CHAPTER 1
Introduction
1.1 Introduction
Each year, millions of people suffer from spinal cord injury and diseases such as myocardial infarction, diabetes, and leukemia. In the past, therapeutic approaches have been limited to the removal of injured parts by surgery and medical treatment.
Human pluripotent stem (hPS) cells have high differentiation ability relative to adult stem cells such as adipose-derived stem cells and bone marrow-derived stem cells. Several studies have demonstrated that hPS cells can be differentiated into specific cell lineages derived from three germ layers. Thus, hPS cells are a promising source for the replacement of damaged or lost cells in regenerative medicine. However, it is necessary to control the proliferation and differentiation of stem cells in xeno-free culture conditions for the clinical use of stem cells. In this case, cell culture biomaterials play an important part in the stem cell fate of proliferation as well as the stem cell fate of differentiation into specific lineages of cells that are going to be used for drug discovery and regenerative medicine.
1.2 Stem Cells
Stem cells are capable of self-renewal, proliferation, and differentiation to various cell lineages, making them advantageous for regenerative medicine applications. Importantly, self-renewal and cellular proliferation are not synonymous, because the former term encompasses both the differentiation and future mitotic potential of the daughter cells in addition to cell division.
Depending on the type and maturity of stem cells in the tissue, stem cell potency and capacity for self-renewal can be varied. There are two main types of stem cells, embryonic and non-embryonic cells. Embryonic stem (ES) cells are pluripotent and ES cells can differentiate into the cells derived from all three germ layers (ectoderm, mesoderm, and endoderm). Non-ES cells are multipotent. Their potential to differentiate into different cell types seems to be more limited and more importantly, multipotent stem cells have an aging problem (limited proliferation) which ES cells do not have (infinite proliferation). The differentiation potential can be classified into four levels: totipotent, pluripotent, multipotent, and unipotent stem (progenitor) cells (Figure 1.1).
Totipotent stem cells can differentiate into embryonic and extra-embryonic cell types, which can construct a complete and viable organism. Totipotent cells are produced from the fusion of an egg and a sperm cell. The fertilized egg and the cells produced by the first few divisions of the fertilized egg are totipotent. Totipotent stem cells give rise to somatic stem/progenitor cells and primitive germ-line stem cells.
Pluripotent stem cells can differentiate into nearly all cell types of the adult organism, because they have the ability to differentiate into the cells derived from three germ layers: endoderm, mesoderm, and ectoderm.
ES cells, which are pluripotent stem cells, are derived from totipotent cells of the early mammalian embryo and are capable of differentiating into cells representing the three embryonic germ layers, namely ectoderm, mesoderm, and endoderm or any of more than 100 cell types present in the adult body, and are characterized by self-renewal, immortality, and pluripotency. ES cells are unlimited and show undifferentiated proliferation in vitro.
Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of the cells. These are stem cells but can only differentiate into a limited number of cell types. For example, the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but not to other types of cells (hematopoietic stem cells), as well as bone marrow-derived stem cells, which are typical mesenchymal stem (MS) cells. Adipose tissue also contains a source of multipotent stem cells (adipose-derived stem cells).
Unipotent stem (progenitor) cells denote a state lineage plasticity to differentiate into only a few cells. Unipotent stem cells are those such as lymphoid or myeloid stem cells. The corneal epithelium is a squamous epithelium that is constantly renewing and is regarded as an unipotent stem cell. Unipotent progenitor cells can produce only one cell type (their own), but have the property of self-renewal, which distinguishes them from non- stem cells. Most epithelial tissues self-renew throughout adult life due to the presence of unipotent progenitor cells.
There are ethical difficulties regarding the use of human embryos, as well as the problem of tissue rejection following transplantation in patients. One way to circumvent these issues is the generation of pluripotent cells directly from the patient's own cells. Somatic cells can be reprogrammed by transferring their nuclear contents into oocytes or by fusion with ES cells, indicating that unfertilized eggs and ES cells contain factors that can confer totipotency or pluripotency to somatic cells.
Through analyzing the gene expression profiles of ES cells, many highly expressed genes in ES cells have been identified. In 2006, Yamanaka and his colleague successfully introduced 24 transcription factors (pluripotent genes) that are highly expressed in ES cells into the fibroblast cells derived from fetal mice. Surprisingly, some ES-like colonies appeared in the culture dish within 2 weeks of retroviral infection. Moreover, these ES-like cells could be propagated in vitro and resemble ES cells morphologically after many cell passages. They found ultimately that four transcription factors including Oct4, Sox2, Klf4, and c-Myc (nowadays, Oct4, Sox2, and Klf4 or less transcription factor) were essential for converting the fibroblast cells into induced pluripotent stem (iPS) cells by reducing the factors one by one in the process of retroviral infection. The iPS cells were created by inducing the specialized cells to express genes that were normally present in ES cells and that control cell functions. However, iPS cells could be successfully derived from the differentiated somatic cells simply based on the morphology changes and no genetic selection was needed, which indicated that human somatic cells without genetic modification could be reprogrammed successfully.
Since their discovery in the mid-2000s, newer generations of iPS cell lines have been created through various non-integrating reprogramming strategies, such as approaches using mRNA, episomes, minicircles, piggyBac transposons, recombinant proteins or Sendai virus.
In 2012, Shinya Yamanaka and Sir John Gurdon were awarded a Nobel Prize for their combined efforts in discovering that "mature cells can be reprogrammed to become pluripotent". iPS cells were created by inducing the specialized cells to express pluripotent genes that were normally present in ES cells and that controlled cell functions. In addition, iPS cells have advantages over ES cells: iPS cells are capable of generating autologous and non-immunogenic patient-specific therapies and can more easily provide cell-based disease models from genetically predisposed patients. These newer generations of iPS cell lines avoid the tumorigenicity risks associated with the genomic integration of reprogramming factors and are a powerful way of creating patient- and disease-specific cell lines for research.
1.3 The Extracellular Matrix
The extracellular matrix (ECM) is the extracellular part of animal tissues, which maintains structural back-up for the stem cells, as well as inspiring many key biological properties. ECM proteins can ascertain whether stem cells will multiply or undergo growth retardation, differentiate or remain static, and expand or undergo apoptotic death (programmed death by cell). Then, the ECM proteins are significant causes in recreating the biological roles of stem cells in vitro that help stem cells to induce into various heredities, for example, β-cells, hepatocytes, neural cells, cardiomyocytes, adipocytes, chondrocytes, and osteoblasts. The differentiation of stem cells in culture relies upon the origin, structure, components, and amount of ECMs that are used. Because ECMs are used as matrices or hydrogels for the arrangement of cells in tissues, ECMs are the major cell cultivation ingredients used to checkmate the differentiation and expansion of stem cells in regenerative medicine and translational medicine, both in vivo and in vitro. Therefore, we will discuss the differentiation of stem cells cultivated on materials composed of appropriate ECMs and on the chemical and biological contact between stem cells and ECMs in Chapter 2.
1.4 hPSC Culture on Biomaterials
Human PS (hPS) cells including human iPS (hiPS) cells and human ES (hES) cells have promise for drug discoveries, disease modeling, and regenerative medicine. In order to fully utilize hPS cells in tissue engineering and cell therapy, the advancement of a well-defined microenvironment for culturing hPS cells is needed. The present highest quality level for proliferation and maintenance of hPS cells is typically cultured on feeder cells (e.g., mouse embryonic fibroblasts or human feeder layer cells) or on Geltrex and Matrigels. The use of feeder layers (cells) to cultivate hPS cells is a laborious process, which varies depending on the specific lots of feeder cells or skill of preparation of feeder cells. In contrast, Geltrex and Matrigel are made of molecules extracted from mice sarcomas of Engelbreth–Holm–Swarm mice. hPS cell culture on the coating dishes with Geltrex or Matrigel is the typical and most reliable method to keep the pluripotent states of many hiPS and hES cell lines in feeder-free conditions. However, these culture conditions are not chemically defined and contain xeno-derived molecules. Their xeno-derived molecules hinder the clinical use of hPS cells cultured on Geltrex or Matrigel coating dishes.
It is critical to develop cell culture biomaterials that support large-scale production of hES cell and hiPS cell lines under xeno-free and feeder-free systems, which are compliant with cGMP (current good manufacturing practice). The use of feeder layers restricts the use of hPS cells in clinics. Studies have reported in detail several alternative hPS cell cultivation methods, which have no feeder layers.
Recently, several cell cultivation matrices were reported to cultivate hPS cells that support their pluripotency in chemically defined media. The recently developed biomaterials need a combination of specific cultivation media, and specific hPS cells may specifically support their pluripotent state on the cell cultivation biomaterials. Moreover, it is currently difficult to choose the ideal and best biomaterials for hPS cell cultivation, although rVN and LN-521 are starting to be used as a gold standard of cell culture matrices for hPS cell culture.
Chapter 3 discusses in detail the current developments in hPS cell cultivation biomaterials and discusses the material-assisted regulation of hPS cells under xeno-free and feeder-free and cell cultivation conditions.
Some strategies can be considered for development of materials for hPS cell cultivation under chemically defined, xeno-free and feeder-free systems. The strategies are hPS cell cultivation (1) on two-dimensional (2D) biomaterial immobilized natural extracellular matrices (ECMs), (2) on or within 2D or 3D hydrogels made from polysaccharide such as GAG (glycosaminoglycan), (3) on 2D biomaterial immobilized synthetic oligopeptides derived from ECMs, (4) on 2D plates made from synthetic polymers, (5) in porous or hydrophilic 3D microcapsules, and (6) on 3D microfibers with or without ECM immobilization.
1.5 hPSC Differentiation on Biomaterials
It is evident that people frequently experience the loss or damage of tissues or organs as a result of birth defects, accidents or disease. Regenerative medicine and tissue engineering may be greatly aided by the use of stem cells, such as hES cells and hiPS cells. The use of synthetic and natural polymer materials to mimic stem cell microenvironments and niches can help generation of significant numbers of stem cells and help produce the widely differentiated cells necessary for in vitro translational medicine. Hormones, ECM, and small chemical molecules are biological cues that determine the pluripotency of stem cells and their differentiation fates. However, researchers have only recently started to consider how external forces (e.g., light signals, magnetic forces, and electrical forces), mechanical force (e.g., shear stress imposed by cultivation medium and cyclic stretching of biomaterials), polymeric biomaterial stiffness, cell culture, cell shape, and other such physical cues impact on the induction of stem cells. The protocols and differentiation methods employed with hiPS and hES cells are significantly different from those employed with adult stem cells because of the much higher pluripotent state of hPS cells. Furthermore, hiPS and hES cells cannot generally be cultivated in conventional polystyrene cultivation plates, because these induce random (spontaneous) differentiation of hiPS and hES cells. On the other hand, adult stem cells can expand and differentiate in polystyrene plates and they can be controlled by the induction medium.
Therefore, Chapter 4 discusses the physical cues of synthetic and natural polymeric materials that lead to the differentiation of hES cells and hiPS cells into several different lineages. Such lineages include dopamine-secreting neurons, neural cells, insulin-secreting beta cells, hepatocytes, osteoblasts, chondrocytes, and cardiomyocytes. In this book, the physical cues of materials that we focus upon are (1) the elasticity of polymeric biomaterials, (2) the topography of polymeric biomaterials, and (3) the mechanical forces associated with materials (electrical stimulation via materials and stretching of materials) used for hPS cell cultivation.
1.6 Biomaterials Control hPS Cell Differentiation Fate
Pluripotent stem cells generated from iPS and ES cells have the potential to induce into several cell types, which are derived from the three germ layers: ectoderm cells (epidermal cell, retinal pigment epithelium, dendrocyte, astrocyte, and neuron), mesoderm cells (blood cell, cardiomyocyte, chondrocyte, and osteoblast), and endoderm cells (lung cell, hepatocyte, and β cells, hepatocyte). However, it is challenging to control the induction of pluripotent stem cells, especially hPS cells, into targeted cell lineages because of their variety of induction ability of differentiation.
The stem cell induction is regulated by some independent factors in the hPS cell microenvironment: (1) cell–material interactions in cell cultivation; (2) physical factors, such as cell shape and size, oxygen concentration, shear stress, and the stiffness of the cell cultivation materials; (3) cell–cell interactions, such as in co-cultivation; and (4) bioactive molecules, such as vitamins, cytokines, growth factors, and small molecules. An excellent strategy is to mimic the stem cell microenvironment for the induction of hPS cells into desired cell lineages using appropriate materials for hPS cell cultivation. The protocol for induction of hPS cells is more complicated because of the high differentiation potential and high pluripotency of hPS cells as well as different cultivation protocols for hPS cells in comparison to human adult stem cells, although human adult stem cells, such as amniotic fluid stem cells, adipose-derived stem cells, dental pulp stem cells, and bone marrow-derived stem cells can be differentiated using simple protocols, such as stem cell culture on materials in differentiation media. Typically, hPS cells are cultivated (1) on Matrigel coating dishes, (2) on feeder cells, such as MEF (mouse embryonic fibroblasts, feeder layer), or (3) on appropriate materials to keep their pluripotency; while human adult stem cells can be cultivated on typical TCP (tissue culture polystyrene) plates. Subsequently, the hPS cell induction method is extensively different from the method for human adult stem cells. Chapter 5 describes several protocols for inducing hPS cells cultivated on materials and considers the appropriate materials for hPS cell induction into targeted cell lineages.
1.7 Stem Cell Therapy Using Biomaterials
The hPS cells are valuable cell sources to cure injured organs or tissues because of their potential to induce differentiation into many cells derived from the three embryonic germ layers in the human body.
At present, clinical trials of stem cell therapy using hPS cells have only been reported for four cases according to the ClinicalTrials.gov database. These cases are (1) macular degeneration (namely Stargardt macular dystrophy and age-related macular degeneration), (2) acute myocardial infarction (AMI), (3) diabetes, and (4) spinal cord injury. Recently, hPS cell-based therapy in clinical trials has been studied. We discuss the current situation of stem cell therapy using hPS cells for patients with (1) myocardial infarction (MI) and (2) macular degeneration, considering the bioengineering points of the therapy in Chapter 6. Moreover, we consider clinical trials using adult or human fetal stem cells such as human mesenchymal stem (hMS) cells that are prepared to cure patients with these diseases.
