, USA
Justin R. King, MD Indiana Center for Vascular Biology and Medicine, Indiana
University School of Medicine, Indianapolis, IN, USA
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA
Center for Regenerative Medicine, Richard L. Roudebush Veterans Administration
Medical Center, Indianapolis, IN, USA
Nicolle Kränkel, PhD Department of Cardiology, Campus Benjamin Franklin,
Charité – Universitätsmedizin Berlin, Berlin, Germany
John Langford, MD Department of Surgery, Yale University School of Medicine,
New Haven, CT, USA
Shin-Rong Lee, MD, PhD Yale School of Medicine, New Haven, CT, USA
Lianming Liao, MD, PhD Department of Laboratory Medicine, Fujian Medical
University Union Hospital, Fuzhou, China
Roman Liberson, MD, PhD Sanz Medical Center, Laniado Hospital,
Netanya, Israel
Zhao-Jun Liu, MD, PhD University of Miami Leonard M. Miller School of
Medicine, Miami, FL, USA
Contributors xi
C. Matouk, MD Department of Neurosurgery, Yale University School of Medicine,

New Haven, CT, USA
Dylan McLaughlin, MD Emory University, Department of Surgery, Vascular
Surgery and Endovascular Therapy, Atlanta, GA, USA
Lara Lellis Navarro Minchillo Lopes, MD Federal University of Minas Gerais,
Belo Horizonte, Minas Gerais, Brazil
Sanjay Misra, MD Department of Radiology, Mayo Clinic, Rochester, MN, USA
Michael P. Murphy, MD Indiana Center for Vascular Biology and Medicine,
Indiana University School of Medicine, Indianapolis, IN, USA
Center for Regenerative Medicine, Richard L. Roudebush Veterans Administration
Medical Center, Indianapolis, IN, USA
Tulio Pinho Navarro, MD, PhD Vascular Biology and Therapeutics Program and
the Departments of Surgery and Cellular and Molecular Physiology, Yale University
School of Medicine, New Haven, CT, USA
Marcio Bittar Nehemy, MD, PhD Department of Ophthalmology, Federal
University of Minas Gerais, Belo Horizonte, MG, Brazil
Mark Niven, MD Sanz Medical Center, Laniado Hospital, Netanya, Israel
Yael Porat, PhD BioGenCell, Ltd., Laniado Hospital, Netanya, Israel
Benjamin Press, MD Yale School of Medicine, New Haven, CT, USA
Hallie J. Quiroz, MD University of Miami Leonard M. Miller School of Medicine,
Miami, FL, USA
Caio Vinicius Regatieri, MD, PhD Department of Ophthalmology, Federal
University of São Paulo, São Paulo, Brazil
Department of ophthalmology, Tufts Medical School, Boston, MA, USA
S. M. Robert, MD, PhD Department of Neurosurgery, Yale University School of
Medicine, New Haven, CT, USA
Louis Shenkman, MD Tel Aviv University, Tel Aviv, Israel
Galit Sivak, MD Rabin Medical center, Tel Aviv University, Tel Aviv, Israel
Elisabeth Tamara Straessler, MD Department of Cardiology, Campus Benjamin
Franklin, Charité – Universitätsmedizin Berlin, Berlin, Germany
Gregory T. Tietjen, PhD Department of Surgery, Yale University School of
Medicine, New Haven, CT, USA
xii Contributors
Department of Biomedical Engineering, Yale University, New Haven, CT, USA

Department of Surgery – Transplant Section, Yale School of Medicine, New
Haven, CT, USA
Edith Tzeng, MD VA Pittsburgh Healthcare System and University of Pittsburgh,
Pittsburgh, PA, USA
Division of Vascular Surgery, University of Pittsburgh Medical Center,
Pittsburgh, PA, USA
Frank Veith, MD NYU-Langone Medical Center, New York, NY, USA
The Cleveland Clinic, Cleveland, OH, USA
Omaida C. Velazquez, MD DeWitt Daughtry Department of Surgery, University
of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
Augusto Vieira, MD Department of Ophthalmology, Federal University of São
Paulo, São Paulo, Brazil
Jie Xie, MD, PhD Indiana Center for Vascular Biology and Medicine, Indiana
University School of Medicine, Indianapolis, IN, USA
Chapter 1
Introduction to Stem Cell Therapy and Its
Application in Vascular Diseases
Lara Lellis Navarro Minchillo Lopes, Tulio Pinho Navarro, and Alan Dardik
1.1 Introduction
1.1.1 Brief History of Stem Cells
Stem cells are undifferentiated cells capable of both self-renewal and differentiation
into various specialized cells [1]. Stem cell therapy is the therapeutic administration
of stem cells to repair or replace tissue function [2]. The term “stem cell” was pro-
posed by Alexander Maksimov in 1908 when developing “the unitarian theory of
hematopoiesis,” which proposed a common stem cell progenitor for all blood ele-
ments (Fig. 1.1) [3]. However, it was only in 1961 that the existence of murine cells
capable of self-renewal was proven by Till et al. while assessing radiation sensitivity
of bone marrow tissue [4].
In 1962, John Gurdon performed a classic experiment in frogs, in which he
replaced the immature cell nucleus in an egg cell with the nucleus from a mature
intestinal cell, resulting in a normal tadpole; this experiment showed that the DNA
of the mature cell had all the information needed to develop all cells in the organ-
ism, challenging the dogma that the specialized cell is irreversibly committed to its
fate [5].
In 1968, Friedenstein et al. reported the discovery of a human bone marrow cell
population with high proliferative potential and osteogenic activity in vivo [6]. In
the same year, Thomas et al. performed the first hematopoietic stem cell
L. L. N. M. Lopes · T. P. Navarro (*)

Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
A. Dardik
Vascular Biology and Therapeutics Program and the Departments of Surgery and Cellular and
Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA
© Springer Nature Switzerland AG 2021 1

T. P. Navarro et al. (eds.), Stem Cell Therapy for Vascular Diseases,
https://doi.org/10.1007/978-3-030-56954-9_1
2 L. L. N. M. Lopes et al.
• The term “stem cell” is proposed by Maksimov.

1908
• Murine bone marrow stem cells are first isolated by Till et al.
1961
• Gurdon et al. demonstrates that mature cells’ nucleus keep information to develop all cell types.
1962
• Human bone marrow-derived stem cells are reported by Friedenstein et al.

1968
• Murine pluripotent stem cells are isolated by Evans and Kaufman.

1981
• Human embryonic stem cells are first isolated Thomson et al.

1998
• Murine somatic cells are induced to a pluripotent stem cell-like state by Takahashi et al.
2006
• Human somatic cells are induced to a stem cell-like state by Takahashi et al.
2007
• Gurdon and Yamanaka won the Nobel prize for their discoveries regarding cell reprogramming.
2012
Fig. 1.1 Timeline with the main events related to stem cell therapy history
transplantation for the treatment of leukemia [7]. Since then, theoretical implica-
tions of human stem cells and their potential clinical applications have been exten-
sively studied.
In 1981, Evans and Kaufman reported the isolation of murine pluripotent
embryonic stem cells, and this was followed by the report of Thomson et al., who
first isolated human embryonic stem cells in 1998 [8, 9]. Shinya Yamanaka and
Kazutoshi Takahashi identified several genes that kept cells immature while per-
forming research on murine embryonal stem cells in 2006. After that, they were
able to reprogram fibroblasts into immature stem cells; these resulting induced
pluripotent stem cells (iPSC) could develop into mature cell types such as fibro-
blasts, nerve cells, and gut cells, demonstrating that intact, mature cells could be
reprogrammed into pluripotent stem cells [10]. Later, in 2007, they reported
induction of human fibroblasts into a pluripotent state [11]. Takahashi’s experi-
ments reinforced the concept of using iPSC as a novel technological frontier in
stem cell therapy perspectives. In 2012, the Nobel Prize was awarded to John
B. Gurdon and Shinya Yamanaka in recognition of their findings showing that
mature, specialized cells could be reprogrammed, creating new opportunities to
study diseases and to develop new methods for diagnosis and treatment [12].
1.1.2 esenchymal Stem Cells vs Mesenchymal Stromal Cells

M
and Cell Markers
In 1991, the term “mesenchymal stem cell” (MSC) was proposed by Caplan et al. to
designate adult stem cells capable of differentiating into cells of mesodermal origin
[13]. However, as these cells showed limited capacity for self-renewal, thus they
1 Introduction to Stem Cell Therapy and Its Application in Vascular Diseases 3
failed to meet the criteria to be called stem cells. As such, the term “mesenchymal
stromal cell,” also abbreviated MSC, was then suggested as a more appropriate
designation to these regenerative cells [14].
In 2005, the International Society for Cell and Gene Therapy (ISCT) declared
that the terms “stem cell” and “stromal cell” were not equivalent [15]. Furthermore,
the term “stem cell” should be limited to a population of cells with demonstrable
self-renewal and differentiation capacities, while the term “stromal cell” referred to
cells with notable secretory, immunomodulatory, and homing features [16]. In addi-
tion, the ISCT provided the following minimal criteria to identify mesenchymal
stromal cells: being adherent to plastic; capable of differentiation into adipocyte,
chondrocyte, and osteoblast lineages; expression of CD73, CD90, and CD105; and
lack of expression of CD11b, CD14, CD19, CD34, CD45, CD79a, and HLA-DR
(Fig. 1.2).
Since then, stromal cell surface markers were shown to be plastic and influ-
enced by microenvironmental conditions and stem cell origin among other vari-
ables [16–18]. Therefore, there are no specific and unambiguous cell surface
markers to distinguish stem cells from stromal cells [16]. Ironically, since the
aforementioned ISCT statement, the interchangeable use of the terms “stromal”
and “stem” cells by the scientific community has spread. A search in the US
National Library of Medicine database (clinicaltrials.gov), performed in November
21, 2019, reported 1009 clinical trials related to the term “mesenchymal stem cell,”
while only 211 results were related to the term “mesenchymal stromal cell” [19].
Few authors have reported complete mesenchymal stromal cell characterization in
both preclinical and clinical publications, and the use of the term “mesenchymal
stem cell” remains controversial [16]. Hence, for the purpose of this chapter, the
abbreviation “MSC” will be employed to designate populations of regenerative
adult mesenchymal cells.
Most of the available data on clinical stem cell therapy relies on adult stem cells
and most frequently MSC. Moreover, the use of embryonal stem cells and induced
Adherence to plastic Marker expresssion Differentiation potential
MSC
MSC MSC
Positive Negative
CD73 CD11b
CD90 CD14
CD105 CD19
CD34
CD45
CD79a
HLA-DR Adipocyte Osteoblast Chondrocyte
Fig. 1.2 MSC criteria according to the International Society for Cell and Gene Therapy
pluripotent stem cells raises important ethical and safety concerns that impair the
use of these two cell types in clinical situations. Therefore, MSC are the main focus
of this chapter.
1.2 Differentiation Potential
Stem cells can be classified according to their differentiation potential into totipo-
tent, pluripotent, multipotent, and oligopotent cells. Totipotent stem cells are found
in the zygote and can differentiate in both embryonic and extraembryonic cells.
Pluripotent cells are also capable of giving rise to any type of embryonic cell, but
not to extraembryonic tissue. Multipotent cells give rise to multiple cells of the
same lineage whereas oligopotent stem cells have a narrower differentiation spec-
trum within a same lineage [1].
1.3 Stem Cell Types
Stem cells can be classified into embryonic stem cells (ESC), adult stromal cells,
and iPSC according to their origin (Fig. 1.3). The diverse characteristics, advan-
tages, and limitations of these groups (Fig. 1.4) lead to different clinical
applications.
1.3.1 Embryonic Stem Cells (ESC)
ESC are stem cells derived from the embryonic inner cell mass and possess unlimited
self-renewal capability as well as the ability to differentiate into any type of somatic
cell. Even though these cells have potential clinical application, clinical use of these
cells is scarce due to ethical conflicts involving the harvest and manipulation of
human embryos to obtain these cells. Additionally, embryonic stem cells’ unlimited
Fig. 1.3 Stem cell types

Stem cells
Adult Induced
Embryonic
pluripotent
Bone marrow Adipose tissue Peripheral blood Umbilical cord

1
Stem Cell Type

Adult Stem Cells
Embryonic IPSC
BM-MSC PB-MSC UC-MSC ADSC
• Donor-specific therapy
• Lower malignancy risk • Future donor-specific
therapy • High differentiation
• Cell-lineage • Donor-specific therapy
• Lower malignancy risk potential (pluripotent)
committed (targeting • Lower malignancy risk
• Cell-lineage • Somatic-cell memory
• Donor-specific therapy differentiation) • Cell-lineage
committed (targeting (targeting
• Lower malignancy risk • No ethical conflict committed (targeting
differentiation) • High differentiation differentiation)
Advantages • Cell-lineage committed • Relatively disposable differentiation)
• Disposable tissue potential (pluripotent) • Donor-specific therapy
(targeting differentiation) tissue • No ethical conflict
• UC tissue harvesting • No ethical conflict
• No ethical conflict • Vein puncture has low • Disposable tissue
has low surgical risk • Disposable tissue
surgical risk • Liposuction has low
• Donor UCB banking • Low cell harvesting
• Simple cell harvesting surgical risk
storage procedure risk
protocol
• Cell lineage committed • Cell lineage

• Biopsy high surgical risk • Cell lineage committed committed • Increased malignancy
• Cell lineage • Increased malignancy
• Non-disposable tissue • Cell concentration and • lmmuno risk
committed risk
performance influenced incompatibility • Complex induction
Disadvantages • Low stem cell • Cell concentration and • Immuno
concentration by comorbities • Ethical conflict protocol
performance influenced incompatibility
• Cell concentration and • G-CSF administration • Low stem cell • Somatic-cell memory
by comorbities • Ethical conflicts
performance influenced by needed concentration (biased differentiation)
comorbities • Need of UCB banking
Introduction to Stem Cell Therapy and Its Application in Vascular Diseases
Fig. 1.4 Stem cell types, advantages and disadvantages [36]

5
differentiation potential lead to a higher risk of malignancy and requires immuno-

modulation as the use of allogeneic cells is mandatory for clinical therapy [20, 21].
1.3.2 Adult Stem Cells (ASC)
ASC are partially undifferentiated cells that can be found within nearly any organ or
tissue. Peripheral blood, umbilical cord, bone marrow, and adipose tissue are the
most common sources of such cells. Endometrium, amniotic fluid and membrane,
placenta, dental tissues, thymus, and spleen are unconventional sources of ASC. ASC
may be isolated from the same patient in whom the cell therapy will be applied
(donor-specific therapy) with no risk of immune rejection. There is no ethical con-
flict regarding the origin of these adult-derived cells or their isolation; however,
isolation protocols for ASC can be laborious, and a significant quantity of tissue is
frequently needed to obtain high stem cell counts [20].
ASC are lineage-committed multipotent cells, being able to differentiate
only into cells of the same germ layer [20]. This feature accounts for the lower
malignancy potential of ASC, and therefore these cells may be desirable to be
used in therapies that target specific cell differentiation, such as host tissue
replacement.
1.3.3 Induced Pluripotent Stem Cells (iPSC)
iPSC are stem cells generated through somatic cell reprogramming into an embry-
onic stem cell-like state, first reported in 2006 [10]. In theory, iPSC can be generated
from any somatic cell, and thus the use of these cells is emerging as an abundant and
accessible source of donor-specific stem cells free of ethical conflicts. These totipo-
tent cells demonstrate a broad differentiation potential, being capable of generating
any somatic or trophoblastic cell, but they also have a high malignant potential [22].
Because iPSC show advantages of both ASC, e.g., donor-specific therapy and
absence of ethical conflicts, and ESC, e.g., differentiation potential, this novel
source of stem cells is considered very promising. Nonetheless, strategies to
enhance the yield of cell induction into the stem cell-like state and simultaneously
control cell differentiation with absence of malignancy are still hurdles to be
overcome.
1.4 Stem Cell Origin
Stem cell therapy can be classified according to the cell origin as either autologous,
allogeneic, or xenogeneic transplantation (Fig. 1.5).
Stem cell origin
Autologous Allogeneic Xenotransplantation
• Healthy stem cell source • No ethical conflict

• lmmunoincompatibility
• No cell harvesting risk for the • Heathy stem cell source
• No ethical conflict
Advantages host patient • No cell harvesting risk for the
• No infection transmission risk
• Donor banking creation host patient
• Donor baking creation
• Lower stem cell concentration
• Relative
and limited healing potential
immunoincompatibility • High immunoincompatibility
Disadvantages • Cell harvestmg procedural
• Need of disease screening • Need of disease screening
risk
• Ethical conflict
Fig. 1.5 Stem cell origins, advantages, and disadvantages [36]
1.4.1 Autologous Transplantation
Autologous transplantation involves the administration of the recipient’s own cells,

typically using either ASC or iPSC. Autologous cells have the advantages of being
immunocompatible, having no risk of infectious disease transmission and involving
no ethical or legal issues. On the other hand, donor characteristics such as advanced
age, diabetes, obesity, and atherosclerosis exert negative impact on stem cell func-
tion, potentially decreasing the effectiveness of cell therapy [23–27]. Therefore,
past medical history and advanced patient age may be some of the limitations to the
use of autologous ASC for cell therapy.
1.4.2 Allogeneic Transplantation
Allogeneic cell transplantation is the exchange of cells between a donor and a recip-
ient of the same species. The use of cells isolated from a healthier or younger donor
could enhance the efficacy of stem cell therapy. However, allogeneic transplantation
has the drawbacks of lower immunocompatibility, risk of disease transmission, and
potential legal issues regarding exchange of biomaterials.
It has been hypothesized that allogeneic MSC are immunoprivileged. Nonetheless,
further research has shown that allogeneic MSC are not privileged, but demonstrate
diminished immunogenicity compared with other allogeneic cell types [28]. The
therapeutic implications of allogeneic MSC immunogenicity are controversial as
cell apoptosis is crucial for immunomodulatory effects but reduces therapeutic cell
longevity and engraftment [29].
Strict donor screening is needed to avoid disease transmission by allogeneic cell
therapy [2]. The use of cadaveric cells may be an alternative to enhance donor
availability and to avoid the risks associated to tissue harvesting therapeutic
cells [30].
1.4.3 Xenotransplantation
Xenotransplantation involves the administration of either nonhuman live cells or

human biological material that has had ex vivo contact with live nonhuman animal
cells [31]. The use of animal stem cells has the potential to increase availability of
donors and to reduce the financial burden related to stem cell transplantation.
However, this source raises concerns regarding immunologic rejection and disease
transmission.
1.5 Stem Cell Isolation and Induction Protocols
The tissue source of stem cells is the main determinant of the required isolation
protocol and influences MSC phenotypes and function [14]. Here we discuss basic
aspects of stem cell isolation and induction protocols and their clinical implications.
1.5.1 Adult Stem Cells
1.5.1.1 Bone Marrow-Derived MSC (BM-MSC)
BM-MSC are isolated from the bone marrow by density gradient centrifugation,
washing, and seeding on culture dishes. BM-MSC can be selected by survival in
minimum essential media with fetal bovine serum and by their adherence to plastic
[14, 32]. However, these isolation protocols usually result in a heterogeneous cell
population, contaminated by other cells [33].
Bone marrow tissue is not a disposable tissue and is necessarily obtained by bone
marrow aspiration. The aspiration is an invasive procedure, which leads to higher
risks to the donor when compared to other MSC isolation protocols.
1.5.1.2 Adipose-Derived Stem Cells (ADSC)
Adipose tissue is a relatively disposable cell source and can be easily accessed by
lipoaspiration, which is considered a minimally invasive procedure. Adipose tissue
processing leads to a heterogenic population termed the “stromal vascular fraction”
(SVF). The SVF is composed of adipose-derived stem cells (ADSC), pericytes,
endothelial cells, pre-adipocytes and immune cells as well as other cell types [34].
Isolation of the SVF may be performed either by enzymatic or mechanical pro-
tocols that generally comprise washes, agitation, centrifugation, and collagenase
digestion (in enzymatic protocols) [35]. Currently, semi-automated SVF isolation
devices are commercially available [34]. ADSC and SVF have been used in clinical
trials, but ADSC isolation protocols frequently fail to exclude the other components
of the SVF, leading to questions of reproducibility and translatability [14, 34].
1.5.1.3 Peripheral Blood-Derived MSC (PB-MSC)
Peripheral blood is a relatively disposable stem cell source and can be obtained by
venipuncture, a minimally invasive procedure. Peripheral blood-derived MSC
(PB-MSC) are isolated by centrifugation and dilution protocols. PB-MSC usually
circulate in low concentrations; therefore, bone marrow stimulation by granulocyte
colony-stimulating factor (G-CSF) is an important adjunct to mobilize PB-MSC
into the circulation before collecting blood [36].
G-CSF administration itself is effective in the treatment of diabetic foot ulcers
[36]. Thus, the administration of G-CSF may be a confounding variable in study
interpretation as well as its associated financial cost.
1.5.1.4 Other Adult Stem Cell Sources
Umbilical cord blood, placenta, Wharton’s jelly, amniotic fluid, dental pulp, syno-
vial fluid, and skin are also MSC sources, although less commonly used.
1.5.2 Embryonic Stem Cells (ESC)
The isolation of human embryonic stem cells (hESC) implies the destruction of the
embryo. Due to associated ethical and political concerns, research involving the use
of these cells has been deferred if not banned. Nevertheless, isolation, culture, and
characterization protocols for hESC have been developed [37].
hESC are found in the inner cell mass of both fresh and frozen embryos. Several
isolation techniques are described in the literature including mechanical dissection,
laser dissection, and immunosurgery [9, 38, 39]. There is no consensus regarding
the best method as each of these techniques have specific advantages and disadvan-
tages regarding success rate and financial and time constraints [37].
1.5.3 Induced Pluripotent Stem Cells (iPSC)
iPSC are stem cells induced from somatic cells; as such they are not isolated per se.
The use of iPSC has obviated ethical conflicts regarding the use of hESC and has
provided insights into early embryo development. The first method to successfully
induce human iPSC was the use of four transcription factors Oct3/4, Sox2, Klf4,
and c-Myc, under ES cell culture conditions [11]. Currently, several induction
methods have been used to generate iPSC including integrating vectors, non-
integrating vectors, non-DNA reprogramming, and small molecules [40].
Pluripotency induction and maintenance are based on the interaction of both extrin-
sic and intrinsic factors [41]. The extrinsic factors involve cytokines, growth factors,
and extracellular matrix; extrinsic elements influence intrinsic pathways that reverse
epigenetic programming of differentiated somatic cells, such as octamer-binding
transcription factor 4 (Oct4) and sex-determining region Y-box 2 (Sox2) [42].
The low cell reprogramming yield (0.01–0.02% when first described), expression
of transgenic viral genes, and tumorigenesis by overexpression of oncogenes (c-Myc,
kfl4) are major concerns of iPSC induction protocols [11, 43]. Further advances on
cell reprogramming techniques are required to enable iPSC use in clinical scenarios.
1.6 Stem Cell Culture and Priming
Culture conditions such as media composition, oxygen tension, and extracellular

structure affect stem cell survival, differentiation, and function [14, 43]. Furthermore,
several cell priming techniques have been proposed as strategies to enhance stem cell
therapeutic effects. Understanding and electing the best cell culture and priming pro-
tocol is important not only to enhance their regenerative potential but also for repro-
ducible cell expansion after low yield isolation protocols and target differentiation.
1.6.1 Culture Media
Medium containing either fetal bovine serum (FBS), human AB serum (HABS),
human platelet lysate (HPL), or chemically defined media (CMD) is commonly used
in MSC culture [14]. FBS is the most common supplement, although it carries the
risk of xenogeneic immune reaction and has a variable composition [44]. HABS and
HPL are derived from peripheral blood that have been proposed as human-derived
immunocompatible alternatives to FBS. However, their composition is also highly
variable, and there is a risk of infection transmission [45]. In addition, HPL has pro-
inflammatory factors and may alter MSC cell markers, affecting its functionality [14,
46]. Serum-free, xenobiotic-free CMD can improve cell expansion and differentia-
tion and is considered a desirable alternative for MSC culture for clinical use [47].
1.6.2 Hypoxia
Hypoxic culture conditions (1–10% O2 partial pressure) mimic hypoxic areas within
the bone marrow where BM-MSC can be found physiologically (4–7% O2 tension)
[14]. However, hypoxia affects stem cells from other sources by influencing cell
metabolism, proliferation, differentiation, and the secretome [48]. Since hypoxia

intensity is variable among stem cell studies that use hypoxic conditioning, conclu-
sions regarding the effects of hypoxia need to be cautiously interpreted.
Low oxygen availability lowers metabolism and production of reactive oxygen
species (ROS), a mechanism that is thought to be responsible for reduced stem cell
injury [48]. In addition, the decrease in ROS added to hypoxia-mediated upregula-
tion of c-jun leads to delayed stem cell senescence and increases immunosuppres-
sion [49].
Upregulation of hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α), vas-
cular endothelial growth factor (VEGF), and angiopoietin-1 (Ang-1) lead to increase
angiogenic potential [50, 51]. This feature is especially desirable when treating
ischemic and inflammatory pathologies. Moreover, HIF-2α inhibits p53, increasing
the regenerative potential of stem cells [51].
Hypoxic-cultured MSC (2% O2 tension) were reported to be more proliferative
when compared to MSC cultured in normoxic conditions (20%) [52]. The increased
cell proliferation compensates for the initial hypoxia-mediated MSC apoptosis in
subsequent cell passages. Furthermore, hypoxia favors maintenance of stem cells’
undifferentiated state and increases cell motility [50, 53].
1.6.3 Culture Matrices and Devices
Stem cells are often cultured in plastic containers such as T-flasks and well plates.
However, these two-dimensional devices are associated with alteration of cell mark-
ers as well as reduced capacity for differentiation and proliferation [54, 55]. As a
result, a variety of three-dimensional culture systems and matrices composed of
collagen, hyaluronic acid, glycosaminoglycan, polyethylene glycol, and alginate
have been investigated as alternative matrices for stem cell culture [14, 56].
In an attempt to reproduce the three-dimensional native environment of MSC,
spinner flasks, wavy-walled cultures, and bioreactors were proposed and developed
as three-dimensional culture systems. Bioreactors demonstrate important advan-
tages besides cost: faster cell expansion, reduced risk of contamination, higher rate
of cytokine production, rigorous monitoring, and control of culture parameters
[57–59].
Besides three-dimensional structure, the inherent material properties of the
cell culture system, such as rigidity and composition, directly influence expres-
sion of MSC markers, the cell secretome, and cell viability and are considered as
part of the strategy to target stem cell differentiation into specific cell populations
[60]. MSC marker expression shifts from neurogenic toward myogenic or osteo-
genic markers as rigidity increases from low to intermediate and high stiffness,
respectively [61]. In addition, the use of extracellular matrix as a surface for cell
growth enhances VEGF and glial-derived neurotropic factor production while
decreasing synthesis of interleukin-6 when compared to the use of tissue-culture
plastic [62].
Besides the aforementioned inherent properties of the culture systems, the mech-
anism of cell release from the matrix and the route of cell administration need to be
considered when choosing an optimal culture matrix, as they affect cell yield and
viability. Stem cells cultured in biocompatible microcarriers, such as poly-ε-
caprolactone, can be directly administrated without the need of enzymatic release
[63]. The administration of cells associated with these types of carriers preserves
yield and functionality while providing a microarchitecture design for tissue regen-
eration [63, 64].
Another trypsin-free alternative is the use of thermoresponsive polymers, such as
poly-N-isopropylacrilamide, which are able to reversibly expand and adhere accord-
ing to the temperature. Hydrogels composed of these polymers are able to transit in
aqueous solutions upon temperature increase [65]. Thermoresponsive polymers
have been investigated as an alternative to increase stem cell yield, homing, and
engraftment.
1.6.4 Stem Cell Priming
Cell priming refers to techniques used to trigger stem cell “memory,” inducing cell
activity toward a specific therapeutic purpose. Stem cells may be primed for differ-
ent therapeutic purposes such as to promote immunomodulation, cell homing, target
differentiation, and decrease apoptosis [14]. Priming techniques include the use of
various stimuli such as pharmaceutical (valproic acid, progesterone), interleukins
(IL-1, IFN-γ), genetic (dsRNA), and environmental (three-dimensional structure,
hypoxia), among others [14].
Priming is an important strategy to enhance the overall effectiveness of stem cell
therapy. Nonetheless, it is especially important when guiding and limiting ESC and
iPSC proliferation and differentiation as these pluripotent stem cells demonstrate
increased oncogenicity.
1.7 Routes of Stem Cell Administration
Cell delivery is a crucial step in stem cell therapy since administration routes can
enhance cell survival and functionality, increasing stem cell therapy effectiveness in
hostile microenvironmental conditions [66]. Intravascular administration is consid-
ered systemic therapy, whereas injections into a particular site and topical adminis-
tration are local delivery methods (Fig. 1.6). Both systemic (intravascular) and local
delivery methods have demonstrated effectiveness in the treatment of vascular
pathologies [67]. Various diseases and clinical scenarios require different cell mech-
anisms of action, and there are a lack of studies comparing cell delivery methods
[68]. Accordingly, no consensus regarding the optimal administration route has
been achieved.
Administration routes
Local Systemic
Intra-arterial Intra-venous
Intranasal
Intrathecal
Intravitreal
Transepicardial
Transendocardial
Topic Injection
Scaffold
Intradermal Direct to
Spray Drops Hydrogel Intramuscular
Subcuteneous organ
Nanocarrier
Fig. 1.6 Routes of stem cell administration
Since treating different diseases requires several cell-induced regenerative path-

ways and proper cell homing, understanding the advantages and disadvantages of
different cell delivery methods is critical to better address the disease and tissue of
interest (Fig. 1.7).
1.7.1 Local Administration Routes
Injections into the diseased site and topical administration are the main local stem
cell delivery techniques. Local delivery methods enhance cell homing, a key feature
in the treatment of patients with peripheral vascular diseases, which by definition
prevents robust intravascular cell delivery [69].
Topical delivery typically consists of administration of a cell suspension directly
to the target lesion using a spray or drops. This is a local, simple, and painless deliv-
ery method that has been used as tissue replacement strategy for chronic wound and
burn treatment [68]. Topical cell delivery is a lower-risk alternative to local injec-
tions and systemic routes and enhances engraftment at the site of interest [68].
However, preliminary procedures to optimize cell homing, such as debridement,
may be unavoidable [66]. Moreover, inaccurate cell density and spacing and the
lack of extracellular protective environment lead to premature cell differentiation
and increased cell mortality [66].
Intramuscular, intradermal, and translesional injections are safe and simple
local administration routes [36]. Intramuscular administration is associated with a
higher cell dwell time and provides a highly vascular support for the therapeutic
14
Stem cell administration route

Local
Systemic
Topic
Hydrogel, scaffold or Injection
Intraarterial Intravenous Spray or drops nanocarrier
• Low risk • Simple

• Can be performed during • Can be performed during • Cell density and spacing
angioplasty angioplasty angioplasty angioplasty • Low risk
• Painless control • Inexpensive
• Possible • Possible • Simple • Better retention and
immunomodulation and immunomodulation and • Better vascular support
Advantages • Low risk engraftment • Crosses anatomical
glucose homeostasis glucose homeostasis • Inexpensive • Mechanical anchorage
optimizing effect optimizing effect barriers
• Stem cell behavior
modulation
• High cell death

• High cell death • Low addressing and poor
• High surgical risk • High surgical risk
• Low addressing and poor engraftment
• Low addressing and poor • Low addressing and poor
engraftment • High protocol complexity • No cell density and
engraftment engraftment
Disadvantages • No cell density and • Expensive spacing control
• Thrombosis/embolia • Thrombosis/embolia
spacing control • Infection risk
• Expensive • Expensive
• Premature differentiation • Ectopic stem cell
• Lung entrapping
transformation
Fig. 1.7 Stem cell administration routes, advantages, and disadvantages [36]
L. L. N. M. Lopes et al.
cells, enhancing local and systemic flow of cytokines [70]. Direct injections into
the target tissue have the potential to overcome delivery obstacles such as the
blood-brain barrier. Nonetheless, ectopic stem cell transformation and increased
surgical risk can be associated with direct injections. Additionally, intraparenchy-
mal cell injection leads to decreased systemic response and can result in tissue
trauma [68].
Bioscaffolds, fibrin, nanofibers, and other exogenous support systems have
been extensively investigated as local stem cell therapy adjuvants [71, 72]. The use
of these components is a strategy to modulate stem cell behavior and provide
mechanical anchorage, ultimately enhancing the efficiency of local administration
[69, 73].
1.7.2 Systemic Administration Routes
Intra-arterial and intravenous infusion are the main routes of systemic stem cell
administration. Systemic cell delivery leads to enhanced interaction with the host
immune system and regeneration signaling pathways and is appropriate to treat sys-
temic conditions or large areas of pathology not amenable to local treatment [68].
Intra-arterial infusion enables cell delivery to the site of interest with lower cell
loss when compared to intravenous delivery [74]. Thrombi and emboli formation
raise concerns regarding the intra-arterial route, especially in intra-coronary and
intra-carotid infusion [68]. These complications can be avoided by controlling infu-
sion speed, cell dosage, and size [75].
Intravenous infusion may be a more accessible and safer route compared with
intra-arterial infusion, as microthrombi can be captured by the lungs with fewer
significant clinical consequences [68]. However, intravenous infused cells are
retained in lung vasculature, at up to 90% in animal models, and therefore may be
removed by the host immune system [76]. After being entrapped in the lung, stem
cells are phagocytosed by monocytes, which switch toward immunoregulatory phe-
notype and are redistributed systemically [77]. As a result, a reduced number of
therapeutic cells may reach the organ of interest by this administration route, even
though there is the potential to have systemic immunomodulation.
1.7.3 Other Administration Routes
Intranasal and intrathecal stem cell administration have been investigated in the
treatment of neurologic pathologies such as subarachnoid hemorrhage and neuro-
pathic pain [78, 79]. Intravitreal stem cell infusion was described as therapeutic
alternative to diabetic retinopathy and retinal vein occlusion [80]. Transepicardial
and transendocardial routes are important local administration routes used in stem
cell therapy for ischemic cardiomyopathy [81, 82].
1.8 Stem Cell-Mediated Mechanisms of Regeneration
The therapeutic potential of stem cells was originally accredited to the cells’ broad
differentiation capability and potential for host tissue replacement by cell engraft-
ment. However, substantial evidence regarding direct cell interaction and trophic
paracrine effects has challenged the importance of stem cell differentiation in tissue
regeneration [83, 84]. This evidence was further supported by studies demonstrating
low MSC engraftment in both clinical settings and in vivo models [85–87]. As such,
the focus of stem cell therapeutic potential has shifted from direct differentiation
toward secondary signaling effector cells. Cytokines, growth factors, and chemokines
released by stem cells induce angiogenesis, immunomodulation, neuroregeneration,
and extracellular matrix production while reducing cell apoptosis and fibrosis [83, 88].
In recent years, investigations demonstrated that inactivated, apoptotic, and frag-
mented mesenchymal stem cells retain some immunomodulatory capacity and are
potentially regenerative [89–91]. Nonetheless, direct stem cell differentiation con-
tinues to play an important role in specific therapeutic scenarios such as tissue engi-
neering and tissue replacing therapies [92, 93].
1.8.1 Immunomodulation
Stromal cells are associated with a spectrum of different immunomodulatory mech-

anisms, attained via both soluble factors and direct cell-cell interaction [77]. Host
microenvironmental characteristics, stromal cell source, culture, and administration
conditions influence the expression of surface markers and the profile of secreted
cytokines [94–96]. Although a clear picture of stem cell-induced immunomodula-
tion is not well understood, some pathways are consistent.
1.8.1.1 Monocytes and Macrophages Interaction
Mesenchymal stem cells inhibit monocyte and macrophage differentiation into the
type 1 phenotype and dendritic cells, inducing type 2 anti-inflammatory and immu-
noregulatory differentiation by secreting interleukin-1 receptor antagonist (IL-1
RA) [97]. These induced anti-inflammatory monocytes play an important role in
MSC-mediated beneficial effects in the treatment of sepsis and induction of toler-
ance against alloimmunity and autoimmunity by secreting high levels of IL-10 and
suppressing T-cell activity, respectively [98, 99].
Recent investigations in asthma and peritonitis animal models show that MSC
phagocytosis by host monocytes induces anti-inflammatory type 2 differentiation in
a cytokine-independent pathway [100, 101]. Furthermore, stromal cells increase
monocyte count and phagocytic activity besides inhibiting dendritic cell maturation
and migration [100, 102, 103].
1.8.1.2 T-Cell, B-Cell, and Natural Killer Cell Interaction
Mesenchymal stromal cells suppress T-cell proliferation and induce a shift from
Th1 pro-inflammatory to Th2 anti-inflammatory subtypes [104]. MSC induce con-
ventional T-cell differentiation into regulatory T-cells (T-reg), important mediators
of graft immune tolerance and prevention of autoimmunity [105, 106]. Stromal cells
inhibit proliferation of alloreactive CD4+ and CD8+ T-cells [107].
MSC suppress plasmablast production and induce regulatory B-cell (B-reg) for-
mation, which promotes immunological tolerance [108, 109]. These effects are
thought to be promoted by direct cell-cell contact, whereas inhibition of B-cell pro-
liferation and differentiation by MSC is ascribed to soluble factors such as IFN-γ
and IL-1 RA [94, 110]. Natural killer cell activity and proliferation are also inhib-
ited by MSC via secretion of prostaglandin E2, TGF-β, nitric oxide, and other fac-
tors [111–113].
1.8.1.3 Complement and Coagulation Systems
MSC exert procoagulant activity by triggering both host coagulation and the innate
immune system, increasing C3 activation, D-dimer, and thrombin-antithrombin
complex formation while decreasing platelet counts [68, 114, 115]. Christy et al.
demonstrated increased tissue factor expression in MSC that varies according to the
cell source [116]. BM-MSC express less tissue factor compared to ADSC, which
are highly procoagulant [116]. Thus, theoretically, BM-MSC may be preferable in
the treatment of ischemic conditions and in systemic MSC infusion, whereas ADSC
may be preferable in the treatment of hemorrhagic conditions.
Procoagulant MSC activity increases with increased cell passages and can be
reduced by cell dilution, heparin, or tissue factor blockers [117, 118]. Adverse
thrombotic events reported by clinical studies will be discussed below.
1.8.2 Angiogenesis
Angiogenesis is the growth of blood vessels from pre-existing vessels. The angio-
genic potential of MSC has been the focus not only in investigations involving dis-
eases caused by limited angiogenesis, such as peripheral arterial disease, myocardial
ischemia, and stroke, but also in pathologic angiogenesis associated with tumors
[87]. Since MSC engraftment is typically low, the pro-angiogenic effects of MSC
are generally attributed to paracrine activity of secreted factors. The MSC secre-
tome is composed of various soluble factors such as vascular endothelial growth
factor (VEGF), fibroblast growth factor 2 (FGF-2), angiopoietin-1 (ang-1), interleu-
kin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), among others.
Investigations using the chicken chorioallantoic membrane and mouse matrigel
plug assay showed that BM-MSC induce angiogenesis in vitro [119, 120].
Another random document with
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This would benefit civilisation!
The form of Government in Morocco is the worst in the world. No officials
except the Customs Officers are paid. The consequence is that all live by
peculation, extortion, and bribery; or, in the words of a Moor describing the system,
‘We are like fishes—the big live by eating up the small.’ The population is reduced
to misery by the avarice of the Governors, and the latter, who have to send twice a
year large sums of money to satisfy the rapacity of the Ministers, are constantly
killing the geese (the farmers) to get the golden eggs. No security for life or
property, no encouragement to industry—and it is only a matter of wonder that the
whole country is not allowed to lie fallow.
The people are a fine race; but, since the days when they were ejected from
Spain and returned to Morocco to be subject to the rule of Sultans who are Pope-
Kings, they have degenerated gradually and become a degraded people.
I am described by Mr. Allen as being all-powerful. If so, the inference naturally
is that I have neglected to do my duty in requiring the Sultan and his Government
to introduce reforms and improvements.
I have never ceased for nearly forty years to preach and pray, to urge and beg.
My archives are full of notes addressed to Ministers of the Moorish Government,
with suggestions and propositions for improving commerce, introducing railways,
roads, telegraphs, mining operations, removal of restrictions on commerce, &c.,
&c. All this, however, to little purpose, for the venal advisers of the Sultan have no
interest in reforms or improvements when they do not see a direct means of filling
their own pockets. Promises are frequently made to me, but rarely fulfilled. I have
lately received promises that the prohibition on the exportation of barley and
wheat, now lying rotting in granaries, will be removed, and yet they hesitate and
delay; and so it is with everything.
Yet I may conscientiously declare that the few improvements which have been
effected in this country have been brought about through my representations and
acts.
I have worked hard of late to obtain the revision of the Treaty of Commerce (of
1856), which has been agreed to by the Sultan; but still the old story of promise,
pause, postpone, and then leave the matter alone.
I have frequently pointed out to my masters at home that if we consider it
desirable that the independence and integrity of a neutral Sovereign like the Sultan
should be upheld, so that the passage of our shipping through the Straits should
remain free in time of peace or war, it is our duty, it is the duty of all those Powers
who desire to maintain the status quo, to take a more active and decided part than
they have done hitherto in requiring the Sultan and his Ministers to introduce
reforms and improvements, and that the people of this country, who can be almost
seen from the shores of Europe, should not be allowed to remain in their present
degraded state—a disgrace to civilisation. But this is a totally different view of the
question from that of allowing France to become the mistress of the great gut of
commerce, where all our shipping must pass when bound for the East or for India,
and to say to us Ne plus ultra.
Again on June 13, 1884, Sir John returns to the subject of French
designs and British apathy:—
Papers will tell you much of passing events here, some correct, others,
especially French, full of mis-statements. Did you see the Standard of June 3?
It contains an admirable article and a letter from ‘One who Knows.’
John Bull ought to know what our insidious neighbours are about, though
singing to our Government ‘Lullaby, lullaby,’ whilst preparing the mine which will
explode when it suits their purpose to make themselves masters of the Straits and
Southern coast of the Mediterranean from Spartel to Tripoli!
You will have seen in the papers that Ordega returned in an ironclad and
demanded that the fort should salute the French flag with twenty-one guns before
his landing, and that the acting Minister for Foreign Affairs and all the authorities
should come down to the pier to meet him. ‘To hear is to obey,’ with the heavy
guns of the ‘Redoutable’ pointed at this wretched town; and all asked for was
conceded.
Yesterday a squadron of eight ships (!) arrived here; they remain, I am told, at
the disposition of Ordega.
P.S.—Just as I closed my letter the French squadron left, and I got a note to
say some arrangement has been made about protection to the Sheríf, and that the
question of frontier is deferred. It will come on, however, before long.
For the time the danger, as the following letter shows, was
averted:—
I said to a colleague the other day that man was prone to attribute to the
machinations of the devil anything that was adverse, whereas the poor devil is the
victim of his traducers; thus, I said, it is with me. Whatever goes wrong in Morocco
is attributed to that bête noire—Drummond Hay.
I know not whether you have seen another clever letter of ‘One who Knows’ in
the Standard of 30th ult.
With reference to the last paragraph, I have to say that a great change has
come over Ordega since the hurried departure of the French squadron (ordered by
telegraph). He has altered his tone with the Moorish Government and the local
authorities, and has told the Sheríf he cannot support the tribes who seek for his
protection against the authority of the Sultan. The question of rectifying the frontier
has also been abandoned, and the most solemn assurances are given to Italy,
Spain, and England that France will not disturb the status quo, unless a state of
anarchy takes place in Morocco compelling her to interfere. That is, however, the
question. Insurrection has been prevented, and the Sultan has given orders for the
chastisement of the disaffected tribes. This system of ‘eating up’ rebels, which you
can remember in the time of the old Sultan, renders of course the Sovereign most
unpopular with his unfortunate subjects.
With reference to the calumnious article in the Gaulois to which he

had called Lord Granville’s attention, he writes from London on
October 18, 1884:—
I have just received a courteous private letter from Lord Granville, saying he
had delayed replying to my letter as he has been in communication with
Waddington; he asks to see me on Monday.
The result of the interview is given in a letter three days later:—
Lord Granville was very civil and kind.

Ferry shirked getting justice done by publishing a disclaimer. His Lordship
agreed that a question should be put in the House of Lords. He only asked that
Zouche should give him notice, and promised to reply in a manner that would be
satisfactory to me. I gave him a full dose; outpouring all that was in my heart, both
about abuse and my having been passed over in the course of my career by
juniors—being told my ‘services were too useful in Morocco to be dispensed
with’—and now, I said, ‘the public press declares that I am useless and stop the
way,’ &c.
Lord Granville looked blandly at me, now and then making encouraging
remarks, such as, ‘Your character stands too high to be affected by the attacks of
men like Monsieur Ordega, and that bankrupt fellow,’ meaning ———.
Before the subject was mentioned in the House of Lords, Her

Majesty’s Government had given proof that they did not underrate
Sir John’s integrity and good service, thereby affording him sincere
satisfaction.
‘I think you will be glad to hear,’ he writes from Ravensrock in November, 1884,
‘that I have just received a note from Lord Granville announcing that Her Majesty
has been pleased to confer upon me the G.C.M.G., “in recognition of my long and
good service.” I confess I care little to add some letters of the alphabet after my
name, but I am pleased at the discomfiture of enemies who have been plotting
against me. My French colleague will have an attack of the English malady,
“spleen.” He is now treating with these unfortunate Moors at the cannon’s mouth.
‘An ironclad is in the bay to support his demands. He seeks for revenge, on
account of the humiliation suffered by his protégé and dupe the Sheríf, who is now
treated almost as an outcast by the Moors of Tangier, and is called the Sheríf
“francés.”’
The question to which Sir John referred in his interview with Lord
Granville was asked by a personal friend in the House of Lords. It
elicited replies which completely exonerated him from all the blame
which had been cast upon him, and was made the occasion for the
strongest expressions of satisfaction with his long and arduous
services. The following passages are taken from the Times of
November 22, 1884:—
Lord Zouche asked Her Majesty’s Government whether any official denial had
been published by the French Government to an article which appeared in the
Gaulois newspaper in the spring of this year wherein the editor accused several of
the Foreign Representatives at Tangier of corrupt practices, and among them the
British Minister, Sir John Drummond Hay, stating that he (the editor) had obtained
this information from the French Minister at Tangier, M. Ordega, who was at that
time in Paris on leave of absence; and, as it would appear that, owing to the fact of
no denial having been given to those grave charges, other accusations were made
by French journals which were referred to in English journals to the effect that Sir
J. D. Hay had obstructed British enterprise and commerce, and had encouraged
the Sultan of Morocco in his policy of resistance to all reform and improvement,
whether there were any grounds for such grave charges having been put forward.
Sir John Hay had been passed over by many of his juniors, and had now been
upwards of forty years in his present post, and he and his friends thought it
incumbent upon them to have some sort of public contradiction of these most
unfounded charges and some sort of public vindication of his character.
Earl Granville.—My Lords, I think the noble lord has correctly stated the facts of
the case. The editor of the Gaulois, it appears, accused Sir John Hay and his
colleagues of most intolerable practices, and gave M. Ordega as his authority.
Now, I am not sure that if I read such an article as this concerning myself I should
not treat it with contempt and trust to whatever character I had. But it is a different
thing when men serving their country in distant countries are thus unjustly
attacked, for, as in this case, the extract from the French paper is copied not only
into other foreign newspapers, but into English newspapers. However, after what
has occurred I thought it necessary, at the request of Sir J. D. Hay, to make an
application to M. Ferry, in courteous terms, that M. Ordega should be called upon
either to substantiate, or retract, or to say that he had not communicated the article
to the Gaulois. M. Ferry, in the first instance, said the Gaulois was perfectly wrong,
that no such report had been circulated by Ordega himself, and that he thought
that it was hardly worth while to contradict a statement made in a newspaper
which was well known to be so strongly opposed to the existing French
Government. M. Ordega was, however, applied to, and he telegraphed to Paris
entirely denying that he had communicated or inspired any such article in the
Gaulois. M. Ferry took the view that a great deal of time had elapsed, and that it
was really better not to call attention to the matter now. I have been in
correspondence with Sir J. D. Hay, and the last letter I received from him, only a
day or two ago, was to the effect that he was perfectly satisfied and that he should
trouble his head no more in the matter. I am glad to be able to add that I believe
there is no man in the diplomatic service more honourable or more energetic in the
discharge of his duties than Sir J. D. Hay. The noble lord says that Sir J. D. Hay
has been passed over for promotion; but I remember instances where persons
employed in the diplomatic service have been, to use a homely phrase, kicked
upstairs to get them out of a place where they were doing mischief instead of
good. I believe it to be exactly the contrary in the case of Sir John Drummond Hay.
He is most fit for the post he has held, and for that reason he has lost some
chances of personal advancement. I really can only repeat in the strongest way
that Sir Drummond Hay was quite justified in dismissing from his mind any
imputation made against him, and I have great pleasure in adding that a short time
ago the Queen granted him the Grand Cross of St. Michael and St. George.
The Marquis of Salisbury.—As the youngest and most recent of the foreign
secretaries the noble earl has referred to, I have very great pleasure in joining with
him in expressing the high estimation which was always entertained for Sir
Drummond Hay by his superiors. Not only was the charge against him ridiculous,
as it would have been against any representative of the Crown, but he is a man of
singular integrity and patriotism, and a more able, progressive, and intelligent
adviser does not exist in the diplomatic service. I always thought it a weak point in
our diplomatic arrangements that a class of men like Sir Drummond Hay, of whom
there are several in the service, who have special qualities for the particular post
they occupy, cannot be rewarded as they should be rewarded without detriment to
the public service, because by the rules of the service their rank cannot be
increased where they are, and because they cannot be removed from the post
they occupy without doing harm to the public service. I think Sir Drummond Hay
has been more than repaid by the universal confidence with which he is looked up
to and the very high esteem in which he has always been held. I think it is
unnecessary to vindicate any English statesman against foreign newspapers,
because their statements are, as a rule, absolutely phenomenal. I remember one
statement in a foreign newspaper which informed us that the noble duke for whose
eloquence we are waiting to-night was about to go abroad to spend the winter in
the South of France with his well-known greyhounds; and I remember another
such statement which informed us that a well-known statesman, and English Lord
Chancellor, was about to receive some high honour from the Crown for his
services as President of the Berlin Congress.
The Earl of Malmesbury and Lord Napier of Magdala also bore their testimony
to the high integrity and character of Sir Drummond Hay, and,
The Earl of Derby said that he did not know any person in any branch of the
public service more utterly incapable of such conduct as that imputed to him than
Sir Drummond Hay. He had always known him as an active and able public
servant.
RAVENSROCK
CHAPTER XXV.
LAST YEAR OF OFFICIAL LIFE. 1885.
Early in 1885 Monsieur Ordega was recalled by the French

Government and succeeded by Monsieur Féraud. Of the new French
Minister Sir John writes on March 30:—
Féraud has arrived, and is all that he has been described—very friendly and
desirous to please, liked by every one.
I gave him a dinner, and we have had many chats. He disapproves entirely of
Ordega’s proceedings, especially of his conduct towards me and of his
contributing venomous articles to journals regarding me and my acts.
He is a first-rate Arabic scholar and even poet, a good artist, a great
archæologist, and is writing a work on Tripoli. In two affairs I have tested his
assurances of good-will, and have good grounds for being satisfied.
I told him positively that there was no reason why there should not always be a
perfect ‘accord’ between us, except on one point, viz. if either of our Governments
desired to take possession of Morocco. ‘Kick it out,’ I said, ‘into the Atlantic a
hundred miles, and then the sooner Morocco was colonised by a civilised people
the better.’
Subsequent intercourse confirmed the favourable impression. In a

later letter Sir John writes again of M. Féraud:—
I think I told you that Féraud complained the other day of inaccurate and
malevolent reports, about his doings, in local papers, and said he hoped I did not
believe them. I told him I was the last man to put any faith in newspapers; that I
had been the butt of their shafts, which, at first, had stung; but I had grown so
accustomed to abuse that now, when not held up as the author of evil, I feel it and
wonder whether I have ceased to be of any importance in the eyes of my revilers.
‘You,’ I said, ‘will soon be accustomed to this also, and find it pleasant.’ ‘Charmes,’
the contributor of Débats, who has been with Féraud to Fas, was in the room, and
had been introduced to me. Last year he wrote virulent articles against me,
inspired, I think, by Ordega. He was sitting on my right, a little behind me, so I took
an opportunity of letting go my shaft, and added, ‘Why, even leading papers in
France chose last year to publish virulent and untruthful articles about me; but, far
from my having any rancorous feeling against the writers, I am grateful to them.
They drew public attention to me and my conduct in such a manner that it was
taken up in our Senate, and my conduct and character were vindicated by the
Ministers of all parties, and a mark of Her Majesty’s approval conferred upon me. I
am grateful to my revilers in England and France.’
When leaving, I gave C. my hand, and my eye, I dare say, twinkled. C. has
lately written an article in the Débats on the policy of keeping the status quo in
Morocco and disapproving of all the late policy. Féraud evidently inspired it.
In June, 1885, he writes:—
Now I am an old man, having entered my seventieth year. How time glides by.
Next year, if I live till then, we shall be quitting this for good. . . .
Féraud is still at the Court. He has made a good name by rejecting the
trumped-up and usurious claims of protected Jews. He denounced them to the
Sultan, and complained of public notaries who, in league with claimants, had
drawn up false documents.
Though he told me and other colleagues he had no affair of importance at Fas,
we know better. He aims at obtaining what France wants by cajolery and presents.
He eschews menace and force. He is more dangerous and far more able than his
predecessor. I shall, I think, get on well with him; I cannot blame him for playing
the game which suits his country. If England had been as contiguous to Morocco
as is France, I think ere this we should have annexed this misgoverned country;
but it would never do for us that France should hold the Straits—the gut of
commerce, the passage to India and the East. It is far more likely to be injurious
than if she held the Canal. As a sentinel of the Straits, I fire my gun, as a warning,
when I know of a move to obtain that object.
An article in Débats says, with some reason, that England, in consequence of
her failures in the East, is no longer looked up to by the Moorish Government as
before, and Italy is the rising sun. There is some truth in this. . . .
Oh! I shall be so glad to be at rest next year, if I live. I am sick of this
Government and its stupid, blind policy. As I said to Torras[63], ‘What is the use of
a fair lady saying she loves you better than any one in the world, and yet, while
refusing to allow you to embrace her, she showers kisses on the man whom she
declares she detests?’ Moor shut up by the sillygism.
His letters become at this time to an increasing degree full of
expressions showing that he was weary of the hopelessness of his
task. Thus he writes:—
Every day as the time draws nearer I sing, ‘Oh be joyful!’ I am sick of the
bother, and the dirty work of British subjects that I have to attend to. I am tired also
of writing and talking to this fossil Government, who cannot, or will not, understand
their true interests.
The same note is struck in a letter written from Ravensrock on

July 3, and September 7, 1885:—
We are well. Air here delightful, only 78° up till now, in the shade. Cholera
striding fast in a deadly march on the other continent.
Weber[64] just left. This time next year I shall have gone also, and go without a
pang, except to leave this lovely spot and the kindly peasantry who always
welcome me with bright faces and affectionate words. Civilised men are getting too
independent to be demonstrative of good-will and gratitude.
Sept. 7.
The Sultan has sent orders for the settlement of various long-pending affairs,
but nothing about the Convention of Commerce. Their last mot on this is, ‘How is it
possible that the Sultan’s treasury can be benefited by a reduction of duties on
exports?’ and, ‘If we export all that the English Minister suggests, in the revised
Tariff, the price of food will rise and the Moslem will be starved!’ These Moors are a
parcel of children; but we can hardly be surprised at their holding these absurd
views when a restrictive policy is pursued in commerce by the greatest nation in
the world.
As to the cable between this and Gibraltar, the Sultan’s advisers tell him that,
once it is laid, ‘Every day some Representative will telegraph for a ship of war!’
One does not know whether to laugh or to cry at such tomfoolery. I think, however,
jealous folk drop poison in the ear of the Sultan, and din in His Sherifian Majesty’s
ears that England has fallen from her high estate, and that she barks but can no
longer bite. The French papers in Arabic from Algeria sing this loudly.
The existence of slavery in Morocco called forth now and again

articles or letters which appeared in British journals, and in this and
the previous year the subject was much discussed in the
newspapers. At Sir John’s suggestion, all natives,—who as
employés of the officials attached to the Legation or Consulates
enjoyed British protection,—were required to liberate their slaves. He
believed, however, the form of slavery in that country to be lenient,
and though always urging on the Moorish Government the
desirability of abolishing an institution so obnoxious to modern ideas,
he foresaw difficulties that might, and did, prove insuperable in his
day. The following extracts from his letters on this topic, written at
different times, will show the attitude which he adopted towards the
question:—
We have no Slave Treaty with Morocco.

The British Government has at times called upon me for reports upon slavery. It
is of the mildest description. There is no slave trade by sea; five or six hundred
slaves are brought yearly by land, I believe. The men are bought for servants in
the houses of wealthy Moors, and the women as handmaids or servants. They are
very kindly treated, and when their masters die are given their liberty and a portion
of the estate.
With one exception, the only cases where I have been appealed to, to
intervene in behalf of slaves, have been to beg that the masters should not give
their slaves liberty! They preferred, they said, a comfortable home! Of course it is
desirable that slavery should be abolished even in Morocco; but it would be a
hopeless task to urge upon the Moorish Government the abolition of a domestic
institution, admitted by the laws of the Prophet, unless England had an opportunity
of rendering Morocco some great service, such as preventing her being attacked
by a stronger Power. Hitherto we have given her no aid but much advice in the
hour of need, and then deserted her.
When England has done as much for Morocco as she has done for Turkey and
Egypt, by preventing unjust aggressions, &c., &c., then she may hope to persuade
the Sultan to abolish slavery. Do you remember the long correspondence between
our dear father and the Sultan on the subject, which finished by His Sherifian
Majesty quoting Scripture in favour of slavery? I also have had a fling on the
subject. But slavery in Morocco exists in the mildest form. Slaves are not used for
agricultural purposes—not transported, like pigs, in vessels—and are generally the
spoilt children of the house.
I am not going to tell all this to the world, and thus appear to be defending
slavery.
However, I hammer away at the Moors on the subject, and in my last note
hinted that if they do not seek to satisfy public opinion by abolishing the
objectionable institution, they may be finally abolished—or something to that effect.
...
The Anti-Slavery Commissioners came to me to say good-bye, thanked me for
courtesy to them, and volunteered to say, ‘You are much belied, Sir John, but we
have taken care to sift for truth and shall make it known.’
The story about the Jewess who was flogged last year by the Governor of Dar-
el-Baida, in the presence of the native employed as British Interpreter, is most
exaggerated. I dismissed the Interpreter as soon as I heard he had been present
at the flogging of a woman.
Esther is a pretty girl of a dissolute character. The sons of the Interpreter had
been wasting their father’s patrimony on her, and when the old father remonstrated
with his sons, caught with Esther, one of them fired a pistol at him, so the
Interpreter rushed off to the Governor to demand the arrest and punishment of the
woman and of his sons. The Governor arrested and flogged all three, in
accordance with the law of this country, but there was no brutal punishment of the
girl. What nonsense to talk about the Interpreter having left without giving
compensation! Who was to give it, and who receive it? The Governor did his duty
according to their tyrannical law. The Interpreter did not punish the woman or his
sons; it was the Governor; and the Interpreter got dismissed by me from his
employment, a very severe mode of showing my disapproval of his being present
at the flogging of a woman. Subsequently I got the Sultan to abolish the flogging of
women by Governors for immorality, and to ordain that it shall be inflicted by a
Kadi only. Now a Kadi cannot order a woman to be flogged for adultery unless six
honourable men of spotless character declare they witnessed her misdeed! So no
woman will henceforward be flogged in Morocco. This I obtained in black and
white, and Esther got a warming with a beneficial result to all females of her class.
Féraud is to join the German Minister and me in negotiation. He is one of the
best Frenchmen I ever had to deal with. I expect the Sultan will kick hard against
the reduction of the Tariff.
Another subject with which Sir John was much occupied during
the closing years of his residence at Tangier was a scheme of prison
reform and the restriction of the period of incarceration for debt.
Writing on this point in March, 1886, he says:—
As to prisons, they are no doubt very bad; and so, Mrs. Fry tells us, were ours
fifty years ago. I obtained an order from the Sultan for cleaning them, and for
bread for those who have no means to buy food; but such orders, though given, if
they entail any expense, are soon disregarded. At Tangier I send a soldier of this
Legation now and then to inspect the bread. The quality and quantity diminish, and
the profits go into the pocket of the person in charge, so I have a constant battle
with the authorities.
Unless the whole administration of the Government were reformed, it is a
hopeless task trying to sweep this Augean stable.
English humanitarians are shocked to find no beds provided for prisoners. They
do not bear in mind that the poorer classes can always take up their bed and walk
—their bed, i.e. a rug, or piece of mat. They say how horrid it is that the prisoners
should have fetters. At Tetuan the fetters were removed; one hundred and fifty
prisoners rushed to the door, knocked over the guards, and fled into the
mountains. This has often occurred when prisoners are free of fetters.
I have taken steps to put a stop to the arrest and imprisonment of debtors of
British subjects without trial. Great cruelties have been practised upon debtors at
the demand of the Foreign Representatives, often for claims that are either
fraudulent or unjust. There is an outcry against me by British creditors because I
do not back the Government in extorting money from wretched debtors, too often
the victims of usurious Christians and Jews. I have just sent in a report to the
Government on this subject.
The Jews are certainly an oppressed race, but many of those who have
obtained protection conduct themselves in such an arrogant manner, and are guilty
of such infamous proceedings in forging false documents about debts of Moors, or
in putting forward preposterous claims based upon the grossest usury, that the
Mohammedans are exasperated, and some day, when a revolution takes place or
the Sultan dies, there will be a massacre and pillage of Jews in the interior.
When the chief Jews of Fas requested Féraud, during his mission, to obtain for
them a grant of ground to add to the Mellah[65], they especially requested that
protected Jews should not be allowed to inhabit the new quarter, as they said they
expected some day an onslaught of the Mohammedans on these persons, and
they wished to be separated from them. Féraud told me this.
The revision of the Commercial Treaty of 1856 might, perhaps,

have been forced upon the Moorish Government by the united
Representatives of the Foreign Powers. But, though on this point the
various Ministers joined hands, the hope entertained by Sir John that
a Convention might be framed which would abolish the system of
irregular protection was not realised. Under the terms of the
Convention of 1882, protection is still afforded to the numerous
agents of European traders and agriculturists, who therefore are not
immediately amenable to the jurisdiction of the Moorish authorities.
On this point Sir John had been defeated by the action of his
colleagues. But the wisdom of his proposals was abundantly justified
by the course which was taken by the negotiations for a new
Commercial Treaty in 1886. On the advantages of revising the
Commercial Code of 1856 all the Representatives of Foreign Powers
were agreed, and made common cause together. But their efforts
resulted in failure, and this failure was principally due to their
previous refusal to surrender or restrict their privileges of protection.
The Moorish Government showed a natural reluctance to encourage
European trade by an improved treaty, fearing that a greater influx of
European merchants and agriculturists would only multiply the
number of irregularly protected Moorish subjects as agents, and
remove more natives from the direct control of the Moorish
authorities.
Though Sir John might reasonably derive some satisfaction from
this practical proof of the wisdom of his advice to his colleagues, his
failure to obtain a revision of the Commercial Treaty deepened his
sense of the impossibility of reforming the Moorish Government. He
was weary of the hopeless struggle which he had carried on for more
than forty years. In spite of his personal regret at severing his
connection with Morocco, he longed to throw off the official harness
under which he had so often chafed. His letters in the summer of
1886 are filled with expressions of his delight at his freedom:—
July 2, 1886. Eve of departure. The Jews have sent a deputation and address.
Moors pour in with lamentations. Torras weeps in a letter. Even British subjects join
in the wail, whilst I continue to sing, ‘O be joyful.’
Alas! dinners and lunches are the disorder of the day; and speeches, which
being pathetic about our departure, choke me and prevent a fitting response.
July 4, 1886. Here I am, with my harness off, kicking my heels like an old horse
turned out to grass. So glad to send dispatches and letters to my address to the
Chargé d’Affaires.
I had a very flattering letter from Lord Rosebery’s private secretary, Villiers, to
say that his Lordship, in ‘recognition of my long and distinguished services,’ will
meet my wishes, &c., &c. . . .
We have been fêted successively by the diplomats here, and speeches were
made laudatory of me. In a circular, each vied in saying flattering things, such as
that I had been looked up to for my experience and clear-headedness as the
guide, &c. of the Diplomatic Corps.
We are on the look out for the s.s. Mogador. I think I told you the Forwood
Company have placed her at my disposal. . . .
Disgusted at the last proof of Moorish apathy and obstinacy, Sir

John declined to pay a formal visit of farewell to the Moorish Court,
and the Sultan’s Prime Minister addressed him a valedictory letter on
behalf of himself and his colleagues in office, a translation of which is
subjoined as a curious specimen of Oriental phraseology:—
Praise be to God!
The beloved and judicious Counsellor, who strives to promote good relations
between the two friendly Sovereigns, the Minister of the exalted Queen of Great
Britain and Empress of India, in the dominions of Morocco.
We continue to make inquiries regarding you and regarding your condition, and
we trust that you may always be prosperous.
Which premised, we have received your letter in which you inform us that, your
term of office having expired, you are about to quit this country, and you express
your regret that you are unable to have an audience of His Majesty, exalted by
God, in order to take leave of His Sherifian Majesty, and express your gratitude for
the marks of good-will, confidence, and friendship that His Majesty has shown
towards you, and you observe that you have served for forty years in these happy
dominions, and that our Lord and Master, the grandsire of our present Lord and
Master (assisted by God), and our Master the sire of His Majesty (may God
sanctify them both), bestowed on you their confidence, friendship, and trust, and
that our Lord and Master (may God assist him) has likewise held you in the same
regard, and that the friendship between the two Governments has remained in the
same state as formerly, it has neither altered nor been disturbed; and that you will
never grow weak in your devotion to the welfare of His Majesty and of his subjects,
for you are convinced of the friendship of His Majesty and of his subjects towards
you: you request us also to bid farewell in your name to the Uzirs and chief officers
of the Sherifian Court, whom you name, and you further state that, should God
prolong your life, you will return to this country after the lapse of a year, and will
reside here for a time during the winter months, and that, should it meet with Her
Majesty’s approval and your Government grant its consent, you would then visit
the Court in a private capacity with the view of taking leave in person of His
Majesty, exalted by God.
I have laid your letter before our Lord the Sultan, and His Majesty has taken
into consideration all you state in it, and (may God render him powerful) has
commanded me to reply to it and to state that your departure from these blessed
dominions causes great grief and sorrow, as it was sure to do, for you are one of
the wise and judicious persons of your illustrious Government, who have from
ancient times mediated between them and the Sherifian Government with
friendship, sincerity, and consideration, as is known to all, and about which there
can be no dispute, and which at all times has been continuously renewed, proved,
and confirmed by the strength and power of God. And the fact that your exalted
Queen selected a sagacious person like yourself, of excellent social qualities,
pleasant to have relations with, and seeking to do good, for service in this country
for so long a time, is a proof of her sincere friendship and of her desire to promote
good feeling and to strengthen the bonds of union between the two friendly
Sovereigns, and is a sign whereby is known Her Majesty’s extreme judiciousness,
wisdom, and judgment; for a person gives proof of his judgment and condition by
one of these things, viz. his envoy, his letter, or his present. His Sherifian Majesty
(may God render him powerful) has commanded me to convey the expression of
his sincere thanks and best acknowledgments to your beloved Queen, and to
yourself also, O friend, and invoked on Her Majesty an increase of power,
greatness, dignity, and grandeur; and on you, blessings on yourself, on your family,
children, relatives, and posterity.
I am to add that what you state regarding the confidence that was reposed in
you by our Lord and Master, His Sherifian Majesty’s sire (may God sanctify him), is
true and well known to every one, and His Sherifian Majesty (may God render him
powerful) likewise reposes confidence in you and regards you as a sincere friend,
and that your remark that the friendship between the two Governments has
undergone no change during the term of your office is also true, for the friendship
between the two Governments is the result of your services, verifying the opinion
held of you by your illustrious Government, the soundness of their judgment and
the accuracy of their discernment regarding yourself, and it (the friendship) has
through your assistance increased in purity, constancy and growth, in love and
affection, in word and deed. And as to what you say that you will not grow weak in
your devotion to His Sherifian Majesty and to his subjects, this is in accordance
with the opinion formed of you, and is what is confidently expected of you, for such
is the disposition of persons of a friendly and affectionate character, whether they
be near or far.
I have taken leave in your name of the Uzirs and officers of the Sherifian Court
whom you mentioned, and they all reciprocated your affection and gave
expression to it, and praised you, and invoked blessings on you, and were not
sparing in their expressions of sorrow and grief at your departure, and recited the
lines of the ancient poet:—
‘Though severed in body we suffer no hurt; for our hearts are united, welded by
pure love.’
With regard to your statement that if God prolongs your life, and it is agreeable
to His Sherifian Majesty, you will visit the Sherifian Court (exalted by God), and
that, should your Government approve, you would come with the object you
mention, our Lord (may God make him glorious) has commanded me to reply that
he prays your life may be prolonged by the power of God, and that you may
continue in happiness and health, leading an agreeable life; and if your
Government sanction your coming with this object, you are welcome, and such
sanction will be agreeable to His Sherifian Majesty (may God assist him), for it
(your Government) desires for you and for His Sherifian Majesty only what is good,
and you seek only to promote the welfare of them both, and how indeed could your
Government refuse to grant its sanction for what is beneficial? Our friendship for
you is everlasting, and its freshness will never fade day or night. May God be
gracious on the leave-taking, and not forbid the meeting.
Finished the last day of Ramadan, 1303 (July 3, 1886).
Mohammed Mefadal Ben Mohammed Gharrit.
On his retirement, Her Majesty was pleased to make Sir John a

Privy Councillor, and, though no longer holding a responsible post,
he was constantly appealed to on Morocco affairs by the British
Government.
‘The Foreign Office,’ he writes, in December, 1886, to his daughter, Mrs.

Brooks, ‘continue to send me dispatches about Morocco to be reported on, and,
when I make suggestions as to actions, they are adopted. This is pleasing to me,
and Government, though they rather bother me with their consultations, flatter me
by their continued confidence in my counsels.’
The Emperor of Austria sent Sir John his portrait set in brilliants
on the lid of a golden casket or snuff-box, and by special permission
of Her Majesty he was allowed to accept the order of the Grand
Cross of the Danebrog from the King of Denmark—for whom as for
Austria he had so long acted as Agent in Morocco. The Danish order
was the only one he was permitted to accept of the many foreign
decorations bestowed on him during his long career.
Until the end of Sir John’s life, it may be added, his name and
personal influence retained their ascendency over the natives, as will
be seen from such passages as the three following extracts from
some of his letters, written from Ravensrock in 1891 and 1892:—
The Basha, Hadj Mohammed Ben Abd-el-Sadek, called to make known to me

an order he had received from the Sultan to tell me that His Sherifian Majesty
looked upon me as a true friend of himself and of the people of this country, and
the Basha said he was directed, should any serious question arise, to ask for my
advice, as His Sherifian Majesty felt persuaded that I would always be actuated by
feelings of justice and friendship in giving counsels, as in the time of his sire and
grandsire.
I informed the Basha that I had withdrawn from intervention in official affairs,
and that some of the Foreign Representatives might be disposed to resent such
interference, even if my counsels happened to be beneficial to them in bringing
about settlements of vexatious questions.
The Spaniards are making lime at the caves of Ashkar, and live there. The
caves are Government property, and the stone has been used for making mill-
stones for two thousand years. The poor villagers of Medióna and Jebíla
complained to me, saying that they are afraid some day that mountaineers who
visit Ashkar to buy mill-stones may kill or rob these Spaniards, and then an
indemnity will be demanded by the Spanish Government, and they (the villagers)
will be thrown into prison. (I told the villagers to complain to the Basha.) . . .
Or again, in January, 1892:—
The ‘Thunderer’ remains here, as the mountaineers belonging to the Tangier

province have revolted against the Basha, and troubles are expected. I think the
Sultan will remove the Basha, who is unfit to govern. Happen what may, I and
mine are quite safe, for the Moors on mountains and plains look upon me as their
friend; and so indeed have I been. I remained during the Spanish war, when every
Christian and Jew bolted, and no barbarian harmed me or mine. . . .
Or, once more, the following written in February, 1892:—

When you arrive, all will be settled with the tribes. The Fahs are coming in with
presents of oxen, &c. Jebála follow. The new Basha, a good fellow, has written me
a letter, received yesterday, to say he is coming up here to pay me a visit as soon
as all the tribes have come in (and looks upon me as Baba[66]). The fact is, I was
appealed to by the tribes, &c. whether they should accept him, as he is a relative
of the late Basha. I said certainly—and told them to come. Ships of war are
leaving. All’s well that ends well.
CHAPTER XXVI.
OUT OF HARNESS.

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Stem Cell Therapy for Vascular

Diseases: State of the Evidence and

Pluripotent Stem Cell Therapy for Diabetes 1st Edition

Digestive System Diseases Stem Cell Mechanisms and

Concepts and Applications of Stem Cell Biology A Guide

Evidence based Therapy in Vascular Surgery 1st Edition

Advances in Stem Cell Therapy Bench to Bedside 1st

Personalized Computational Hemodynamics: Models,

Stem Cell Genetics for Biomedical Research Raul

Stem Cell Oncology : Proceedings of the International

State of the Evidence

Stem Cell Therapy for

ISBN 978-3-030-56953-2 ISBN 978-3-030-56954-9 (eBook)

© Springer Nature Switzerland AG 2021

Belo Horizonte, Brazil Tulio Pinho Navarro, MD, PhD

1 Introduction to Stem Cell Therapy and Its Application

10 Mesenchymal Stem Cell and Hematopoietic Stem

Brian H. Annex, MD Division of Cardiology and Department of Medicine,

Lucíola da Silva Barcelos, PhD Physiology and Biophysics, Federal University

C. Matouk, MD Department of Neurosurgery, Yale University School of Medicine,

Department of Biomedical Engineering, Yale University, New Haven, CT, USA

1.1.1 Brief History of Stem Cells

L. L. N. M. Lopes · T. P. Navarro (*)

© Springer Nature Switzerland AG 2021 1

• The term “stem cell” is proposed by Maksimov.

• Human bone marrow-derived stem cells are reported by Friedenstein et al.

• Murine pluripotent stem cells are isolated by Evans and Kaufman.

• Human embryonic stem cells are first isolated Thomson et al.

1.1.2  esenchymal Stem Cells vs Mesenchymal Stromal Cells

Adherence to plastic Marker expresssion Differentiation potential

1.2 Differentiation Potential

1.3 Stem Cell Types

1.3.1 Embryonic Stem Cells (ESC)

Fig. 1.3 Stem cell types

Bone marrow Adipose tissue Peripheral blood Umbilical cord

Stem Cell Type

• Cell lineage committed • Cell lineage

Fig. 1.4 Stem cell types, advantages and disadvantages [36]

differentiation potential lead to a higher risk of malignancy and requires immuno-

1.3.2 Adult Stem Cells (ASC)

1.3.3 Induced Pluripotent Stem Cells (iPSC)

1.4 Stem Cell Origin

Stem cell origin

Autologous Allogeneic Xenotransplantation

• Healthy stem cell source • No ethical conflict

Fig. 1.5 Stem cell origins, advantages, and disadvantages [36]

1.4.1 Autologous Transplantation

Autologous transplantation involves the administration of the recipient’s own cells,

1.4.2 Allogeneic Transplantation

Xenotransplantation involves the administration of either nonhuman live cells or

1.5 Stem Cell Isolation and Induction Protocols

1.5.1 Adult Stem Cells

1.5.1.1 Bone Marrow-Derived MSC (BM-MSC)

1.5.1.2 Adipose-Derived Stem Cells (ADSC)

1.5.1.3 Peripheral Blood-Derived MSC (PB-MSC)

1.5.1.4 Other Adult Stem Cell Sources

1.5.2 Embryonic Stem Cells (ESC)

1.5.3 Induced Pluripotent Stem Cells (iPSC)

Belo Horizonte, Brazil Tulio Pinho Navarro, MD, PhD

1 Introduction to Stem Cell Therapy and Its Application

10 Mesenchymal Stem Cell and Hematopoietic Stem

1.1.1 Brief History of Stem Cells

1.1.2 esenchymal Stem Cells vs Mesenchymal Stromal Cells

1.2 Differentiation Potential

1.3 Stem Cell Types

1.3.1 Embryonic Stem Cells (ESC)

1.3.2 Adult Stem Cells (ASC)

1.3.3 Induced Pluripotent Stem Cells (iPSC)

1.4 Stem Cell Origin

1.4.1 Autologous Transplantation

1.4.2 Allogeneic Transplantation

1.5 Stem Cell Isolation and Induction Protocols

1.5.1 Adult Stem Cells

1.5.1.1 Bone Marrow-Derived MSC (BM-MSC)

1.5.1.2 Adipose-Derived Stem Cells (ADSC)

1.5.1.3 Peripheral Blood-Derived MSC (PB-MSC)

1.5.1.4 Other Adult Stem Cell Sources

1.5.2 Embryonic Stem Cells (ESC)

1.5.3 Induced Pluripotent Stem Cells (iPSC)

1.6 Stem Cell Culture and Priming

1.6.1 Culture Media

1.6.3 Culture Matrices and Devices

1.6.4 Stem Cell Priming

1.7 Routes of Stem Cell Administration

1.7.1 Local Administration Routes

1.7.2 Systemic Administration Routes

1.7.3 Other Administration Routes

1.8 Stem Cell-Mediated Mechanisms of Regeneration

1.8.1.1 Monocytes and Macrophages Interaction

1.8.1.2 T-Cell, B-Cell, and Natural Killer Cell Interaction

1.8.1.3 Complement and Coagulation Systems