Stem Cell Engineering

Aspiring to comprehend and control regeneration – the ability to recreate lost or damaged cells, tissues, organs or even limbs – has been the mind-boggling challenge for over 250 years. Regeneration is a common feature in many animal species, whereas its capacity in mammals is notoriously limited. The partial or complete loss of digits or limbs and the deformation of facial injuries profoundly affect the quality of life of the wounded and present a set of challenges for the medical community. This chapter is devoted to some of the problems and prospects of human limb regeneration. It briefly reviews the appearance of regenerative abilities in different tribes across the animal kingdom as well as the genes and cellular signaling pathways involved. Special emphasis is placed upon blastema and scar formation as well as on morphogenetic pattering.

The analysis of evolutionary manifestations of regeneration ability, the apparent relation to the embryonic development mechanisms, as well as consideration of some existing clinical approaches suggest the possibility to “awaken” the regeneration ability in mammals and even in human beings. The ultimate goal of further research will be to identify ways for enhancing the capacity for wound healing and tissue restoration in humans. This would open new exciting prospects for future regenerative medicine.

In recent years there has been an explosion of interest in stem cell research, given their promising medical applications in cell-based tissue regeneration, drug testing and of course basic research. A decade of restless experimental and clinical research has demonstrated that the routine use of stem cells to repair solid organs is not at hand in spite of recent excessively enthusiastic announcements in the press and even serious scientific journals. Indeed, biologists only partially comprehend cell-differentiating mechanisms and have mapped only a few of the extrinsic and intrinsic factors involved. Even less is understood the complex qualitative, quantitative and temporal orchestration of these factors in the different steps featuring the whole differentiating process.

Most of the current research is centred on the identification of soluble ligands which regulate and control signalling pathways, and our knowledge on the role of the physical and structural microenvironment is still scarce. In this chapter, we focus only on cues which can be controlled externally using mechanical and structural parameters, and so can be easily defined using appropriate engineering and design. Firstly, the influence of the single parameters on cell behaviour is described, and then we discuss how technological tools such as biomaterials, scaffolds and bioreactors, as well as well-constructed and defined multiscale classification models can be best employed to engineer artificial biomimetic in vitro systems.

Mesenchymal stem cells (MSCs) can be found in various tissues of the adult organism. These cells display a multilineage potential and can be in vitro differentiated toward the osteogenic, adipogenic, chondrogenic, and myogenic lineage, which makes them promising candidates for future tissue replacement strategies.

For stem cell research the focus in the past has been on embryonic stem cells, cells from which humans are initially constructed, but which are limited in availability. A new direction in stem cell research is looking at a different kind of pluripotent cells – adult stem cells, which are responsible for maintenance and repair of tissue. To retrieve and grow stem cells remains an important challenge in medicine, but carries the hope to cure many diseases. Pluripotent adult stem cells have been found in many organs and also in cancer tumors. Adult stem cells can both self-renew and differentiate to replace compromised tissue in the organ where they reside. If single adult tumor stem cells can re-grow into a new tumor, just as adult stem cells can rebuild a physiological organ, the potential of adult stem cells would be significant in new cancer therapy and organ engineering approaches.

This chapter introduces the methods behind isolation of adult stem cells, in vitro culturing, characterization, genetic manipulation for imaging purposes, and the promising results of re-implantation into healthy tissues. Adult stem cells derived from human early-stage prostate tumors are used in a newly developed, novel research model. Since the environmental niche is an important factor for stem cell growth ex vivo, stem cell isolation is achieved via culturing in a characterized environment that mimics the stem cell niche for these types of adult stem cells. The human prostate tumor stem cell niche is described and prostate tumor stem cells (PrTuSCs) are used to show the so-called stem cell center (SCC) growth in cell culture. It is also demonstrated how PrTuSCs are epigenetically altered with eGFP for tracking and imaging purposes. Cultured PrTuSCs are characterized as adult stem cells by stem cell marker analysis and expression of stem cell-associated transcription factors. To further demonstrate the pluripotent potential of PrTuSC, the effects of re-implantation into healthy tissues in vivo are presented in the orthotopic xenografting and tissue recombination methods. When implanted into immune-suppressed SCID mice, results show that cultured PrTuSC not only can re-grow a cancerous prostate tumor, but, depending on the implantation site and its microenvironment, also have the ability to generate normal benign human prostate glandular structures in vivo. The dorsal mouse-skinfold window chamber method is introduced as an experimental model that allows direct observation of implanted stem cells and their behavioral characteristics, e.g., promotion of healthy or tumor-associated angiogenesis, which is critical in tissue renewal and cancer metastasis.

Cardiovascular diseases account for more deaths than any other illness. Cardiac tissue engineering has turned to embryonic stem cells as a renewable source of myocytes for use in tissue replacement. Existing methods for stem cell differentiation toward the cardiac lineage are relatively non-specific, yielding low numbers of myocytes with varying contraction frequencies and strengths. Here we describe novel experimental approaches, utilizing an electrical stimulation regimen, aimed at increasing the efficiency of cardiac differentiation from mouse embryonic stem (mES) cells. These methods generate cardiac myocytes with functional characteristics that more closely resemble native tissues. The amplitude, duration, and frequency of the electrical stimulus as well as the timing of its onset are some of the critical experimental parameters that determine the enhancement of cardiac differentiation.

In order to form embryoid bodies, an optimum differentiation regime was followed incorporating the hanging drop method followed by suspension culture and subsequent post-plating on conductive slides with electrical stimulation. Approximately three times more stimulated mES cells exhibited evidence of cardiac differentiation than their non-stimulated counterparts, as determined by the expression of ventricular marker myosin light chain-2v. Spontaneous contractions of the stimulated cell populations began up to 1 day earlier and had an average beat frequency close to that of the stimulus applied during differentiation. The spontaneously contracting regions had larger areas of contraction, which beat more rhythmically, as determined by real-time digital imaging analysis.

Our results suggest that appropriate electrical stimulation generates greater numbers of more robust cardiac myocytes, which in turn may be better suited for repairing or regenerating an ailing heart and for use as 3D model systems for drug discovery.

Blood transfusions and bone marrow transplantations are cell-based therapies that have been used to treat diseases of the haematopoietic system for decades. However, such therapies are completely reliant on the availability of appropriate donors and fraught with the risk of transmissible infection. Can recent advances in pluripotent stem cell technology alleviate these problems? We review the development of protocols used in haematopoietic differentiation of mouse and human embryonic stem (ES) cells and the progress made in the production of haematopoietic stem cells capable of long-term reconstitution. Relatively pure populations of mature haematopoietic cells including erythrocytes, macrophage and dendritic cells have also been generated from ES cells and their potential use in the future treatment of disease is considered. However, even assuming the successful production of a desired cell type in the research laboratory, we are faced with significant challenges in the translation of these protocols into clinical grade processes and in the scale-up of these strategies that will allow the economic production of vast quantities of cells. We discuss briefly some of the technology that has been applied recently to the ES cell system as a first step in overcoming some of these challenges.

Human embryonic stem cells (hESCs) and induced pluripotent cells (iPSCs) are well suited for translational cell therapy. For hESC, the pluripotent phenotype, naturally occurring within the inner cell mass of the early embryo, bestows the capability to differentiate into any cell type of interest and this, coupled with their ability to remain in an undifferentiated state with indefinite proliferative capacity, means that essentially unlimited numbers of identical, well-defined and genetically characterised stem cells can be produced in culture for therapeutic applications. An understanding of the regulatory mechanisms responsible for pluripotency and differentiation potential of hESCs is critical for translating their potential in vitro to therapeutic use in vivo. Harnessing of this therapeutic potential in conjunction with modern genetic modification tools promises great advancement in the study of developmental and adult physiology and pathophysiology with a view towards implementation of genetically modified hESCs to advance regenerative medicine.

Human embryonic stem cells (ESC) provide a great hope for regenerative medicine in different diseases like neodegenerative disease, diabetes, heart, or liver failure. Immune rejection was not thought to be a major issue for cell therapy because of a low immunogenicity of fetal or embryonic cells in preliminary animal studies. However, increasing evidence suggests that this is not true, and controlling the immune response will be crucial for the success of ESC transplantation. The source of ESC is of crucial importance with regard to genetic difference between donors and recipients. Immune reaction against genetically identical origin (autologous or from identical twin) would be fully absent or negligible in contrast to immune response against allogenic transplanted cells. However, in the situation of ESC the origin of the cell would be necessarily allogenic and the immune reactivity against allogenic cells is expected to be similar to what has been learned from decades of research in the field of cell, tissue, or solid organ transplantation. In this chapter, we will first describe the risk of potential immune reactivity against ESC according to the origin of the cells. Second, we will review the immune mechanism of rejection and the current literature on the topic not only in animal models but also in humans. Finally, we will come with therapeutic approaches that can allow crossing genetic barriers of donor cells by preventing immune reaction that could lead to irreversible loss of the graft function.

Oxygen is vital for cellular metabolism in higher organisms. Explanted somatic cells are regularly cultured at ambient oxygen conditions, which often appear stressful for primary cells, thereby leading to accelerated aging or premature senescence in vitro. Therefore, sophisticated instrumentation for ex vivo cell manipulation has been introduced and is now increasingly being used for the propagation and subsequent differentiation of various types of stem cells. In this particular context also primary mesenchymal stromal cells (MSC) have been investigated. Their developmental fate greatly depends on oxygen availability.

In this contribution, we describe the current knowledge about MSC properties according to varying oxygen tension in vitro: while reduced oxygen tension supports their proliferation, differentiation potential is reversibly attenuated. This finding is relevant for various in vivo situations such as wound healing or distinct pathologic alterations. Inappropriate oxygenation is actually not impacting on MSC viability, but greatly diminishes their differentiation capabilities, in due course leading to irreversible changes in tissue structure including functional alterations involved.

Regeneration of damaged tissue by embryonic or adult stem cells has been and still is a topic of highest interest in experimental and clinical medicine. Adult stem cells have the ability to differentiate into a number of different cell types in dependence on their environment. Endothelial progenitor cells (EPCs) represent a population of adult stem cells. They contribute to the renewal of the largest organ of the body: the endothelium which is the inner layer of the vessel wall.

After an introduction into the topic of EPCs presenting the origin, fate, and biology of EPCs, we describe techniques of cell culture and bioassays for in vitro and in vivo testing of EPCs. Their applications in animal models and in humans are described to demonstrate the relevance of EPCs in cardiovascular medicine. On the other hand, nitric oxide (NO), a key player in cardiovascular biology, is introduced and the first findings describing the apparently powerful interactions between EPCs and NO are discussed. EPCs and NO seem to be the “Yin and Yang” of endothelial function and regeneration. We would like to illustrate recent findings of EPC biology with a special interest in their interaction with NO. Finally, we open the wide and interesting spectrum of further research activities in this field of biomedicine. If the differentiation of EPCs could be controlled in the laboratory, these cells may become the basis of cell-based therapies for cardiovascular disease. An interdisciplinary approach and the cooperation between scientists, engineers, and physicians will be able to transfer the findings of basic science into daily clinical application. Initial ideas and first steps for the realization of new concepts in EPC–NO research and therapeutic strategies with a special reference to bioengineering concepts are discussed.

Cells respond strongly to the shape of their environment and this is called contact guidence. For many years this has been studies at the microscale and with terminally differentiated cell types. With developments in materials technology studies at the nanoscale are now possible. Hand-in hand with these advances are developments in stem cell culture. This review looks at the adult stem cell/nanoscale interface considering cell adhesion, motility, gene expression (mechanotransduction) and ultimately differentiation. Particularly of interest is the reorganisation of the interface nucleus and possible links to differentiation.

Cardiovascular disease is the leading cause of death worldwide. As such, vascular reconstruction and graft bypass surgery are in high demand to slow the rate of morbidity following the onset of this pathology. However, the use of non-biocompatible synthetic grafts, especially those of small diameter (6 mm or less), frequently leads to thrombosis and occlusion of the vessel. The field of vascular tissue engineering provides an alternative method to generate small (and large)-diameter bypass grafts that can support cell growth and are expected to exhibit long-term patency. Following the generation of the first in vitro blood vessel over 30 years ago, there has been considerable progress in this area in terms of scaffold availability, construction of the vessels and application of stem cells. This chapter will specifically focus on the use of stem cells in the generation of vascular grafts. Here we will highlight the current scaffolds available for seeding cells, the alternate stem cell sources and their isolation, the methods used to differentiate stem cells into vascular lineages and their application in generating blood vessels in vitro. The future hurdles that must be overcome before tissue-engineered blood vessels can be applied to a clinical setting will also be discussed.

Endothelial progenitor cells (EPCs), present in the blood and bone marrow, represent a potential source of endothelial cells for repair of injured blood vessels, neovascularization, and tissue engineering. EPCs are present at low levels in peripheral blood, although their numbers increase in response to cytokines, VEGF, and statins. There are at least two types of EPCs characterized following in vitro culture: colony-forming unit ECs (CFU-ECs) and endothelial colony-forming cells (ECFCs). CFU-ECs appear early in culture, have limited ability to proliferate, and share markers for endothelial cells and monocytes. In contrast, ECFCs appear later in culture, grow rapidly, and to large numbers express only endothelial cell markers. This chapter examines the properties of these EPCs, in vitro and in vivo studies using these two cell types, and the potential of these EPCs for therapeutic applications.

Stem/progenitor cells are in the focus of regenerative medicine for a future therapy of acute and chronic renal failure. However, broad knowledge about parenchymal regeneration in kidney is lacking. For that reason developmental pathways leading from stem/progenitor cells to newly formed tubules have to be investigated. A new technique promotes renal stem/progenitor cells to form numerous tubules between layers of polyester fleeces. This artificial interstitium replaces coating by extracellular matrix proteins, supports spatial extension of renal tubules, and can be used with chemically defined Iscove’s modified Dulbecco’s medium (IMDM) during a long-term culture period of 13 days. The development of tubules is stimulated by aldosterone and depends on the applied hormone concentration. The tubulogenic effect cannot be mimicked by precursors of the aldosterone synthesis pathway or by other steroid hormones. Antagonists such as spironolactone or canrenoate prevent the development of tubules, which indicates that the mineralocorticoid receptor (MR) is involved. Administration of geldanamycin, radicicol, quercetin, or KNK 437 in combination with aldosterone blocks development of tubules by disturbing the contact between MR and heat-shock proteins. Transmission electron microscopy (TEM) further demonstrates that generated tubules exhibit a junctional complex between the apical and the lateral plasma membrane. At the basal aspect a continuously developed basal lamina is present. Immuno-label for Troma I (cytokeratin Endo-A) shows isoprismatic cells, while label for laminin γ1, occludin, and Na/K-ATPase α5 confirms typical features of a polarized epithelium. Finally, the introduced system makes it possible to pile and pave renal stem/progenitor cells, so that the spatial development of tubules can be systematically investigated.

The development of a safe and effective bio-synthetic encapsulation system will potentially improve the treatment of diseases known to benefit from cell implantation therapies. This is of particular importance in the progression towards a permanent treatment for type 1 diabetes. Cell-based therapies for insulin-dependent diabetics ultimately aim to eliminate the need for exogenous insulin through the implantation of donor islet cells or, alternately, stem cells differentiated into glucose-sensitive islet-like cells. Encapsulation devices are required to protect implanted cells from destructive host immune factors. A successful design will be bio-synthetic, having well-defined permeability and providing appropriate biomolecules for physiological functionality and survival of the encapsulated cells.

Due to their great potential, mesenchymal stem cells (MSC) are of special interest for regenerative therapies. One of the most investigated areas in this field is the regeneration of bone tissue using tissue engineering strategies. This chapter focuses on osteogenic differentiation and cultivation of human MSC in vitro and their utilisation for tissue engineering of bone tissue, discussing a number of protocols to induce and influence the osteogenic differentiation of MSC into the osteoblastic lineage in vitro.

According to the tissue engineering approach, 3D porous biomaterials (scaffolds) are seeded with autologous cells, followed by a period of in vitro cultivation which can be carried out under differentiation-inducing conditions and finally by implantation of the cell-matrix constructs into the defect. Here we describe as an example the in vitro cultivation and osteogenic differentiation of human MSC in porous scaffolds consisting of biomimetically mineralised collagen. This artificial extracellular bone matrix that resemble the nanocomposite made of fibrillar collagen type I and hydroxyapatite crystals in natural bone tissue was furthermore used to establish an in vitro model of the bone remodelling process: osteoblasts derived from human MSC and osteoclast-like cells which were also differentiated from their natural precursors were co-cultured on membranes of mineralised collagen to learn more about the cellular cross talk between both cell types.

The concept of in situ tissue engineering, a novel strategy to accelerate bone defect healing, aims at the utilization of natural chemotactic mechanisms to colonise scaffolds with MSC in vivo. Exemplarily, the chemoattraction of human MSC into porous 3D scaffolds of mineralised collagen using the stromal-derived factor 1α (SDF-1α) is demonstrated.

Cultivation of mammalian cells holds promise for generating cell and tissue models and transplants, but the shortcomings of current cell culture methods are limiting such use. In order to provide biological material that can represent cells and tissues of the body in drug development and regenerate organs and systems impaired by disease in the clinic, it is important to recognize that the extracellular integrity, the interphase between the cultureware surface and the cells, and the cell-to-cell contacts are an integral part of the cell culture. One example would be the deposition of type IV collagen, the main structural component of the basement membrane, by human endothelial cells, which can be manipulated by varying the cultureware surface and/or the level of oxidative stress in the culture. Even if culture protocols were optimized to make the cell environment more like the environment in the body, the integrity of the culture would be destroyed during cell harvesting, as matrix molecules are digested by enzymes used for dissociating the cells, and cannot be accumulated from one passage to another. The Thermo Scientific Nunc^TM UpCell^TM Surface enables dissociation of cells from the cultureware upon a simple change in temperature. Being slightly hydrophobic at 37°C, the surface allows cells to attach and grow, but hydrophilic when the temperature is reduced to below 32°C, the surface will bind water and swell, resulting in the release of adherent cells with their underlying matrix molecules. The preservation of matrix molecules enables the harvesting of contiguous cell sheets, which can be grafted onto another cell layer in vivo or in vitro without the use of fibrin glue or sutures. This chapter gives examples of how extracellular culture integrity can be built, preserved, and applied in cell analysis and tissue engineering.

In regenerative medicine, there are several issues directly related to the production of cell-based therapeutics. As pluripotent embryonic or multipotent adult stem cells are committed and differentiated to specific tissue cells and proliferate in number, it is easily conjecturable that there may be internal and/or external cellular factors involved in such a biological event, and that internal factors depend on genetic characteristics while external cellular factors mainly exist within the extracellular matrix.

Stem cells are characterized by their ability to self-renew and to generate a diverse range of physiological cell types (Singec et al., Annu Rev Med. 2007; 58:313–328). In mammals, stem cells found in the mature organism (somatic stem cells) generate progeny of a specific cell lineage or tissue type (multipotency). For example, a hematopoietic stem cell gives rise to the cell lineages found in bone marrow and blood, while a central nervous system (CNS) neural stem cell generates neurons, oligodendrocytes, and astroglia (Murry and Keller, Cell. 2008; 132:661–680). At an earlier developmental stage, embryonic stem (ES) cells obtained from the inner cell mass of blastocyst-stage embryos exhibit limitless capacity to self-renew and can give rise to any cell type of the organism, thereby defining pluripotency (Murry and Keller, Cell. 2008; 132:661–680; Singec et al. Annu Rev Med. 2007; 58:313–328). The term “totipotent” is reserved for cells that can also give rise to the trophoblast and extraembryonic tissue in vivo, such as the fertilized egg (zygote). These designations exemplify how differentiation toward a specific mature cell phenotype is accompanied by an increasingly limited spectrum of potential descendant cell types and by a diminished proliferative capacity (Yeo et al., Hum Reprod. 2008; 23:67–73) (Fig. 1 ). In addition, recent advances in reprogramming of adult somatic cell types into pluripotent cells (Takahashi et al., Cell. 2007; 131:861–872) are aimed at controlling the regulatory mechanisms that govern stemness and pluripotency, which may soon enable even more refined modulation of cell fate (Yeo et al., Hum Reprod. 2008; 23:67–73; Pruszak and Isacson, Development and engineering of dopamine neurons. Austin, TX: Landis Bioscience; 2008; Jaenisch and Young, Cell. 2008; 132:567–582). To achieve translation of stem cell biology into clinical applications, somatic, embryonic, and reprogrammed stem cell sources alike are presently being investigated. Despite the fast-paced progress of stem cell research as a field, many aspects of cell development in the dish are still not fully understood. To ensure appropriate patterning, signaling parameters for cell lineage specification need to be identified, and generating the phenotype of interest requires close monitoring and controlled modulation of the microenvironmental conditions in the dish.

We summarize universal principles, advancements, and ongoing challenges in deriving and characterizing therapeutic cell types from pluripotent and multipotent stem cells for clinical and scientific biomedical scenarios. Specifically, this overview illustrates the paradigm of neural stem cell differentiation and the application of microphysiometer systems to monitor and control conditions fostering the generation, maturation, and survival of specific neural cell populations aimed at treating neurological disease.

Significant advancements in stem cell research have provided important information on stem cell biology and offer great promise for developing novel successful stem cell-based medical treatments. Further investigations appear to be necessary to translate the basic knowledge into clinical therapeutic applications in humans. The identification of specific biomarkers to each type of adult stem/progenitor cells relative to their more committed and mature progeny is important for the characterization of their specific physiological functions.

Embryonic stem cells (ESCs) are recognized by the general public mainly as a possible source of cellular replacement therapy and transplantation. However, the usefulness of ESCs and ESC-derived tissue cell types for cell-based in vitro test systems in drug screening and toxicity was already recognized early after the discovery of ESCs in the late 1980s.

In the present chapter, the employment of ESCs in cell-based test systems is described in the light of state-of-the-art methods in the field of drug discovery, and the advantages and disadvantages of the applications are discussed. The possible use of ESCs in the drug development process is illustrated by three examples: (1) the use of ESCs for basic research in early developmental biology, (2) the embryonic stem cell test for embryotoxicity, and (3) the use of ESC-derived cardiomyocytes for detection of cardiac toxicity. Potential applications of human ESCs, induced pluripotent stem cells (IPS cells), and personalized medicine are described in the last part.

Heart organogenesis is sensitive to both genetic and environmental factors and heart malformations account for the majority of birth defects. The development of the mammalian heart is a complex morphogenetic process that ultimately results in a contractile, four-chambered organ that is formed from progenitor cells of mesodermal and neural crest origins. Studies in animal models with primitive hearts such as drosophila, zebrafish, and xenopus have provided important insights into the way the heart begins to form and the molecular cues that guide the early stages of cardiac morphogenesis. Much of what we know about the development of the heart in higher vertebrates comes mostly from embryological investigations in chick and mouse. In addition, genetic studies in zebrafish and mouse, including loss- and gain-of-function approaches, have been especially valuable for gaining information about the role of individual genes in heart development. However, the various animal models also have a number of limitations regarding lack of genetic tools (chick, xenopus), inaccessibility to observation and embryological manipulation (mouse), or unsuitability for high-throughput screens to identify pharmacologic compounds to treat cardiac defects. Mouse embryonic stem (ES) cells give rise to a wide variety of organ-specific cell types, offering an accessible and relevant system to investigate how cell lineages emerge and grow. The ES cell model is particularly pertinent for studying the molecular mechanisms regulating the specification and differentiation of cardiovascular progenitor cells, because these cells appear relatively early during embryonic development and ES cell differentiation. This chapter reviews a selective number of studies that have applied the in vitro differentiation of embryonic stem cells toward understanding the regulatory steps of cardiac development.