STEM

CELLS
A REVOLUTION IN MEDICINE
PRESENTATION
Stem cells are increasingly being used in many research applications,
including tissue engineering and regenerative medicine, drug discovery,
toxicology, and disease modeling. These cells present many interesting
properties, such as self-renewing and differentiation into specialized cell
types, which make them very attractive for these applications. This e-book
provides an overview of the concept of stem cells, history and evolution;
their classification according to origin and potency; as well as their
properties and possible applications when associated with other
technologies, such as 3D bioprinting. We have brought together various
resources to help you better understand the functionalities of stem cells
and their great potential for the future of human health.

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© TissueLabs SAGL, 2021. No part of this document may be copied,
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or machine-readable form, in whole or in part, without prior written
consent of TissueLabs SAGL. Any other reproduction in any form without
the permission of TissueLabs SAGL is prohibited.

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 PAGE INDEX
INTRODUCTION ............................................................ 3
TYPES, CHARACTERISTICS AND
POTENCY OF STEM CELLS ............................................ 5
Embryonic Stem Cells............................................ 5
Adult Stem Cells (Somatic or Tissue-
specific Stem Cells) .............................................. 6
Induced Pluripotent Stem Cells (iPSCs) ................ 7
Totipotent Cells .................................................... 8
Multipotent Cells ................................................. 9
Oligopotent Cells ................................................ 9
Unipotent Cells .................................................. 10
PROPERTIES AND APPLICATIONS ............................... 11
REFERENCES ................................................................ 14

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INTRODUCTION
Stem cells are unspecialized cells capable of replicating or
self-renewing themselves, as well as differentiating into specialized cells.
They retain the ability to differentiate into other cell types, acting like a
repair system for the body and replenishing other cells as long as the
organism is alive. Due to these properties, stem cells are used as research
tools to study cell differentiation signals and mechanisms, thus allowing
understanding causes of disease and helping develop new therapies. They
also allow to evaluate the safety of new medicines, reducing the need for
animal testing.

Stem cells can provide a continuous supply of new cells that make
up human tissues and organs, becoming of great interest as a therapeutic
tool for various diseases and conditions. For example, cancer stem cells are
used to screen for potential anti-tumor drugs, and non-cancer stem cells
are essential platforms for replacing cells lost in degenerative diseases.

Researchers working on embryonic development during the

1950s were the first to propose the concept of a stem cell by observing
that this type of cell acts as the starting point for some biological
processes. Thenceforth, stem cells began to be seen as the source of
different kinds of blood cells. Their first therapeutic application was in
bone marrow transplant, a medical procedure introduced as a treatment
for cancer and genetic blood disorders. In 1961, James A. Till and Ernest
A. McCulloch described the first stem cells, called pluripotent stem cells,
following an observation that mouse bone marrow cells could differentiate
into various cell types (Figure 1).

Two decades later, scientists cultivated the first embryonic stem

cells from mice blastocysts. From that, several discoveries in this field
began to be introduced, such as the first human embryonic stem cells
being isolated by James Thomson in the USA in 1998. In 2012, Shinya
Yamanaka and John Gurdon were co-recipients of the Nobel Prize in
Physiology or Medicine for their discovery that mature cells could be
reprogrammed to a pluripotent state, which today has become the gold

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 standard when it comes to stem cell technology, known as induced
pluripotent stem cells (iPSCs).

Figure 1: Remarkable events in the evolution of stem cells.

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TYPES, CHARACTERISTICS
AND POTENCY OF STEM
CELLS
Stem cells can be identified and named according to their
potencies, which reflect their differentiation potential or “plasticity”; or
according to their origin, which refers to their stage of development. Cells
from different sources may present varying potencies, self-renewal
potential and mechanisms of action.

Based on their origin, stem cells can be grouped into three broad
categories: embryonic stem cells (ESCs), adult stem cells, and induced
pluripotent stem cells (iPSCs).

EMBRYONIC STEM CELLS

Embryonic stem cells (ESCs) are a group of cells present in an

embryo’s inner cell mass at a very early stage of development, a blastocyst,
which is reached within 4-5 days after fertilization. They divide and
differentiate inside the embryo into germ layers as they become
specialized. The blastocyst has two layers of cells: the inner cell mass,
which will form the embryo, and the outer cell mass, called trophoblasts,
that will form the placenta. The culture of embryonic stem cells has
become increasingly important due to their performance as a new source
for regenerative medicine, and as a tool for studying genetic diseases.

ESCs are identified by the presence of transcription factors, such

as Nanog and Oct4, that can maintain the stem cells in an undifferentiated
state and capable of self-renewal. The conditions to maintain ESCs in an
undifferentiated state during freezing and thawing cycles are critical,
demanding a feeder layer of embryonic fibroblast cells (MEFCs) or medium
that contains the anti-differentiation cytokine leukemia inhibitory factor
(LIF). The removal of LIF from the medium or removing ESCs from the

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 feeder layer leads to the formation of "embryoid bodies” present in all the
three germ layers - the endoderm, mesoderm, and ectoderm.

Despite the outstanding characteristics of ESCs for the use in the

regenerative field, the research on human embryonic stem cells raises
ethical and political controversies due to their origin, which end up
generating debates regarding the moral status of the human embryo.

ADULT STEM CELLS (SOMATIC OR TISSUE-SPECIFIC STEM CELLS)

Adult stem cells, also called somatic stem cells, are the cells found
in specific adult tissues that have the function to repair and originate
limited types of cells. They are considered less potent than ESCs and have
a restricted differentiation capacity. This type of stem cell increases the
repair of animal models of injury and preserves homeostasis by replacing
cells lost through the harm. Several tissue types have already been
identified as a source of adult stem cells. They are classified as
hematopoietic, mesenchymal, epithelial, muscular and neuronal.

Mesenchymal stem cells (MSCs) have the ability to differentiate

into various types of cells, such as adipocytes, chondrocytes, myocytes and
osteoblasts. The adipose tissue and dental pulp are non-invasive sources of
access for MSCs, as well as amniotic fluid, placenta and the Wharton's jelly
of the umbilical cord. Other sources of MSCs are the bone marrow, the
periosteum and hematopoietic stem cells can be found in blood from the
umbilical cord. They differ in multiple hematopoietic strains, NK cells,
macrophages, dendritic cells, platelets and erythrocytes, and are
commonly used to treat blood disorders and restore hematopoietic
function after radiation and chemotherapy.

In 2001, the first population of cells called lipoaspirate cells

isolated from human adipose tissue was reported by Mizuno et al. These
cells were separated from adipose tissue with the aid of digestion by
collagenase and became known later as adipose-derived stem cells (ASCs).
ASCs are mesenchymal stem cells that can differentiate into adipocytes,
osteoblasts, chondrocytes, myocytes, neurocytes, endothelial cells,
smooth muscle cells, and other cell types.

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 All tissues of the three germ layers can be a source to obtain adult
stem cells. Many studies have demonstrated that transplantation of adult
stem cells restores damaged organs in vivo, including bone tissue repair
and revascularization of the ischemic cardiac tissue orchestrated by
differentiation of the stem cell and generation of new specialized cells.
Cultured adult stem cells can also secrete various molecular mediators with
anti-apoptotic, immunomodulatory, angiogenic, and chemoattractant
properties that promote tissue repair. A key advantage of using adult stem
cells is their lack of rejection and their lack of ethical controversies.

The lack of rejection is related to the non-immunogenicity of

MSCs. They express low levels of class I human leukocyte (HLA)
histocompatibility complex (MHC) molecules and very low levels of class II
HLA, thus, not expressing co-stimulatory molecules, such as the CD40,
CD80 and CD86, which is important to divert immune surveillance.

INDUCED PLURIPOTENT STEM CELLS (iPSCs)

Limitations of adult stem cells, such as their finite potential of

differentiation, led to the development of a new type of stem cell, named
as induced pluripotent cells (iPSCs). These cells are obtained from adult
somatic cells by reprogramming the genes, seeking to reach a state similar
to ESCs. In 2006, Takahashi and Yamanaka were the first ones to report
iPSCs, more specifically mouse iPSCs, by transducing mouse fibroblasts
with four genes encoding transcription factors such as the octamer-binding
transcription factor 3/4 (OCT3/4), SRY-related high-mobility group box
protein-2 (SOX2), the oncoprotein c-MYC, and Kruppel-like factor 4 (KLF4).
In 2007, they were able to generate human iPSCs from adult human dermal
fibroblasts with the same four factors. The newest focus of scientists
worldwide is researching new methods to generate safe iPSCs without
genomic manipulation.
iPSCs are similar to ESCs in terms of morphology, proliferation,
surface antigens, and gene expression. These stem cells have significant
potential in therapeutic medicine, as doctors can generate cells of
practically all body organs in a personalized manner. Therefore, the
technology enables the use of iPSCs in areas beyond regenerative
medicine, such as drug development and disease modeling.

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 Apart from their origin, stem cells can also be categorized
according to their differentiation potential into five groups: totipotent,
pluripotent, multipotent, oligopotent, and unipotent stem cells (Figure 2).

Figure 2: Scheme representing the potential and the differentiation

hierarchy of stem cells.

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TOTIPOTENT CELLS

The most undifferentiated cells found in the early stage of

development are called totipotent or omnipotent stem cells. Totipotent
cells are the cells of the first two divisions after the oocyte is fertilized - the
zygote - and they can develop into a complete organism or differentiating
into any of its cells or tissues, including the placenta and extraembryonic
attachments.

PLURIPOTENT CELLS

Pluripotent stem cells can differentiate into cells that arise from
the ectoderm, endoderm, and mesoderm, the three germ layers. They
were initially derived from the internal cell mass of the blastocyst. Still,
scientists have already generated pluripotent cells by reprogramming
somatic cells, which became known as induced pluripotent stem cells
(iPSCs) and share similar characteristics with ESCs. Essential features that
define pluripotent stem cells include growth as multicellular colonies,
regular and stable karyotypes, the ability to replicate in passages
continuously, and also the ability to differentiate into cells derived from all
three embryonic germ layers.

MULTIPOTENT CELLS

Multipotent stem cells can be found in most human body tissues

and differentiate into cells of a single germ layer, being mesenchymal stem
cells the best-known example of multipotent stem cells. Tissues where
multipotent cells can be found include the bone marrow, adipose tissue,
bone, Wharton's jelly, umbilical cord blood, and peripheral blood. MSCs
adhere to cell culture plates and are characterized by specific surface cell
markers; they can be isolated from many adult tissues and can differentiate
into cells of the mesodermal lineage, such as adipocytes, osteocytes, and
chondrocytes, as well as cells from other embryonic lineages. This type of
cell is also known for interacting with cells of the innate and adaptive
immune system, leading to the modulation of various effector functions.

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 OLIGOPOTENT CELLS

Oligopotent stem cells are able to self-renew and form two or

more strains in a specific tissue; they can differentiate into just a few cell
types. Known examples of these cells are lymphoid or myeloid stem cells.
Another example of oligopotent cells in the corneal epithelium cells, a
squamous epithelium that is continuously renewing.

Oligopotent cells are generally known as progenitor cells, they

can divide only a limited number of times and have the ability to
differentiate only into a specific type of cell. They are more specific than
stem cells and can only be pushed to differentiate into their target cell.

UNIPOTENT CELLS

Unipotent stem cells can self-renew and differentiate into just one specific
cell type and form a single lineage, such as muscle stem cells, which
originate only from mature muscle cells and not any other cells. However,
it is the property of self-renewal that distinguishes the unipotent stem cells
from non-stem cells.

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PROPERTIES AND
APPLICATIONS
Stem cells are present in the human body from the early stages of
development until death, being a vital part of the functioning of all organs
and tissues, promoting their development, growth, maintenance and
repair. Therefore, studying stem cells in the laboratory is important to
discover the mechanisms behind stem cell self-renewal, allowing scientists
to grow stem cells more efficiently. Unraveling stem cell’s functions is also
essential to understanding how normal embryonic development is
regulated and even understanding how cancer development works.

The treatment of incurable or challenging diseases has recently

relied on tissue engineering and regenerative medicine with stem cell
applications. The stem cell sources for this application can be autologous,
allogeneic, or xenogeneic. Autologous refers to the use of the host's cells,
while allogeneic is related to the use of cells from another individual. In
xenogenic transplants, the cells are obtained from an animal other than
human, which is not an ideal source since it can present immunogenic risks
restricting its use in tissue engineering, unlike autologous and allogeneic
sources.

Tissue engineering approaches to culture and implantation of

stem cell lines show great potential and have an outstanding contribution
to regenerative therapies. Although major improvements have occurred in
this area, there is still a need for advanced studies to develop fully
functional organs and tissues since the lack of vascularization in 3D tissues
end up being a significant cause of cell death.

For the therapeutic application of stem cells, they can be

employed either at the undifferentiated or differentiated state. The focus
of therapy can be on the differentiation potential of stem cells or
concentrating on their regulatory effects, such as the immunomodulatory
properties of MSCs. Regarding MSC isolation technologies, adherence to

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 a plastic surface, followed by cytofluorimetric analysis to determine the
presence of markers that are classified as mandatory properties of
mesenchymal stem cells; these expressed markers include CD44, CD73,
CD90, CD105, and CD166.

Other properties that are marked as criteria for identifying MSCs,

stipulated by the International Society for Cell and gene Therapy (ISCT), in
addition to adherence to plastic and specific surface antigen expression
patterns, are the differentiation of these cells in chondrogenic, osteogenic,
and adipogenic lineages. These differentiations can be proven through
essays such as the oil red staining for chondrogenic differentiation, alizarin
red staining for osteogenic differentiation, and aggrecan
immunofluorescence for adipogenic differentiation.

With the discovery of human-induced pluripotent stem cells in

2006, patient-specific stem cell lines can now be created from adult stem
cell types, such as peripheral blood mononuclear cells by venipuncture, or
fibroblasts from dermal skin by skin biopsy. The ability to reprogram
specific patient cells also allows for the creation of stem cells containing
hereditary mutations, allowing for personalized studies.

3D bioprinting approaches using stem cells can be extremely

advantageous for regenerative medicine applications, including modeling
and treating heart diseases, for example. The ability to accurately
reconstruct native heart valves using stem cells, for example, has
substantial clinical implications. This technology is up-and-coming when
taking into account the regeneration of organs in a personalized way.

At the University of Illinois, in 2019, Eben Alsberg and his team,

using a bioink made up of only stem cells, created a printing platform that
allows 3D bioprinting of cells without classical scaffold support. They used
a temporary hydrogel bead bath in which printing took place. The
approach used in this project consists of printing the cells in the hydrogel
sphere matrix, where they are exposed to ultraviolet light, which crosslinks
the spheres. Then, the spheres are frozen in place, allowing the printed
cells to connect, mature, and grow within a stable structure. When there is
a need to supply fresh nutrients to the cell or discard the waste produced

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by them, the media that bathes the cells flows smoothly through the
crosslinked gel granules.

By combining 3D bioprinting with stem cells, Gu et al. (2017)

evaluated human-induced pluripotent stem cells’ ability to differentiate
along the neural lineage, to generate neurons and glial cells. They used a
printing technology based on cell extrusion within a bioink composed of
polysaccharides such as alginate, carboxymethyl chitosan, and agarose.
The iPSCs were then differentiated in situ for self-assembling of 3D
embryoid bodies that expressed markers from the three germ layers.

Adult stem cells also frequently participate in tissue engineering

studies, as seen in a study by Hirsch and his team (2017), in which they
were able to restore 80% of the skin of a patient suffering from a severe
and lethal genetic disease, known as bullous junctional epidermolysis
(JEB). The procedure consisted of using fibrin culture grafts containing
genetically modified autologous keratinocytes and epidermal stem cells.
The regeneration promoted a robust epidermis, fully supported by
epidermal stem cells, resistant to mechanical stress, and did not develop
blisters or erosions for 21 months.

Finally, it is evident that stem cells’ properties are essential for

maintaining human life. Their use in new technologies, such as
regenerative medicine, tissue engineering, cancer research, and biological
models, to study new pharmacological agents can ensure unique health
benefits. These cells’ potential to replace damaged tissues and regenerate
organs is vital since allogeneic transplantation can solve significant
complications resulting from standard transplants, with potential to
improve the lives of more than 109,000 patients who remain on the organ
waiting list in 2020 in the US alone. However, the therapeutic use of stem
cells presents some challenges since the underlying mechanism of action
of transplanted cells is not yet fully understood. Therefore, some critical
factors such as heterogeneity and inherent differential potency of stem
cells and the heterogeneity of disease progression among patients must
be carefully considered in the association of stem cells with host tissues for
long-term beneficial results.

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