Application of the Scientific Method in Stem

Cell Research 12
Ahmed Gamal Tehamy, Mohamed Atef AlMoslemany, Toka A. Ahmed,
and Nagwa El-Badri

Contents
12.1 The Scientific Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
12.2 The Scientific Method in Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
12.3 Scientific Ethics in Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
12.3.1 Ethical Responsibilities of Scientists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
12.3.2 New Technologies and Ethics in Stem Cell Research . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
12.4 Application of the Scientific Method in Stem Cell Research: Milestones . . . . . . . . . . . . . . . . 354
12.4.1 1745: Parthenogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
12.4.2 1957: Intravenous Infusion of Bone Marrow in Patients Receiving Radiation
and Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
12.4.3 1958–1959: Testicular Teratoma in 129 Mouse Strain . . . . . . . . . . . . . . . . . . . . . . . . . 355
12.4.4 1978: The First Successful In Vitro Fertilization (IVF) . . . . . . . . . . . . . . . . . . . . . . . . 355
12.4.5 1981: The First Cultivation of Embryonic Stem Cells (ESCs) . . . . . . . . . . . . . . . . . 355
12.4.6 1986: Bone Marrow Transplant After the Chernobyl Nuclear Accident . . . . . . 355
12.4.7 1987: Developing Technology for Mutagenesis by Gene Targeting
in Mouse ESCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
12.4.8 1992: Development of Methods for In Vitro Culture of Embryonic Germ Cells
(EGCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
12.4.9 1996: Mammalian Cloning (Somatic Nuclear Transfer) . . . . . . . . . . . . . . . . . . . . . . . . 356
12.4.10 1998: Isolation of Human ESCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
12.4.11 2002: Differentiation and Transdifferentiation of Adult Stem Cells . . . . . . . . . . . 357

A. G. Tehamy · M. A. AlMoslemany
Faculty of Medicine, Menoufia University, Menoufia, Egypt
T. A. Ahmed · N. El-Badri (*)
Center of Excellence for Stem Cells and Regenerative Medicine (CESC), Helmy Institute of
Biomedical Sciences, Zewail City of Science and Technology, Giza, Egypt
e-mail: nelbadri@zewailcity.edu.eg

# Springer Nature Switzerland AG 2020 347

N. El-Badri (ed.), Regenerative Medicine and Stem Cell Biology, Learning Materials in
Biosciences, https://doi.org/10.1007/978-3-030-55359-3_12
348 A. G. Tehamy et al.

12.4.12 2006: Generations of iPSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

12.4.13 2016: CRISPR/Cas9 Genome Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
12.4.14 2017: Mechanoresponsive Cell Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

What You Will Learn in This Chapter

The scientific method aims to discover new reliable knowledge. It limits biases and
subjective tendencies, by following a stepwise process of investigation based on
rationalism, empiricism and/or skepticism. The basic steps of the scientific method
include observation, questioning, hypothesis statement, hypothesis testing,
generating and evaluating data, reaching conclusions, and eventually developing
theories. You will learn in this chapter the basics of the scientific method, and the
role of ethics in the process. The chapter will conclude with important milestones in
stem cell research, and how they relate to the scientific method.

12.1 The Scientific Method

The scientific method is defined as a systematic way of developing certain steps to examine
ideas and build knowledge by making observations, asking questions, formulating and
testing hypotheses, collecting and analyzing data, and developing theories [1, 2]. Following
the scientific method excludes bias risk and subjective tendencies, and the obtained
information is highly probable to be true. When scientific research is performed using
justified reliable methodology, the outcome is considered reliable knowledge, which is
highly distinguishable from the false or unjustified beliefs [3]. People have different
conventions, beliefs, and accumulated bodies of knowledge, and frequently, these
convections are untested and unjustified. The scientific method provides a standardized
process to test these beliefs and prove or disprove them [4]. Scientific thinking is the basis
for the scientific method and is rooted in three essential concepts: the use of empirical
evidence (empiricism), practicing rational logic (rationalism), and skepticism [5]. The latter
is defined as holding a skeptical attitude towards previously known knowledge that leads to
self-inquiry, hold provisional conclusions, and free thinking (willingness to change one’s
beliefs) [5].

• Empiricism: Empirical evidence is a type of evidence that relies on physical senses as

smelling, hearing or touch. Empirical evidence is considered reliable because other
people can experience and repeat it in the same way. Consequently, it can be replicated
several times by different research groups with the same outcomes each time
[5]. Scientists rely on empirical evidence as the main, and only, type of evidence to
make claims about nature and phenomena due to its objectivity and the ability to be
repeated and tested [5].
12 Application of the Scientific Method in Stem Cell Research 349

• Rationalism: Science is based on practice of the rules of logical reasoning. Scientists

use logical reasoning while investigating and understanding nature. Most individuals do
not think logically. Instead, they use emotional and hopeful thinking because it is far
easier to believe something is true; we feel it is true or wish it was true, rather than deny
our emotions and investigate phenomena in a logical and systematic way to find out
what is true [5].
• Skepticism: Skepticism involves developing a skeptical attitude towards supposed
knowledge or beliefs. Because deception is common and close to human nature
compromising the ability to obtain reliable knowledge, scientists must examine the
basis for holding their beliefs in a continuous manner [5]. In addition, scientists have to
ensure the reliability of the surrounding knowledge. If this knowledge matches the
logical consequences of one’s assumption and objective reality, as measured by empiri-
cal evidence, it is then considered safe to conclude that one’s beliefs and assumptions
are true and justified (in other words, reliable knowledge) [5].

12.2 The Scientific Method in Action

The basic components of the scientific method include observation, asking a question,
literature searching, hypothesis formulation and testing, obtaining and analyzing results,
deriving conclusions, and developing theory. These components are not necessarily
followed in that order (Fig. 12.1).

• Observation: Observation is the active acquisition of information by employing the

senses [6]. It can take many forms, such as watching a natural phenomenon like an apple
falling from a tree, an observation that prompted Isaac Newton to ponder his famous law
of gravity [7]. It may also be the result of immersing oneself in an experiment, or in the
form of cumulative evidence observed from the literature [6].
• Developing a Question: A research question is designed to solve a particular knowl-
edge gap [8]. It is essential for the researcher to know how to formulate a good research
question, as a good question is the corner stone of empirical research. Research
questions identify, clarify, focus, and pinpoint the research problem. They help in
building up a good hypothesis. When applying the scientific method, determining a
good question is an important starting point that will significantly impact the outcome of
the investigation [9]. When pondering a research question, it should fulfill certain
criteria as described by Hulley and colleagues [10]. It should be,
• Feasible: affordable in cost and time, adequate logistics and technical expertise, and
manageable.
• Interesting: answers interest the investigators and the community.
• Novel: rejects, confirms, improves, and/or extends previous research.
350 A. G. Tehamy et al.

Observaon

Queson

Literature Searching

Hypothesis

Testable?
Falsifiable?
Raonale?

Predicons

Modify
Tesng

Results

Hypothesis is true Hypothesis is false

Report Results

Reproducible

Corroborated
Theory
Hypothesis

Fig. 12.1 Steps of scientific method process. (Research cycle)

• Ethical: the research is conducted with minimal risk of harm and passable to be
approved by ethics boards.
• Relevant: impacts the scientific knowledge and future research.
12 Application of the Scientific Method in Stem Cell Research 351

Asking a question is followed by reviewing the literature, learning from previous

research and accumulated knowledge in the particular field of specialization [11].

• Formulating a Hypothesis: A hypothesis is the researcher’s argument to guess or

assume a predictive solution or explanation for a research problem [12]. The word
“hypothesis” consists of “hypo” which means “tentative” or “subject to verification,”
and “thesis,” which refers to a statement about problem solution [13]. A hypothesis is a
tentative statement for problem solution [14]. The complete hypothesis must have three
components: variables, population, or elements to be tested, and a relationship between
the variables. The main criteria of a good hypothesis is that it must be empirically
testable, falsifiable, precise, and realistic; otherwise, it will not serve for further
investigations [14]. A hypothesis helps researchers find suggested explanations or
solutions to their problems, drawing meaningful conclusions based on the empirical
data [14]. A hypothesis not only investigates research properly but also contributes in
developing new theories, and linking theories to investigations [14].
• Testing the Hypothesis: Empirical research depends mainly on testing hypothesis. A
hypothesis should be tested empirically to determine whether it is true or not [15]. Test-
ing the hypothesis, in empirical research, is usually achieved by conducting experiments
that help to diminish biases and obtain the most reliable knowledge. When a hypothesis
is empirically verified, it supports drawing meaningful conclusion with reliable infor-
mation and empirical data [14]. A hypothesis is not always completely true, and it is
possible to have results which contradict it [15]. If the hypothesis fails the test, it can be
either modified or changed into other hypothesis based on the results [14, 16]. If the
hypothesis passed, it has to undergo further tests to be corroborated [15]. In testing a
hypothesis, it is crucial that scientists avoid controversial practices that could cloud their
findings. HARKing (Hypothesizing After the Results are Known), which is a post hoc
hypothesis that uses known results to place a hypothesis [17] is frequently used by
scientists for several reasons. HARKing depends on already known results, and rejects a
true null hypothesis (type I errors), propounding hypotheses that would not pass
otherwise, and using post hoc explanations as a priori explanation [17, 18]. There are
various positions on the ethical use of this form of hypothesis. Some positions view the
practice as completely unethical [17], as it is predetermined and averts honest commu-
nication of research. Others view it to be more or less ethical according to circumstances
[19, 20]. Still, some researchers find HARKing to be acceptable provided that
hypotheses are explicitly inferred from prior evidence and theories, and the reader has
the ability to access the research data [18].
• Results and Data Analysis: Tests usually provide scientists with raw data
(observations, descriptions, or measurements) that is necessary to be analyzed and
interpreted to become evidence. Analyzing the data and discussing the results might
initiate further options and assumptions that have to be investigated in further studies
[14]. After publishing, other scientists may carry out different tests to verify the
352 A. G. Tehamy et al.

hypothesis. If it passes subsequent tests, it becomes highly corroborated and is now

considered to be reliable knowledge or a scientific fact that can build a theory [15].
• Theory: A hypothesis, when tested, helps to support or reject an existing theory. But
also, a hypothesis that is successfully tested implies certain facts and helps in developing
new theories based on the empirically tested data [14]. Thus, a theory is a buildup of
reliable knowledge deduced from a process that follows the scientific method about
observed phenomena, and provides explanations and predictions that can be tested [15].

12.3 Scientific Ethics in Research

New technologies and discoveries are generated daily to fulfill the evolving human needs
and aspirations. These novel discoveries must be controlled by a set of guidelines to ensure
a safe process without violating human beliefs on what is right and what is wrong.
Scientific research ethics thus controls scientific conduct within the framework of the
local and natural laws. The word “ethics” is derived from the Greek word “ethos” which
means custom or habit [21]. According to the Research Excellence Framework, research is
“a multi-stage process of investigation leading to new insights, which has to be regulated
by ethical standers” [22]. Indeed, the importance of ethics appears when the community
traditions are being challenged by the new developments [23].

12.3.1 Ethical Responsibilities of Scientists

Scientists have the main responsibility to apply the ethical regulations while conducting
their own research. They have to verify ethics on different levels, including the responsi-
bility towards their peers and the community, and to conduct research honestly and
objectively [24]. In clinical research, the safety of research subjects, the research
participants’ information, and the validity of data present the main ethical concerns.
Researchers—similar to physicians—must do no harm; the efficacy of the new products
must be scrutinized and proven superior to the current best practices. Periodically, scientists
have the responsibility to ask themselves about the importance of the ongoing research and
its benefit to the community [25]. Safety of the community and its engagement are essential
to achieve cultural conversion and to accept and understand the ramifications of new
technologies [26, 27]. Despite these common beliefs, the situations arise that may push
scientists to violate some of these ethical standards, for example, the urgent need of patients
to test the effectiveness of therapies, and the pressures of funding agencies and research
institutes for productivity and publications [28–30].
Ethical principles in human research are naturally different from those in other
disciplines such as engineering and physics [31]. While the laws of physics and engineer-
ing are almost fixed, humans are variable in their physical and psychological makeup; even
within the same population, variability, and the complexities arising from biological and
12 Application of the Scientific Method in Stem Cell Research 353

social differences [31, 32]. Dealing with humans in biomedical research or social studies is
a complex process that requires the input of many entities of scientists, physicians, patients,
and various members of the community [25].

12.3.2 New Technologies and Ethics in Stem Cell Research

“Ethics always says no to the new technologies,” this was one of the most critical responses
that Wolpe received from a group of scientists when asked about the rationale for not
effectively advocating their own work to their communities [24]. In fact, most of new
technologies seem subversive, dependent on challenging tradition and beliefs, and break-
ing the boundaries of the current knowledge. Ethical principles could be applied to prevent
new technologies from causing any kind of harm including physical harm, personal privacy
violation, and environmental damage. Scientists and ethicists are thus expected to collate
their efforts to accomplish valuable scientific research without ethical violations. In
addition, they should work simultaneously to prepare the community to accept new useful
technologies. The acceptance of the public for the research funded from their taxes is
clearly important. However, the majority of the public do not seem to have sufficient
scientific knowledge to make informed decisions [24]. An important example which
reflects the failure to prepare the community to new discoveries was the cloning of Dolly
in 1996 [24, 33]. After announcing this scientific breakthrough of the first mammalian
cloning, public surveys reported that more than 90% of Americans rejected the concept of
animal cloning [24]. Not only the specialized scientific outlets but also the media played a
pivotal role in science communication and spread of scientific knowledge, and in forming
the public opinion. Sometimes scientific news is exported to the public without adequate
clarifications or without preparation for new discoveries, and without educating the public
about the scientific basis or possible applications of the new research. Many discoveries in
the stem cell research field have suffered this lack of preparation. In case of the first
mammalian cloning (see Milestones, 31) the “new” technique challenged many traditions
and beliefs, and perpetuated a series of public reactions that were frequently not based on
understanding of the new methodology, and its ramifications. After cloning of Dolly, the
spotlight was focused on human cloning for reproductive purposes and as a source for
human organs. Although this application was too early and still unexplored, the media
reporting did not provide the opportunity to the scientific community to illustrate to the
public the concept of cloning and all its expansive and valuable applications.
Similar debate followed the publications on embryonic stem cells (ESC) research, both
in animals and humans [34, 35]. ESCs research has become a subject of controversy after
the first publications on the differentiation potential and possible vast clinical benefits of
ESCs. ESCs research was heavily debated, with questions on whether the life of an embryo
was more valuable than the life of an adult patient or a child. Public debate over this
technology started as soon as discoveries were announced without even much understand-
ing of the accuracy of the scientific details, for example, the proper sources of ESCs, and
354 A. G. Tehamy et al.

the possible applications of the new technologies to treat many ill-fated diseases. In both
cases, there was no true public education or understanding of the difference between
embryonic cells and fetal tissues, which face much more restrictions by the scientific
community. There was also no difference in the public eye between reproductive cloning
and therapeutic cloning, and its true potential to save lives. Research on fetal tissue is now
banned in most of the world, and ESC research has also been hampered [34, 35]. Although,
new alternative sources for stem cell research led to the Noble winning work of the induced
pluripotent stem cells (iPSCs) [36–38].

12.4 Application of the Scientific Method in Stem Cell Research:

Milestones

12.4.1 1745: Parthenogenesis

In 1745, Charles Bonnet noticed that the female aphids produced offspring without
fertilization by the male. His observation led to the discovery of “parthenogenesis” [39],
a type of asexual reproduction in which an ovum grows to a new individual without
fertilization. This was followed by several experimentations. In 1899, Jacques Loeb
reported the first case of induced parthenogenesis by artificial fertilization of sea urchin
eggs [40]. Thereafter, Gregory Pincus produced a baby rabbit by inducing parthenogenesis
in a rabbit ovum cultured in a mix of estrone and saline, then implanted in the mother rabbit
[41]. This revolutionary observation led later to animal cloning, and the progress of stem
cell research into induced pluripotent stem cells [41]. Research then evolved to include the
derivations of human parthenogenetic stem cells, due to their ability of unlimited division
and the ability to differentiate into all cell types [42–44].

12.4.2 1957: Intravenous Infusion of Bone Marrow in Patients Receiving

Radiation and Chemotherapy

The basis of the Noble work of Donnel Thomas was to test the hypothesis that bone
marrow transplantation could rescue lethally irradiated patients and restore their
lymphohematopoietic system. Thomas pioneered the intravenous infusion of bone marrow
cells in patients receiving radiation and chemotherapy. In the study reported in The New
England Journal of Medicine, six patients whose bone marrow was ablated by radiation and
chemotherapy received intravenous marrow infusion from healthy donors. The patients
survived, and the donor marrow could reconstitute their hematopoietic cell population with
mature functioning blood and lymphoid cells from donor origin [45].
12 Application of the Scientific Method in Stem Cell Research 355

12.4.3 1958–1959: Testicular Teratoma in 129 Mouse Strain

Another example of experimentation following observation is when Leroy Stevens

observed that mouse strain 129 develops spontaneous teratoma during the early stages of
gonadal differentiation [46]. These studies were the basis for the gold standard testing of
embryonic stem cells [46, 47].

12.4.4 1978: The First Successful In Vitro Fertilization (IVF)

Louise Brown was the first baby to be born after in vitro fertilization of human eggs outside
the body, in Manchester, England. Gynecologist Patrick Steptoe and scientist Robert
Edwards removed a mature egg from the mother and combined it with the father’s sperms
in vitro, where fertilization and normal cleavage proceeded to embryonic development. The
8-cell embryo was then implanted into the uterus after 2.5 days. Few months later, the first
IVF baby was born [48].

12.4.5 1981: The First Cultivation of Embryonic Stem Cells (ESCs)

After observing that mouse strain 129 develops teratoma [46], followed by the develop-
ment of embryonal carcinoma cell lines, it was proposed that early embryos contain cells
which are pluripotent unless they receive differentiation signals for embryogenesis
[49]. Evans and Kaufman were the first to discover and identify mouse embryonic stem
cells (ESCs) [50]. They isolated the ESCs from the inner cell mass of a mammalian embryo
in early embryogenesis (embryoblasts) and grew them in cell cultures [50]. In the same
year, Gail Martin reported similar findings [51]. Evans and Kaufman pointed out in their
paper the possibility of using ESCs as a vehicle for gene modification and gene targeting.

12.4.6 1986: Bone Marrow Transplant After the Chernobyl Nuclear

Accident

In 1986, the Chernobyl nuclear power station accident exposed nearly 200 people to high
doses of total body radiation. After the accident, the hypothesis which links radiation
damage to bone marrow stem cells was used to transplant bone marrow from allogeneic
donors into victims. The results, however, were poor due to the effect of extensive burns,
trauma, and other radiation-related organ toxicity [52].
356 A. G. Tehamy et al.

12.4.7 1987: Developing Technology for Mutagenesis by Gene Targeting

in Mouse ESCs

Thomas and Capecchi reported the first homologous recombination technology to mouse-
derived ESCs for mutagenesis by gene targeting. The researchers isolated and cultured
ESCs, introduced a mutation by a vector containing sequence similar to the gene to be
modified, and replaced the target gene in the chromosome [53].

12.4.8 1992: Development of Methods for In Vitro Culture of Embryonic

Germ Cells (EGCs)

In 1992, studies reported the isolation and culture of embryonic germ cells (EGCs) from
primordial germ cells in mice [54, 55]. These EGCs had similar characteristics and
differentiation potential to ESCs, and the pluripotent cells derived from preimplantation
embryos [54].

12.4.9 1996: Mammalian Cloning (Somatic Nuclear Transfer)

Ian Wilmut and colleagues from the Roslin Institute in Scotland reported that they
produced the first cloned mammal (Dolly) [33]. A nucleus of an adult sheep’s mammary
gland cell was successfully fused, using electric stimulation, with an enucleated egg from
another sheep. The produced cell was transplanted into the uterus of a surrogate mother
ewe. The newborn was an identical copy of the sheep from which the somatic cell nucleus
was obtained, and the cloning technique was named somatic cell nuclear transfer (SCNT)
[33]. The production of Dolly opened new horizons in stem cell research, and was the
foundation for many experiments afterwards, as well as for animal cloning.

12.4.10 1998: Isolation of Human ESCs

In 1998 James Thomson and colleagues reported the isolation and culture of human ESCs
derived from human blastocysts [56], and established the first human ESC line [56]. The
ESCs were derived from donated embryos, after in vitro culture to the blastocyst stage
[56]. Blastocysts become pluripotent upon division. Pluripotent cells of inner cell masses
were isolated, and Thomson’s team cultured five ESC lines that were used in research for
several years [56]. The efficiency of these lines to form derivatives of embryonic germ
layers was verified by the production of the three germ layers: endoderm, mesoderm, and
ectoderm [56].
12 Application of the Scientific Method in Stem Cell Research 357

12.4.11 2002: Differentiation and Transdifferentiation of Adult Stem Cells

It was believed that adult stem cells have restricted potential to differentiate into their
specified organ of origin. As such neural stem cells could only differentiate into cells of
nervous system origin, similarly (hematopoietic stem cells) HSCs would differentiate into
blood cells. New data showed that postnatal stem cells have higher differentiation capacity
than previously thought, and they can differentiate into multi-lineage and unrelated cell
types, resulting in plethora of publications on turning blood into brain and vice versa
[57]. Many efforts have then focused on developing differentiation cocktails to facilitate
the generation of cells that are most challenging to replace in the heart, lungs, nervous
system, etc.

12.4.12 2006: Generations of iPSCs

In 2006, Yamanaka and colleagues reported the ability to generate ESC-like cells from
adult somatic cells. These induced pluripotent stem cells (iPSCs) could differentiate into
almost all other cell types [37]. The extensive experimentation that preceded this milestone
took over a decade to examine over 20 different possible factors/combination of factors to
reprogram fully differentiated somatic fibroblast into a more embryonic, less mature
phenotype [37]. The first reported factors upregulated the pluripotency genes: Oct3/4,
Sox2, c-Myc, and Klf4 [58]. The generation of iPSCs paved the way to wide applications
of stem cell therapy in regenerative medicine, while overcoming the restrictions that
hampered ESC research and its use in the clinic. By availing autologous stem cells, after
reprogramming the patient’s blood, skin, or other cells into iPSCs, growing autologous
neurons, pancreatic cells, and other tissue cells for personalized medicine became possible,
while avoiding much of the risk associated with immune rejection [59].

12.4.13 2016: CRISPR/Cas9 Genome Editing

CRISPR/Cas9 is the abbreviation of Clustered Regularly Interspaced Short Palindromic

Repeats/CRISPR associated protein 9. Scientists at Stanford University developed a
method to correct the sickle cell disease mutations in human HSCs using CRISPR/Cas9-
mediated genome editing technology, followed by autologous transplantation [60]. This
technique works based mainly on two molecules: case 9 and guide RNA (gRNA). Cas 9 is
an enzyme that helps to cut the two strands of the DNA, while gRNA molecules find and
bind a particular sequence in the DNA.
358 A. G. Tehamy et al.

12.4.14 2017: Mechanoresponsive Cell Systems

Scientists at UC Irvine devised a technique called mechanoresponsive cell system (MRCS)

[61], which selectively identifies and destroys breast cancer cells that have metastasized in
mouse lungs by sensing the matrix stiffness in the tumor niche in vivo [61]. MRCS targets
the breast cancer metastases through mechanoenvironmental cues, specifically matrix
stiffness, to deliver cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine
(5-FC) to the active anti-metabolite 5-fluorouracil (5-FU), to kill the cancer cells [61].

Take Home Message

• The scientific method follows a stepwise process of scientific thinking that aims to
build reliable knowledge based on observation and experimentation.
• Achieving reliable knowledge is based on rationalism, empiricism, and/or
skepticism.
• The basic steps of the scientific method include observation, questioning, hypoth-
esis statement, hypothesis testing, generating and evaluating data, reaching
conclusions, and eventually developing theories.
• Scientific ethics aim to conduct research with integrity and respect for human
values and natural laws. They are governed by institutional review boards that
constitute diverse members of the scientific community and other communities.
• Important milestones in stem cell research have followed the scientific method,
and paved the way to current and future applications.

Acknowledgment This work was supported by grant # 5300 from the Egyptian Science and
Technology Development Fund (STDF), and by internal funding from Zewail City of Science and
Technology (ZC 003-2019).

References

1. Ryan JK. An introduction to logic and scientific method. Vol. 9, new scholasticism: Read Books
Ltd; 1935. p. 71–3.
2. D’Attore SM. The Shorter Routledge Encyclopedia of philosophy. Vol. 46, international philo-
sophical quarterly. Routledge; 2006. p. 134–135.
3. Landsberg PT, Passmore J, Ziman J. Science and its critics reliable knowledge: an exploration of
the grounds for belief in science, vol. 13. Leonardo: Cambridge University Press; 1980. p. 248.
4. Gauch HG. Scientific method in practice. Scientific method in practice: Cambridge University
Press; 2015. p. 1–435.
5. Madden EH. The principles of scientific thinking. R. Harré. Philos Sci. 1971;38(2):321–3. https://
doi.org/10.1086/288372
6. Kosso P. A summary of scientific method [Internet]: Springer Science & Business Media; 2011.
p. 41. Available from: https://app.box.com/s/aozfyfka32zchcuye4ythd14umfbfnfs
12 Application of the Scientific Method in Stem Cell Research 359

7. Newton I. Philosophiae naturalis principia mathematica. Vol. 1, Philosophiae naturalis principia

mathematica: G. Brookman; 1687.
8. Bryman A. The research question in social research: what is its role? Int J Soc Res Methodol.
2007;10(1):5–20.
9. Daniel P., Schuster WJP. Translational and experimental clinical research [Internet]. Lippincott
Williams & Wilkins; 2005. p. 490. Available from: https://books.google.com/books?
id¼C7pZftbI0ZMC&pgis¼1
10. Hulley SB. Designing clinical research. Lippincott Williams & Wilkins; 2007.
11. Ecker E, Skelly A. Conducting a winning literature search. Evid Based Spine Care J [Internet].
2010;1(1):9–14. Available from: https://pubmed.ncbi.nlm.nih.gov/23544018
12. Gettys CF, Fisher SD. Hypothesis plausibility and hypothesis generation. Organ Behav Hum
Perform. 1979;24(1):93–110.
13. Devi PS. Research methodology: a handbook for beginners. Notion Press; 2017.
14. Kabir SMS. Formulating and testing hypothesis. In: Basic guidelines for research: An introduc-
tory approach for all disciplines. Chittagong: Book Zone Publication; 2016. p. 51–71.
15. Thornton S. Karl Popper. In: Zalta EN, editor. The Stanford Encyclopedia of philosophy. Winter
201: Metaphysics Research Lab, Stanford University; 2019.
16. Schafersman SD. An Introduction to Science, scientific thinking and the scientific method. 1997.
17. Kerr NL. Harking: hypothesizing after the results are known. Personal Soc Psychol Rev [Inter-
net]. 1998;2(3):196–217. Available from:. https://doi.org/10.1207/s15327957pspr0203_4.
18. Rubin M. The costs of HARKing. Br J Philos Sci [Internet]. 2019. Available from: https://doi.org/
10.1093/bjps/axz050
19. Rubin M. When does HARKing Hurt? Identifying when different types of undisclosed post hoc
hypothesizing harm scientific progress. Rev Gen Psychol. 2017 Dec;21(4):308–20.
20. Leung K. Presenting post hoc hypotheses as a priori: Ethical and theoretical issues. Manag Organ
Rev. 2011;7(3):471–9.
21. Patrão NM. Ethics. In: Encyclopedia of global bioethics [Internet]. Cham: Springer International
Publishing; 2015. p. 1–12. Available from: http://link.springer.com/10.1007/978-3-319-05544-
2_177-1
22. Research Excellence Framework (REF). Guidance on submissions to REF 2021. 2019. p. 7.
23. Iaccarino M. Science and ethics. EMBO Rep. 2001;2(9):747–50.
24. Wolpe PR. Reasons scientists avoid thinking about ethics. Cell. 2006;125(6):1023–5.
25. Parveen H, Showkat N. Research Ethics. 2017:1–12.
26. Miller FG, Wendler D, Swartzman LC. Deception in research on the placebo effect. PLoS Med.
2005;2(9):e262.
27. Buchanan DR, Miller FG, Wallerstein N. Ethical issues in community-based participatory
research: balancing rigorous research with community participation in community intervention
studies. Prog Commun. Heal Partnersh Res Educ Action. 2007;1(2):153–60.
28. DuBois JM, Anderson EE, Chibnall J, Carroll K, Gibb T, Ogbuka C, et al. Understanding
research misconduct: a comparative analysis of 120 cases of professional wrongdoing. Account
Res. 2013;20(5–6):320–38.
29. Hofmann B, Holm S. Research integrity: environment, experience, or ethos? Res Ethics. 2019;15
(3–4):1–13.
30. Tijdink JK, Bouter LM, Veldkamp CLS, van de Ven PM, Wicherts JM, Smulders
YM. Personality traits are associated with research misbehavior in Dutch scientists: a cross-
sectional study. PLoS One. 2016;11(9):e0163251.
31. De Winter JCF, Dodou D. Scientific method, human research ethics, and biosafety/biosecurity.
In: Human subject research for engineers. Springer; 2017. p. 1–16.
360 A. G. Tehamy et al.

32. Meehl PE. Theoretical risks and tabular asterisks: Sir Karl, Sir Ronald, and the slow progress of
soft psychology. J Consult Clin Psychol. 1978;46(4):806–34.
33. Campbell KHS, McWhir J, Ritchie WA, Wilmut I. Sheep cloned by nuclear transfer from a
cultured cell line. Nature. 1996;380(6569):64–6.
34. (US) PC on B. Monitoring Stem Cell Research: A Report of the President’s Council on Bioethics.
President’s Council on Bioethics; 2004.
35. Begum DM, Khan DFA. Ethical issues in the stem cells research- An updated review. Int J Med
Sci Clin Invent [Internet]. 2017 Feb 14;4(2 SE-). Available from: https://valleyinternational.net/
index.php/ijmsci/article/view/707
36. Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, et al. A more efficient method
to generate integration-free human iPS cells. Nat Methods. 2011;8(5):409–12.
37. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult
fibroblast cultures by defined factors. Cell. 2006;126(4):663–76.
38. Ahmed TA, Shousha WG, Abdo SM, Mohamed IK, El-Badri N. Human adipose-derived
pericytes: biological characterization and reprogramming into induced pluripotent stem cells.
Cell Physiol Biochem. 2020;54:271–86.
39. Bonnet C. Traité d’insectologie ou observations sur quelques espèces de vers d’eau douce, qui
coupés par morceaux, deviennent autant d’animaux complets. Seconde partie, vol. 2: Durand;
1745. p. 232.
40. Loeb J. Artificial production of normal larvae from the unfertilized eggs of the sea urchin. J Am
Med Assoc. 1899;XXXIII(18):1106.
41. Pincus G. The parthenogenetic activation of rabbit eggs. Anat Rec. 1936;67(Suppl 1)
42. Tang Y, Yu P, Cheng L. Current progress in the derivation and therapeutic application of neural
stem cells. Cell Death Dis. 2017;8(10):e3108.
43. Schmitt J, Eckardt S, Schlegel PG, Sirén A-L, Bruttel VS, McLaughlin KJ, et al. Human
parthenogenetic embryonic stem cell-derived neural stem cells express HLA-G and show unique
resistance to NK cell-mediated killing. Mol Med. 2015;21(1):185–96.
44. Gonzalez R, Garitaonandia I, Semechkin A, Kern R. Derivation of neural stem cells from human
parthenogenetic stem cells. In: Neural stem cells. Springer; 2019. p. 43–57.
45. Thomas ED, Lochte HL Jr, Lu WC, Ferrebee JW. Intravenous infusion of bone marrow in
patients receiving radiation and chemotherapy. N Engl J Med. 1957;257(11):491–6.
46. Stevens LC. Studies on transplantable testicular teratomas of strain 129 mice. J Natl Cancer Inst.
1958;20(6):1257–75.
47. Stevens LC. Embryology of testicular teratomas in strain 129 mice. J Natl Cancer Inst. 1959;23
(6):1249–95.
48. Steptoe PC, Edwards RG. Birth after reimplantation of a human embryo. Lancet. 1978;2
(8085):366.
49. Karperien M, Roelen BAJ, Poelmann RE, Gittenberger-de Groot AC, Hierck BP, DeRuiter MC,
et al. Chapter 3 - tissue formation during embryogenesis. In: Van Blitterswijk CA De Boer
JBT-TE, Second E, editors. Oxford: Academic Press; 2015. p. 67–109.
50. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos.
Nature. 1981;292(5819):154–6.
51. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium
conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1981;78(12 II):7634–8.
52. Baranov A, Gale RP, Guskova A, Piatkin E, Selidovkin G, Muravyova L, et al. Bone marrow
transplantation after the chernobyl nuclear accident. N Engl J Med. 1989;321(4):205–12.
53. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-
derived stem cells. Cell. 1987;51(3):503–12.
12 Application of the Scientific Method in Stem Cell Research 361

54. Resnick JL, Bixler LS, Cheng L, Donovan PJ. Long-term proliferation of mouse primordial germ
cells in culture. Nature. 1992;359(6395):550.
55. Matsui Y, Zsebo K, Hogan BLM. Derivation of pluripotential embryonic stem cells from murine
primordial germ cells in culture. Cell. 1992;70(5):841–7.
56. Thomson JA. Embryonic stem cell lines derived from human blastocysts. Science (80). 1998;282
(5391):1145–7.
57. Verfaillie CM. Adult stem cells: assessing the case for pluripotency. Trends Cell Biol. 2002;12
(11):502–8.
58. Takahashi K, Okita K, Nakagawa M, Yamanaka S. Induction of pluripotent stem cells from
fibroblast cultures. Nat Protoc. 2007;2(12):3081–9.
59. Ebert AD, Liang P, Wu JC. Induced pluripotent stem cells as a disease modeling and drug
screening platform. J Cardiovasc Pharmacol [Internet]. 2012;60(4):408–16. Available from:
https://pubmed.ncbi.nlm.nih.gov/22240913
60. Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, et al. CRISPR/Cas9
β-globin gene targeting in human haematopoietic stem cells. Nature. 2016;539(7629):384–9.
61. Liu L, Zhang SX, Liao W, Farhoodi HP, Wong CW, Chen CC, et al. Mechanoresponsive stem
cells to target cancer metastases through biophysical cues. Sci Transl Med. 2017;9(400):
eaan2966.