Stem Cell Laboratory Techniques: A Guide for Researchers and Students

By Akon Higuchi

Stem Cell Laboratory Techniques: A Guide for Researchers and Students introduces the reader to stem cell culture, handling techniques and versatile applications used by researchers. Sections introduce stem cells, including definitions, types and basic use of stem cells in biomedical science research. The book explains laboratory procedures and techniques ranging from the extraction of stem cells from animals, cell seeding and culture, harvest and maintenance of stem cells, stem cell characterization, accurate recording, quality control, and more. In addition, it guides researchers on topics such as transcriptome analysis, proliferation study analysis, and microphysiological study.

Final sections cover useful and recent applications in stem cells, such as gene editing techniques and the preparation of stem cells for in vitro study, as well as stem cell lab design and equipment used in the lab. Lastly, human and animal research ethics are discussed.

• Introduces readers to the stem cell culture and moves to handling techniques and versatile applications
• Includes coverage of gene editing techniques for stem cells and stem cells for in vitro study
• Presents stem cell lab design and equipment used in the lab

• Language: English
• Publisher: Academic Press
• Release date: May 27, 2023
• ISBN: 9780128237304

Book preview

Chapter 1: Introduction: Stem cells and their application in research and therapy

Tong Jiabeia; Pooi Ling Moka; Suresh Kumar Subbiahb    a Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

b Centre for Materials Engineering and Regenerative Medicine, Bharath Institute of Higher Education and Research, Chennai, Tamil Nadu, India

Abstract

Stem cells are a group of cells capable of self-renewal and demonstrate restricted differentiation when placed under specific conditions. Based on the differentiation potential, these cells can be classified into totipotent stem cells, multipotent stem cells, and unipotent stem cells. According to the human developmental stage, stem cells can also be divided into embryonic stem cells and adult stem cells. Stem cells have become valuable in cell transplantation, regenerative medicine, and tissue engineering for correcting or managing disorders. In research, stem cells could be used to recapitulate the process of human development, thereby enhancing our understanding in the interplay between genes and surrounding molecules in a normal tissue and during pathogenesis of a disease. This is useful in facilitating novel drug development and toxicity evaluation. It is expected that stem cells will be the substitution of traditional therapy methods in clinical treatment and have become a hot spot in the field of drug screening.

Keywords

Stem cells; ESCs; iPSCs; MSCs; ASCs; Cell differentiation

Abbreviations

ADSCs adipose tissue-derived stromal cells

ASCs adult stem cells

BMSCs bone marrow mesenchymal stem cells

DPSCs dental pulp stem cells

ECVAM European Centre for Alternative Animal Experiments

EPCs endothelial progenitor cells

ESCs embryonic stem cells

EST embryonic stem cell test

HSCs hematopoietic stem cells

HUVECs human umbilical vein endothelial cells

iPSCs induced pluripotent stem cells

MSCs mesenchymal stem cells

Oct-3/4 octamer-binding transcription factor 3/4

SSEA-3/4 stage specific embryonic antigen 3/4

TDGF1 teratocarcinoma-derived growth factor 1

UMSCs umbilical cord mesenchymal stem cells

1: Definition and characteristics of stem cells

Stem cells are a group of cells that possess the unique characteristics of undergoing self-renewal and differentiation. They can proliferate in vitro and also differentiate into specific kinds of cells. During the development of the oosperm, the majority of the cells will undergo differentiation and maturation to form cells with specific phenotypes and functions. A small number of undifferentiated cells will be preserved in various tissues as a reservoir for tissue maintenance. The microenvironment in which stem cells are present is known as stem cell niche. This niche consists of signals that could modulate the proliferation and differentiation of stem cells. When there is a loss of tissue due to physical or chemical injury, for example, the stem cells will be activated to divide and differentiate into specific cells to replace the damaged tissue (Bacakova et al., 2018; Kolagar et al., 2020; Sameri et al., 2020).

In the area of regenerative medicine, stem cells can be used as an important strategy for cell or tissue replacement in the treatment of various conditions such as traumatism, cardiovascular disease, neurodegenerative diseases, endocrine disease, and other diseases. Additionally, human stem cells can also be used as research models to explore the various growth and differentiation mechanisms of cells and to study the processes involved in human development, growth, and diseases (Bourgeois et al., 2021; Elder and Dale, 2019; Fagan, 2019; Zakrzewski et al., 2019). Therefore, stem cell-related research has significant application value in cytotherapy, organ transplantation, and drug screening.

2: Classification of stem cells

2.1: Classification based on differentiation potential

Oosperm cleavage produces morula with 16–32 cells and then continues to develop into a blastocyst. The blastocyst can differentiate into the endoderm, mesoderm, and ectoderm. The cells derived from the different germ layers will differentiate into tissue specific progenitor/stem cells or form diverse adult cells. As tissues are injured by trauma, senility, diseases, or other factors, these progenitor/stem cells can play a role in facilitating the repairing process in the injured tissues. Based on the differentiation potential, stem cells can be classified into the following three types (Fig. 1):

(1)Totipotent stem cells: These are stem cells that can efficiently differentiate and develop into a complete individual. Totipotent stem cells can be generated from the zygote, which can divide into more cells and generate blastocyst. The human cells in the 16-cell stage after zygote division is totipotent (Baker and Pera, 2018; Cai et al., 2022).

(2)Pluripotent stem cells: Unlike totipotent stem cells, pluripotent stem cells have restricted potency to differentiate and develop into a complete individual. However, these stem cells can differentiate and form various cell tissues. For example, embryonic stem cells (ESCs) can proliferate infinitely and differentiate into over 200 types of cells in different tissues and organs, and then further aid in the formation of all the tissues and organs of an organism (Le et al., 2020; Riveiro and Brickman, 2020; Yamanaka, 2020). Other than that, mesenchymal stem cells (MSCs) in the bone marrow can differentiate into adipogenic, osteogenic, and chondrogenic cell lineages.

(3)Unipotent stem cells: Also known as committed stem cells, the unipotent stem cells can only differentiate into one or two closely related types of somatic cells, for example, skin fibroblast, vascular endothelial progenitor cells, and osteoblast. Most precursor cells belong to this class of stem cells (Bahmad et al., 2021; Sundaravadivelu et al., 2021; Xu et al., 2019).

Fig. 1

Fig. 1 Stem cell: Classification based on differentiation potential. Totipotent stem cells include zygotes and the cells of the 4-cell stage embryo, which can develop into the three primary germ cell layers and into extra-embryonic tissues such as the placenta. Differentiation begins at the 8-cell stage embryo. ESCs are the pluripotent stem cells derived from the blastocyst ICM, which can differentiate into any of the three germ layers. DSCs , dermis-derived stem cells; EPSCs , epidermal stem cells; ESCs , embryonic stem cells; ICM , inner cell mass (in blastocyst); MSCs , mesenchymal stem cells; NSCs , neural stem cells.

2.2: Classification based on cell sources

Based on the developmental stage of embryonic development, stem cells can be further divided into ESCs and adult stem cells (ASCs). The somatic cells can also be reprogrammed to achieve pluripotency by forced expression of specific transcription factors, direct nuclear transfer from a somatic cells into an enucleated egg, or somatic cell fusion with pluripotent cells (Laplane and Solary, 2019; Zakrzewski et al., 2019).

ESCs are the cells derived from the inner mass of the blastocyst, which predominantly exists in the undifferentiated state. These cells have developmental potency to differentiate into most types of cells in adult animals, including germ cells. Besides, embryonic germ stem cells, trophoblast stem cells, and some other stem cells can also be isolated from other parts intra and extra of an embryo (Suman et al., 2019; Yamanaka, 2020).

Adult stem cells exist in the tissues and organs of adult animals, such as the nerve, skin, and marrow, and possess the potency of repairing as well as regenerating. In an adult human body, ASCs primarily exist in a specific microenvironment and can maintain a state of dormacy for a long time without entering the cell differentiation cycle. These cells can also generate new stem cells or differentiate by using certain procedures and form new cells with a specific function to maintain the functional state of tissues and organs (Clevers and Watt, 2018; Gurusamy et al., 2018).

2.3: The classical types of stem cells

2.3.1: Embryonic stem cells (ESCs)

After the ovum is fertilized and an oosperm is formed, oosperm goes through a series of cleavage reactions to produce a set of cells called blastomeres. These cells further can effectively divide and rearrange to form a blastocyst, which consists of an inner layer of cells called the embryonic knob and an outer layer of cells called the trophoblast. The cells in the inner cell mass can eventually develop into the different tissues and organs of the body, so it can be an ideal source of ESCs (Kaufman and Thomson, 2019; Muhammad, 2020; Yamanaka, 2020). But isolation of ESCs from inner cell mass will destroy embryos. Since opponents of embryo research consider that deriving the stem cells destroys the blastocyst, an unimplanted human embryo at the sixth to the eighth day of development, this made the application of inner cell mass face ethical issues. So the researchers prefer to obtaining these cells from a single blastomere biopsy (Klimanskaya et al., 2006).

ESCs can form dense spherical cell colonies in the culture medium and express several transcription factors and cell surface markers, which include stage-specific teratocarcinoma-derived growth factor 1 (TDGF1), stage specific embryonic antigen 3/4 (SSEA-3/4), octamer-binding transcription factor 3/4 (Oct-3/4), and Nanog (Mora et al., 2017; Suman et al., 2019).

2.3.2: Mesenchymal stem cells (MSCs)

Mesenchymal stem cells (MSCs) are stem cells that primarily exist in mesenchyma of adult tissues and retain the potency of proliferation and differentiation, including bone marrow mesenchymal stem cells (BMSCs), adipose tissue-derived stromal cells (ADSCs), umbilical cord mesenchymal stem cells (UMSCs), and dental pulp stem cells (DPSCs). MSCs can display the dual functions of self-renewal and differentiation, and they can be differentiated into many kinds of adult cells, which are widely used in stem cell therapy. The dynamic balance of the two abilities can aid to maintain the normal fate of the organization. Otherwise, excessive differentiation and insufficient self-renewal can lead to stem cell depletion. Moreover, excessive self-renewal will hinder differentiation, stem cell expansion, and even lead to formation of teratoma. The prevention of excessive differentiation and insufficient self-renewal depend on the gene expression regulation of stem cells (Liu et al., 2020b; Saeedi et al., 2019; Zhang et al.,