Stem Cell Bioprocessing

For Cellular Therapy, Diagnostics and Drug Development

Tiago G. Fernandes, M. Margardia Diogo, Joaquim M.S. Cabral

• Publisher: Woodhead Publishing
• Year: 2013
• Print ISBN: 9781907568886
• eBook ISBN: 9781908818300
• Topic: Biological Sciences
• Subtopic: Biotechnology in Medicine
• Index: Biological Sciences

Stem cell bioprocessing describes the main large-scale bioprocessing strategies for both stem cell culture and purification, envisaging the application of these cells for regenerative medicine and drug screening. Bioreactor configurations are described, including their applications for stem cell expansion, and stem cell separation techniques such as isolation and purification are discussed. Basic definitions are provided concerning the different types of stem cells, from adult stem cells to the more recent induced pluripotent stem cells. The main characteristics of these different stem cell types are described, alongside the molecular mechanisms underlying their self-renewal and differentiation. The book also focuses on methodologies currently used for in vitro stem cell culture under static conditions, including the challenge of xeno-free culture conditions, as well as culture parameters that influence stem cell culture. Approaches for both stem cell culture and separation in micro-scale conditions are presented, including the use of cellular microarrays for high-throughput screening of the effect of both soluble and extracellular matrix molecules. A further section is dedicated to application of stem cells for regenerative medicine.- Maintains a unique focus on both the basic stem cell biology concepts, and their translation to large-scale bioprocessing approaches- Envisages the use of stem cells in regenerative medicine and drug screening applications- Discusses the application of microscale techniques as a tool to perform basic stem cell biology studies

1

Characteristics of stem cells

Abstract:

Stem cells are characterized by their ability for unlimited or prolonged self-renewal and differentiation into highly distinct cell lineages. Due to these properties they are considered a very attractive source of cells for a wide range of clinical and pharmacological applications. However, significant progress needs to be made to fully realize this enormous potential. Stem cell functions are subject to tightly regulated control mechanisms, so gaining a deeper understanding of these mechanisms is crucial. Only then it will be possible to employ these cells to repair damaged tissue in regenerative medicine and tissue-engineering applications.

Key words

stem cells

pluripotent stem cells

adult stem cells

somatic stem cells

signaling networks

systems biology

1.1 Introduction

Stem cells are usually considered to have both the ability for unlimited or prolonged self-renewal and for differentiation into highly distinct cell lineages. These properties make these cells attractive for a wide range of clinical and pharmacological applications. However, stem cell functions are subject to tightly regulated control mechanisms, and in the fields of tissue engineering and regenerative medicine progress in harnessing these cells to repair damaged tissue will rely on gaining a deeper understanding of these mechanisms. Although great efforts have been made to elucidate some of these aspects, much more basic research must be carried out before new and effective therapies can emerge. Therefore, there are several major challenges in stem cell research, including the identification of new signals and conditions that regulate cell function (Discher et al. 2009) and the application of this information towards the design of stem cell bioprocesses and therapies.

1.2 Stem cell specialization

It is well known that several organisms, like newts and other amphibians, have the capacity to regenerate damaged tissue in their bodies; in limited amounts, this is also the case for humans, as human bodies are constantly regenerating blood, skin and other tissues. The identity of the potent cells that allow the regeneration of some human tissues was first revealed during experiments with bone marrow following the Second World War, which postulated the existence of stem cells in our bodies and led to the development of bone marrow transplantation (Thomas et al. 1957), a procedure now widely used in medicine. This discovery allowed, for the first time, regeneration of damaged tissue with a fresh supply of healthy cells based on the unique ability of stem cells to give rise to many of the specialized cell types in our body.

After recognizing the medical potential of stem cells and regenerative medicine through the success of bone marrow transplantation (Becker et al. 1963), great efforts were made to identify similar cells within embryos. Up to that point, studies of human development had demonstrated that cells of the embryo were capable of producing all the cell types in the human body. This led to the isolation of mouse embryonic stem cells in 1981 (Evans and Kaufman 1981), but it was not until 1998 that human embryonic stem cells were successfully isolated and cultured in vitro (Thomson et al. 1998). These cells could remain unspecialized for long periods of time, while maintaining their ability to transform into a plethora of specialized cell types, including nerve, muscle, bone and cartilage cells.

New therapies that rebuild or replace damaged cells and tissues with stem cells and/or stem cell derivatives, are now being developed. Stem cell research opens up the possibility of achieving major medical breakthroughs, offering hope to people suffering from devastating diseases like cancer, diabetes, cardiovascular disease, spinal cord injury and many other disorders. Both adult and embryonic stem cells may also provide a way for the development of innovative methods of drug discovery and screening. Finally, they are also powerful tools in fundamental research and can lead to a better understanding of the basic biology of the human body (Figure 1.1).

Figure 1.1 The potential use of stem cells and stem-cell progeny for cellular therapies, regenerative medicine, drug discovery and toxicology screening. Stem cells may also be used as model systems to elucidate the fundamental biology of the human body

1.3 Stem cell types and functions

Stem cells are special cells with a set of unique characteristics. They can renew themselves through mitotic cell division (a property known as self-renewal) or by differentiation into specialized mature cells. Some of these properties generate great interest in the scientific community, especially the processes by which unspecialized stem cells become mature cell types in the body. In fact, the human body is made up of over 200 different cell types that can be tracked to a pool of stem cells in the early embryo. During development, and later on in the adult, various stem cell types give rise to specialized, differentiated cells that carry out specific functions, such as skin, blood, muscle, and nerve cells.

Although all known types of stem cells may prove to be useful for different applications, each has both advantages and limitations. Embryonic stem cells, for instance, are isolated from the inner cell mass of the embryonic blastocyst and have the potential to produce all the cells in the organism. However, the adult stem cells found in certain tissues in the adult are limited in that they only produce cells from the tissue in which they reside. Stem cells have also been identified in umbilical cord blood and matrix (UCB/M) and placental tissue. They can give rise to various types of cells and matrix (De Coppi et al. 2007; Prindull et al. 1978; Romanov et al. 2003).

1.3.1 Embryonic stem cells

Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst – an early stage of the developing embryo (Smith 2001). These cells are considered to be pluripotent, which means that they can give rise to cells derived from the three embryonic germ layers, known as the ectoderm, endoderm and mesoderm (Figure 1.2).

Figure 1.2 Pluripotent embryonic stem cells are isolated from the inner cell mass of the blastocyst. These cells can differentiate into all derivatives of the three embryonic germ layers – the ectoderm, endoderm and mesoderm

In normal development, a sperm fertilizes an egg and creates a single totipotent cell – the zygote – which has the ability to produce all the differentiated cells in the organism, including extra-embryonic tissues like placenta. The zygote eventually divides and develops into a blastocyst. The blastocyst then implants in the wall of the uterus to become the embryo that continues to develop into a mature organism. The outer cells of the blastocyst, or trophoblast, form the placenta and the inner cell mass starts to differentiate into the progressively more specialized cell types of the body. Consequently, ESCs have a transient nature in vivo since they rapidly form the three primitive germ layers that will ultimately generate the entire organism: the ectoderm gives rise to the central and peripheral nervous systems, as well as the skin, cornea and lens of the eye and diverse epithelial cells; the mesoderm gives rise to skeletal, smooth and cardiac muscle, bone marrow, blood, fat, bone, cartilage and other connective tissues; the endoderm produces the epithelium of the entire digestive and respiratory tracts, the liver, pancreas, thyroid, parathyroid and thymus glands, as well as the epithelium of the urethra and bladder (National Institutes of Health 2001).

In order to isolate ESCs, the cells of the inner cell mass of the blastocyst are collected and transferred to a culture. However, some researchers claim that these cells are culture artifacts since ESCs are exposed in vitro to a very different environment, which causes them to exhibit properties not usually observed within embryos. In addition, the actual in vivo counterparts of ESCs should be better defined (Silva and Smith 2008). Nevertheless, ESCs seem to be more flexible than adult stem cells, since they have the potential to produce all cell types in the human body. They are also generally easier to isolate, purify and maintain in the laboratory than adult stem cells and are able to replicate themselves in an undifferentiated state for long periods of time, which means that a limited number of cells can build a large stock of stem cells to be used in research.

However, ESCs cannot be used directly for transplantation because they can originate teratomas after implantation. A teratoma is a rare tumor with tissue or organ components resembling normal derivatives of all three germ layers, as well as undifferentiated stem cells (Mummery and van den Eijnden-van Raa...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• List of figures
• List of tables
• Acknowledgments
• About the authors
• Chapter 1: Characteristics of stem cells
• Chapter 2: Stem cell culture: mimicking the stem cell niche in vitro
• Chapter 3: Bioreactors for stem cell culture
• Chapter 4: Stem cell separation
• Chapter 5: Microscale technologies for stem cell culture
• Chapter 6: Stem cells and regenerative medicine
• Conclusions
• Index