Stem-Cell Nanoengineering

By H. Baharvand

Stem Cell Nanoengineering reviews the applications of nanotechnology in the fields of stem cells, tissue engineering, and regenerative medicine. Topics addressed include various types of stem cells, underlying principles of nanobiotechnology, the making of nano-scaffolds, nano tissue engineering, applications of nanotechnology in stem cell tracking and molecular imaging, nano-devices, as well as stem cell nano-engineering from bench to bedside.

Written by renowned experts in their respective fields, chapters describe and explore a wide variety of topics in stem cell nanoengineering, making the book a valuable resource for both researchers and clinicians in biomedical and bioengineering fields.

• Language: English
• Publisher: Wiley
• Release date: Dec 30, 2014
• ISBN: 9781118540725

Book preview

Preface

Nanobiotechnology is a fast growing area of research that aims to create nanodevices, nanoparticles, and nanoscale development in the field of stem-cell and tissue-engineering based therapies. Concepts and discoveries from this field, along with stem cell research, provide exciting opportunities of using stem cells for regeneration of tissues and organs and to address the challenges of disease therapeutics.

In this book, Stem-Cell Nanoengineering, we aim to provide the premier source of reviews in nanotechnology approaches towards stem cells and tissue engineering and regenerative medicine, which will serve as a textbook. This book overviews the fast moving field of stem cells, discusses challenges to the field that can be addressed through nanotechnology, provides information on stem cells, principles of nanobiotechnology, manufacture of nanoscaffolds, nanotissue engineering, changes in stem-cell-fate decisions by micro- and nanoengineering of the microenvironment, application of nanotechnology in stem-cell tracking and molecular imaging, and finally stem-cell nanoengineering from bench to bedside.

The contributions to this book, all written by renowned experts in their respective disciplines, describe and explore various facets of this field. This book will be an especially valuable resource for biomedical and bioengineering researchers and clinicians.

This book could useful for postgraduate students and advanced undergraduate students in cell biology, biochemistry, genetics, developmental biology, biomaterial, medical engineering, nanotechnology, and biomechanics; physicians; life science scientists; biomedical researchers; cell biologists; academics; surgeons; scientists and engineers in the field of regenerative medicine; clinicians and specialists; biotechnology and pharmaceutical industry professionals.

We want to sincerely thank all authors that have contributed to this book for their devoted efforts and their excellent contributions. We hope that you, as a reader, will enjoy this book. We are also grateful to Drs. Hamid Gourabi, Abdolhossein Shahverdi, and Ahmad Vosough Dizaj for having faith in and supporting us throughout this project. We also wish to acknowledge the great support provided by many at John Wiley and Sons. A special thank you goes to our dedicated colleagues at Royan Institute for Stem Cell Biology and Technology, who, with their tireless commitment for stem-cell research and therapy, have become crucial factors in encouraging us to edit this book. We are grateful to Asma Ghodsi and Zahra Maghari for their assistance with collecting the chapters and in follow-up. Our thanks also to the many scientific colleagues and students, Sasan Jalili-Firoozinezhad, Mohammad Kazemi Ashtiani, Hossein Ghanian, Leila Montazeri, and Fahimeh Khayatan, and other members of the Cell Engineering program at the Royan Institute who stimulated our intrests, encouraged us, and supported our efforts.

Hossein Baharvand and Nasser Aghdami

Part 1

An Introduction to Stem Cells

Chapter 1

Adult Stem Cells

Andreas Nussler and Sahar Olsadat Sajadian

Eberhard Karls Universität Tübingen, Department of Traumatology, Tübingen, Germany

Introduction

Humans and many animals share the ability to regenerate missing parts of the body. Although humans are not able to replace missing parts of the body as a whole, like legs or hands for example, the human body is able to perpetually regenerate various tissues and blood. The mysterious cell type that enables the human body to perform this regeneration was discovered in the 1950s and subsequently named stem cell [1]. The first stem cells were discovered in the bone marrow. Therefore, at the beginning, stem cells were almost exclusively isolated from human bone marrow. Later on, the routine isolation and manipulation of bone marrow stem cells led to the development of a method of bone marrow transplantation for the treatment of blood diseases, such as leukemia, that is used all over the world today.

Regardless of their origin, stem cells have the following specific properties: their division and self-renewal capacity over long periods of time, their lack of specification, and their ability to differentiate into specialized cell types. Over the past 50 years, many important discoveries have raised hope that stem cell research will achieve major breakthroughs in medicine. A short history of stem-cell research is presented in Table 1.1, and the different kinds of stem cells with their respective sources are displayed in Table 1.2.

Table 1.1 Important events in the history of stem cell research

Table 1.2 Sources, characteristics, advantages and disadvantages of different types of human stem cells

Adult Stem Cells

Adult stem cells (ASCs) are also called stromal cells. Their regenerative potential has been recognized several decades ago. For instance, it has been demonstrated that hematopoietic stem cells that are derived from adult tissues are able to generate every type of blood cell [2]. However, adult stem cells were thought to have a rather restricted potential for generating new tissue, but recent studies have changed this viewpoint. Recent observations have suggested that, in addition to the production of the derivatives of the blood system, stem cells from the bone marrow of the juvenile and adult organism can create muscle and neuron-like cells in the brain [3].Therefore, if ASCs turn out to have the same potential as embryonic stem cells (ESC), some ethical issues concerning ESCs may be overcome.

Hematopoietic stem cells (HSCs) were the first type of adult stem cells to be discovered, by Becker et al., in 1963 [4]. These cells reside in the bone marrow and are able to renew themselves and to differentiate [5]. Bone marrow contains at least three types of stem cells: HSCs, the first type discovered are able to differentiate into every type of blood cell. The second type discovered a few years later, is known as bone marrow stromal cells, mesenchymal stem cells or skeletal stem cells: they are a mixed cell population capable of generating bone, cartilage, fat and fibrous connective tissue [6, 7]. The third type is known as endothelial progenitor cells (EPCs), and they contain a unique population of peripheral blood mononuclear cells [8].

In the 1990s, Caplan popularized the term mesenchymal stem cells (MSCs) [9]; however, when publishing clinical studies of MSCs, some investigators still prefer not to refer to them as stem cells [10, 11]. The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) proposed a uniform nomenclature for MSCs published in 2005 [12] according to the following guidelines: first, the MSCs must be plastic-adherent when they are maintained in standard culture conditions; second, they have to express surface markers, such as CD105, CD73, and CD90, without expressing the surface molecules CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR; third, they have to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro. In addition, it has been demonstrated that MSCs differentiate into a large variety of specialized MSCs, including myocytes, tendocytes, and ligament cells [13, 14]. The MSCs reside in different locations of the body, for example in bone marrow, around blood vessels as pericytes, in fat, skin, muscles, teeth and other locations [13–15]. Until recently, it had been believed that adult stem cells only differentiate into mature phenotypes of various cells, but within the MSC lineages, it has been demonstrated that chondrocytes are able to differentiate into osteoblasts and adipocytes and change their phenotype to osteoblasts. These findings have demonstrated the plasticity of MSCs [16–19].Adult stem cells are generally thought to be rare, difficult to isolate and to purify, and difficult to maintain in an undifferentiated state when grown in culture. Therefore, developing methods for upscaling adult stem cells outside the body is currently one of the priorities of stem-cell research targeted at further clinical application.

Adult Stem-Cell Plasticity

Until recently, no researcher had seriously considered the possibility of adult stem cells generating the specialized cell types that are necessary for the formation of different tissues, either from the same embryonic germ layer or from a different germ layer. Recently, however, studies have demonstrated that blood stem cells (derived from the mesoderm) may be able to generate both skeletal muscle (also derived from the mesoderm) and neurons (derived from the ectoderm) [20, 21].

The term plasticity refers to the ability of a stem cell that is derived from one adult tissue to differentiate into a differentiated cell type from another tissue. This process is referred to either as unorthodox differentiation or as trans-differentiation [22, 23]. However, many examples have demonstrated that MSCs are able to differentiate into an endodermal phenotype, such as hepatocyte-like cells [24–26]. In addition, it has been demonstrated that MSCs differentiate into epithelial cells, such as retinal pigment epithelial cells [27, 28], sebaceous duct cells [29], skin epithelial cells [30], and tubucular epithelial cells in the kidney [17, 31]. According to the experiments that have been reported to date, adult stem cells may assume the characteristics of the cells that have developed from the primary germ layer. For example, many experiments on plasticity involve stem cells derived from bone marrow, which is considered to be of mesodermal origin. The bone-marrow stem cells may differentiate into another mesodermally derived tissue, such as skeletal muscle or cardiac muscle [20, 32–34].

Classification of Adult Stem Cells

Adult stem cell niches are distributed throughout several regions of the body, including bone marrow, brain, fat, skeletal muscle, retina, liver, and skin. Some of these adult stem-cell types are discussed in detail below. Many tissues that have specific ASC phenotypes have been identified so far. Some examples are mammary stem cells, intestinal stem cells, endothelial stem cells, olfactory stem cells [35], liver-derived stem cells, testicular stem cells, and dental pulp stem cells [8].

Bone Marrow-Derived Stem Cells

Today, bone marrow is one of the most popular sources of stem cells. Bone marrow-derived stem cells (BMSCs) have a self-renewal potential and they are readily available through bone marrow biopsy. The BMSCs contain at least three different types: HSCs, MSCs and EPCs [8]. The HSCs and the MSCs are presented in more detail later, since they are considered to have the highest potential in therapeutic approaches.

Hematopoetic stem cells (HSCs) are involved in the production of blood cells. This process is called hematopoiesis. They give rise to the entirety of the blood cells in the human body. Therefore, HSCs are a potential tool for curing blood diseases, for example leukemia or lymphoma, as well as blood disorders, for example anemia or immunodeficiencies.

Mesenchymal stem cells (MSCs) are derived from bone marrow stromal progenitor cells and can form mesenchyme, a loose connective tissue. The MSCs can be isolated from HSCs by their capability to adhere to tissue culture plastic. They are able to form mesodermal and non-mesodermal tissues, such as bone, cartilage, tendon, adipose tissue, and muscle [36].

Endothelial progenitor cells (EPCs) include a unique population of peripheral-blood mononuclear cells derived from bone marrow that are involved in postnatal neovascularization during wound healing, limb ischemia, post-myocardial infarction syndrome, arteriosclerosis, and tumor development. Both HSCs and EPCs are derived from common a precursor called hemangioblast [37].

Adipose Tissue-Derived Stem Cells or Adipocyte-Derived Mesenchymal Stem Cells

White adipose tissue is one of two types of adipose tissue found in mammals. The tasks of this tissue consist in saving energy and in acting as a thermal insulator. Adipose tissues originate from the mesodermal layer of embryos and can develop pre- as well as postnatally. Three different types of cells are present in adipose tissues: adipocytes, pre-adipocytes, and a heterogeneous population called stromal vesicular fraction. Adipose tissue-derived stem cells (ADSCs) are multipotent cells that are able to differentiate into other types of mesenchymal tissues, such as adipocytes, chondrocytes, myocytes, and osteoblasts [38]. Furthermore, they grow faster and are easier to culture in vitro over a long period of time. In the past decade, abundant evidence has been presented for the fact that the secretion of vascular endothelial growth factor by ADSCs leads to the healing of damaged tissue. Several studies have been presented indicating that adipocyte-derived mesenchymal stem cells (AD-MSCs) have the ability to differentiate to hepatocyte-like cells under specific conditions [39–41]. Therefore, MSCs are expected to be an ideal source for transplantation or liver tissue engineering: however, the hepatic differentiation of MSCs is still insufficient for clinical application. But the proliferation and the differentiation capacity of ASCs also cause changes in their metabolic activity. These changes may, in turn, increase the risk of tumor formation [42].

Neural Stem Cells

Neural stem cells (NSCs) differentiate into three major cell types: neurons, astrocytes, and oligodendrocytes [43]. The NSCs that have been identified in the ventricular zone of the brain include neuroblasts, precursor cells and astrocytes. They all express proteins like GFAP (glial fibrillary acidic protein) and the glycoprotein CD133, which permits the identification of this cell. Most NSCs of the lateral ventricles (ependymal cells) are quiescent and do not perform active division. The NSCs express nestin, which is a specific marker of neural precursors. Furthermore, NSCs of the hippocampus are ciliated and play an important role in the memory function of the brain. They express neuronal markers, such as NeuN, neuron specific enolase, and calbindin [44].

Neural stem cells are commonly cultured in vitro as so-called neurophases. In this case, they assume a free-floating cell cluster configuration. By using the neurophase in cell culture, NSCs are capable of differentiating into glial-like cells [45]. It is believed that they have the potential of curing brain disorders, such as anxiety, depression, memory deterioration, and some brain tumors [8]. Furthermore, there is some speculation that these cells may overcome the paralyzation caused by spinal cord injuries [46, 47].

Neural Crest Stem Cells

A remnant of embryonic neural-crest stem cells has been identified in the hair follicles. Similar cells have also been found in the gastrointestinal tract, in the sciatic nerve, and in the spinal cord. Neural crest stem cells can differentiate into neurons, Schwann cells, myofibroblasts, chondrocytes, and melanocytes [48].

Hematopoietic Stem Cells

Nearly 50 years ago, HSCs were identified by Till and McCullough [49]. The remarkable characteristics of these cells are their ability for continuous self-renewal in the bone marrow and their ability to differentiate to all types of blood cells (Figure 1.1). Hematopoietic stem cells normally reside in bone marrow, but under certain conditions they migrate through the blood in order to settle in other tissues. They are also present in fetal liver, the spleen, placenta blood, and in the umbilical cord. A decade ago, several studies have revealed that HSCs can give rise to a liver-like cell phenotype [23, 50]. One study has demonstrated that HSCs that are transplanted into an irradiated mouse evolve not only into various blood-cell types (from the mesoderm layer of the embryo), but also into epithelial cell phenotypes in the lung, gut (endoderm layer), and skin (ectoderm layer) [51]. If HSCs are truly multipotent, their potential for life-saving regenerative therapies may be considerably expanded in the future.

c1-fig-0001

Figure 1.1 Differentiation of hematopoietic and stromal cells.

There are several problems concerning the implementation of standardized HSC protocols. The identification and characterization of HSCs is difficult, as those with long-term replicating ability are rare and difficult to upscale. Furthermore, HSCs have multiple phenotypes and resemble other blood or bone-marrow cells, which makes them difficult to distinguish from each other [52].

Over the past years, different kinds of surface markers have been used to identify, isolate, and purify HSCs derived from the bone marrow and the blood. Undifferentiated HSCs and hematopoietic progenitor cells express c-kit, CD34, and H-2 K. These cells often lack the Lin lineage marker completely, or only express it at a very low level (Lin−/low). Weissman and his collaborators have focused on surface protein markers of blood cells from mice and have identified the closest common markers for mouse and human HSCs. Moreover, it has been demonstrated that the cell surface markers can no longer be identified during cell development [53]. Indeed, for transplantation purposes, cells that express the surface proteins CD34+, Thy1+, and Lin− are the most likely to contain stem cells [54, 55] (Table 1.3).

Table 1.3 Proposed cell-surface markers of undifferentiated hematopoietic stem cells

There are also different sources of HSCs, for example, bone marrow, peripheral blood, umbilical cord blood, and fetal hematopoietic system. In 1985, Perkins demonstrated that all major lineages of progenitor cells can be obtained from the bodies of mouse embryos, even without adding the hematopoietic growth factor [56]. Several scientists have demonstrated that, at earlier developmental stages, HSCs from different tissues have a great ability of self-replication, show different homing and surface characteristics, and are less likely to be rejected by the immune system. Therefore, they could be used for therapeutic transplantation [57].

Further studies have shown that there are two types of HSCs: long- and short-term HSCs. Long-term HSCs proliferate during their entire lifetime. It has been demonstrated that in young adult mice, 8–10% of these HSCs enter the cell cycle and divide every day. Short-term HSCs proliferate only for a limited time. Long-term HSCs have a higher telomerase activity than their short-term counterparts [58].

Active telomerase is a feature of dividing, undifferentiated cells and is also found in cancer cells. In mice, only 1 in every 10,000–15,000 bone-marrow cells is considered to be a long-term HSC [58].

Mesenchymal Stem Cells

The existence of non-hematopoietic stem cells was suggested by Cohnheim in 1867 [59]. He claimed that bone marrow may be the source of fibroblasts with collagen fibers that are part of the normal wound-healing process [60].

In 1974, Friedenstein and his colleagues performed the first isolation of MSCs from bone marrow. They reported that the adherent cells (the non-adherent cells were HSCs that were removed 4 h after the cells had been seeded on plastic culture dishes) had a heterogeneous appearance, but that most of them were spindle-shaped and formed foci of two to four cells, which remained inactive for 2–4 days and then began to grow rapidly. They also demonstrated that these cells were able to differentiate into colonies that had some similarities to bone and cartilage. Further studies extended Friedenstein’s observations and demonstrated that these cells were multipotent and able to differentiate into osteoblasts, chondrocytes, adipocytes, and even into myoblasts. They are currently referred to as mesenchymal stem cells or marrow stromal cells [61].

Sources of MSCs

Mesenchymal stem cells have the potential to differentiate into chondrocytes, osteoblasts, adipocytes, fibroblasts, marrow stromal cells, and other tissues of mesenchymal origin. The MSCs have various origins and the ability to regenerate specific cell types for several tissues, for example adipose tissue, periosteum synovial membrane, muscle, dermis blood, bone marrow, and teeth. Bone marrow stroma is considered to be the source of a large amount of multipotent cells that have access to various tissues via the blood circulation. It has been established that MSCs from bone marrow stroma are capable of differentiating into adipocytes, osteoblasts, chondrocytes, and also into hematopoiesis supporting stromal cells. The differentiation of MSCs into adipocytes, osteoblasts, and chondrocytes can be significantly increased by the use of specific differentiation cocktails [13, 17] (Figure 1.2).

c1-fig-0002

Figure 1.2 Sources of adult mesenchymal stem cells and their potential of differentiation into multilineage cell types.

Although BMSCs are an option for stem-cell therapies, their use is still subject to some limitations: first, a bone marrow harvest is a painful procedure; second, although MSCs grow well under standard tissue culture conditions, ex vivo expansion is necessary due to the relatively low numbers of MSCs that are present in the harvested marrow. Therefore, and since obesity is becoming increasingly widespread in industrialized countries, adipose tissue has become an attractive alternative source of stem cells for clinical and nonclinical applications [62]. Moreover, adipose tissue yields a much higher amount of MSCs than bone marrow [63]. The two following characteristics of ADSCs have caught the attention of scientists over the past few years: their immune privilege properties that are due to their lack of human leukocyte antigen (HLA) DR (a class II major histocompatibility antigen – MHC II) expression, which enable their use in therapeutic applications [64], as well as their suppression of the proliferation of activated allogeneic lymphocytes [62, 65]. Other studies, however, have demonstrated that these cells also possess immunosuppressive capacities, as MSCs can lead to tumor growth and cell transformation [66, 67]. These contradictory results demonstrate that further studies and protocols are necessary in order to identify the effects of ADSCs on tumor formation [66, 68]. Seeliger et al. have demonstrated that hepatocyte-like cells that are derived from adipose tissue offer a promising alternative approach to the treatment of urgent metabolic liver dysfunctions or for in vitro use. The researchers claim that hepatocyte-like cells have outstanding advantages as they maintain important metabolic functions and many enzymatic activities during cryopreservation, which renders them constantly available. As MSCs have an immunosuppressive capacity, the data obtained by Seeliger et al. suggest that these cells can be applied autologously without any lifelong immunosuppressive therapy being necessary [24].

Identification of MSCs

Mesenchymal stem cells are often identified by their morphology or by their phenotype, if they have a fibroblast-like morphology. They can also be identified by several other means, such as their differentiation potential into colony forming units (CFUs), which underlines their proliferative capacity. In addition, the ability to adhere to plastic is another characteristic marker for MSCs. Phenotypically, MSCs express various markers, but unfortunately none of them is really specific to MSCs. However, it is generally agreed upon that adult human MSCs do not express hematopoietic markers, such as CD45, CD34, CD14 or CD11 [69, 70]. In Table 1.4, different surface markers of mesenchymal stem cells are presented.

Table 1.4 Expression of profile surface markers of mesenchymal stem cells (MSCs) [13, 132, 133]

Immunosuppressive Properties of MSCs

In 2006, it was reported that MSCs possess immunomodulatory properties [71, 72]. Based on these findings, it was speculated that MSCs may play a significant role in the maintenance of peripheral tolerance, transplantation tolerance, tumor evasion, and fetal maternal tolerance [73]. Various studies on human, baboon, and murine MSCs have confirmed the immunosuppressive characteristics of MSCs and have illustrated that these cells are able to suppress the activation and in vitro proliferation of T lymphocytes [74, 75]. In line with these findings, multipotent mesenchymal stromal cells have been intensively studied in regenerative medicine. Besides their effects on T cells, their immunosuppressive effects are attributed to the secretion of a soluble factor by MSCs.

Mesenchymal stem cells have paracrine effects, such as immunomodulation, which occurs through the secretion of soluble mediators, like nitric oxide, cytokines (e.g. interleukin-6), transforming growth factor-β, human leukocyte antigen G5, and prostaglandin E2 [76, 77]. Moreover, MSCs from the bone marrow are in close contact with T and B cells and are able to regulate the immunological memory by setting various survival niches for plasma cells and memory T cells of the bone marrow. In addition, it has been demonstrated that MSCs modulate the function of B cells by inhibiting their proliferation [73]. Furthermore, there is some evidence that adult MSCs suppress the differentiation and functions of dendritic cells (DC) [78].

Since the immunosuppressive characteristics of BMSCs have been reported in vitro and in vivo, clinical trials on allogeneic transplantation through the reduction of the graft-versus-host disease (GVHD) in the recipient have been supported [62]. One possible explanation is the lack of expression of HLA-DR (MHC II) in MSCs and the suppression of the proliferation of activated allogeneic lymphocytes as demonstrated in ADSCs. It has been demonstrated that ADSCs promote engraftment and prevent or treat severe GVHD in allogeneic stem-cell transplantation in vitro and in vivo [62]. Recently, it has been speculated that the use of MSCs in diseases, such as alcoholic liver fibrosis, may suppress the inflammatory response [79]. If this assumption is correct, the use of MSCs could become a very innovative approach for the treatment of progressing liver fibrosis, since, so far, all routine clinical applications for treating this disease have been ineffective.

However, several studies have suggested that the immunosuppressive effects of ASCs may support the growth of tumor cells [80, 81].

Aging

During the aging process, the ability of the human body to regenerate tissues and organs decreases significantly. Some scientists have even stated that humans are as old as their tissue-specific adult stem cells [82, 83]. Aging is related to a functional decrease in various tissues and organs, for example impairment of the immune system, skin fragility, cardiac dysfunction, bone degeneration, and an increased risk of cancer development. Cellular senescence is a complex phenotype that leads to changes in the function and the replication capacity of the affected cells. Different protocols, culture conditions, and cell types lead to different kinds of senescence. However, in vitro, senescent cells generally exhibit a characteristically enlarged, flattened morphology [84]. Senescence is characterized by an irreversible arrest of G1 growth. This arrest leads to the repression of genes that induce the progression of the cell cycle, to the upregulation of cell-cycle inhibitors [85], to an increased global methylation [86], and to a reduced multipotency of ASCs [84].

Aging of ASCs and Possible Consequences

Adult stem cells could be the perfect source for patient-specific cell therapy of a wide range of regeneration defects, including age-related diseases, such as acute bone and cartilage defects, various liver diseases, macular degeneration, acute myocardial ischemia (AMI), stroke, and amyotrophic lateral sclerosis (ALS). There exist numerous clinical trials with MSCs that have already entered phase III [87, 88].

There is increasing evidence, however, that ASCs change their function with increasing age. For example, aging leads to a loss of the self-renewing capacity of stem cells or to their incomplete differentiation into a new cell type [89, 90]. Aged MSCs are reported to be larger than younger ones. These old MSCs exhibit more podia, spread further and possess more actin stress fibers, a decreased number of CFUs, and a lower replicative lifespan [84, 91].

Aged cells exhibit important changes in their DNA, such as telomere shortening. When telomeres reach a certain length, cells stop dividing and enter into senescence. When the telomere has reached its minimal length, the lifespan of primary human cells is limited to 50–70 cell divisions. Age-related telomere shortening has been identified in osteoblasts, chondrocytes, and myocytes. A similar pattern has been detected in older MSCs [84, 92]. These findings prove that adult stem cells undergo the same aging process as primary cells. Further evidence of this process is provided by the observation that, in vitro, MSCs lose their telomeric repeats at the same rate as primary cells (30–120 bp/PD) [92]. It has to be noted, however, that the telomeres of osteoblasts and chondrocytes are longer than the telomeres of MSCs [93, 94].

Our own studies with AD-MSCs have demonstrated an age-dependent decline in cell proliferation as well as a decreased expression of pluripotent associated genes (NANOG, OCT, and LIN28A). These changes are related to an impaired osteogenic differentiation due to age [95]. In the same line of evidence, we have been able to show that the reduction of the global methylation in AD-MSCs improves hepatic differentiation. These findings clearly demonstrate that the epigenetic modification of ASCs improves the differentiation potential of MSCs. The most interesting finding was that 5-azacytidine (AZA), a well-known DNA methyltransferase inhibitor, and BIX01294 (BIX), a histone deacetylase inhibitor, are equally able to improve the differentiation capacity of AD-MSCs towards hepatocyte-like cells. The authors have even been able to demonstrate the partial rejuvenation of old AD-MSCs [24].

The epigenetic modification of the genome apparently plays a significant role as a regulatory pathway in the control of stem-cell aging. These changes are mainly identified in DNA methylation and chromatin remodeling [96]. Increased global DNA methylation has been observed in many aged adult tissue and cell types [86]. Recently, it has been reported that ten-eleven translocation (TET) proteins mediate the conversion of 5-methyl cytosine (5mC) to 5-hydroymethyl-cytosine (5hmC), which plays a crucial role in DNA demethylation in embryonic stem cells [97]. Our own group has been able to demonstrate for the first time that TET proteins also mediate the conversion of 5mC to 5hmC in AD-MSCs. In donors with old AD-MSCs, we have observed a lower level of expression of 5hmC, together with a higher level of expression of 5mC. This finding suggests a DNA hypermethylation pattern in aged AD-MSCs [98]. Furthermore, it has been shown that the growth and the osteogenic differentiation potential of old adult AD-MSCs have been improved by the pretreatment with small molecules AZA and BIX. A gene expression analysis of pluripotent markers (NANOG, OCT-4, lIN28A, SOX2) has revealed age-dependent changes in AD-MSCs which suggest that an increase in age leads to a reduction of DNA demethylation activity. Moreover, it has been demonstrated that TET2 and TET3 gene expressions are increased by treatment with AZA and BIX. This suggests that TET2 and TET3 might be involved in the conversion of 5mC to 5hmC in AD-MSCs [99]. However, the specific role of TET2 and TET3 in the process of aging will require further investigation.

In summary, these data suggest that a reduction of the DNA demethylation activity in AD-MSCs correlates with an increased donor age. Furthermore, the treatment of AD-MSCs with small molecules (AZA, BIX) is able to restore the growth and the differentiation potential of these cells.

Stem Cells in Regenerative Medicine and in vitro Application

Regenerative medicine is a multidisciplinary field of research. It includes the use of biomaterials, growth factors, and stem cells for repairing, replacing, or regenerating damaged tissues and organs.

Due to their self-renewal and differentiation capacity, stem cells are ideal candidates for an application in regenerative medicine [62]. However, many questions about the criteria that should define the application of stem cells in regenerative medicine still have to be answered.

The first and foremost problem that needs to be solved consists in finding a possibility to access the millions or even billions of ASCs that can be found in every human body. Second, it has to be analyzed whether the stem cells can be harvested in an in vitro environment without any risk of transformation. Third, it has to be investigated whether these cells can be differentiated along multiple cell lineage pathways in a controllable and reproducible manner, or whether it is even better to use these cells in an undifferentiated state. The question of whether ASCs can be safely and effectively transplanted to an autologous or an allogeneic host also has to be answered. Finally, it has to be examined whether ASCs can be safely produced on a large scale according to Good Manufacturing Practice guidelines (GMP).

Since James Thomson performed the first isolation of human ESCs, their potential of unlimited self-renewal and differentiation has led to numerous attempts to use them in drug discovery, disease modeling, and regenerative medicine [100]. The purpose of these attempts consists in differentiating human ESCs into hepatocytes [101, 102], cardiomycytes [103], neurons [104], and intestinal tissue [105]. Several pluripotent and multipotent stem cell-based therapeutics have entered clinical trials. Table 1.5 presents a few examples of therapeutics that have already been approved for clinical application.

Table 1.5 Selected stem-cell-based therapeutics currently undergoing clinical trial

The most well-known example of the use of stem cell therapy is the transplantation of HSCs. This procedure has been practiced successfully for decades in order to treat serious hematological diseases. In the framework of this procedure, HSCs are injected directly into the blood of the recipient. This injection is called bone marrow transplantation. The stem cells recognize their pathway through homing, in which chemokines play a crucial role [106, 107].

Different types of studies have indicated the potential of mesenchymal and embryonic stem cells for undergoing similar homing into injured tissue [108–110]. However, there is still a debate as to whether stem cells should be systemically applied or brought to the site of the damage. Systemic application of MSCs in humans has shown beneficial effects in osteogenesis imperfecta and graft-versus-host disease [10, 111].

It is widely accepted that cell therapy alone is not sufficient for the regeneration of large tissue defects. Therefore, a combination of tissue engineering and the differentiated cells from MSCs seems to be required to trigger the healing of damaged tissue [112]. In fact, tissue engineering has already been used for the replacement of some tissues, for example skin, bone, or fat [112, 113] .

Stem cell-based therapy, however, brings about several safety challenges that cannot be addressed by using standard analytical procedures. A particular difficulty is the ability to monitor biological cell distribution, since it may not be possible to distinguish the injected cells from the host cells. The ability to track the therapeutic cells enables the assessment of the risk of the formation of inappropriate ectopic tissue and of tumorigenesis. Moreover, the detection of misplaced or transformed cells may necessitate the development of methods for their removal. However, such a removal of cells is not technically possible at the moment. Furthermore, the delivery of a cell with an unlimited renewal potential and a capacity to differentiate into any human cell type brings about a huge safety concern that is not raised by any other type of treatment. The finding that undifferentiated stem cells that are injected into immunocompromised animals are capable of forming teratomas, emphasizes the importance of addressing this safety issue in the future, and underlines the caution that is necessary in the development of therapies based on the use of stem cells [114].

If cells contain genetic abnormalities, they could potentially develop into teratocarcinoma [115, 116], that is tumors composed of elements of teratomas together with persisting undifferentiated cells that are highly malignant [114]. Another safety issue that should be tackled is the immunogenicity of stem cells [117]. Although there are many reports confirming the immune privilege of human ESCs, any foreign cell that is introduced into a patient will be subjected to immune surveillance [118]. This evidence indicates that our understanding of stem cells is probably not yet good enough to completely evaluate the safety of these therapies in a comprehensive manner. Therefore, further research on this topic is necessary.

Summary

Adult stem cells are a group of cells that are mainly characterized by their place of origin (hematopoietic, bone marrow or tissue-derived stem cells) and by their surface marker. They seem to present an attractive alternative in cell transplantation and in the establishment of in vitro assays, since they raise less ethical concerns than embryonic or tailored stem cells. Nevertheless, there are concerns with regard to the upscaling of ASCs and their possible transformation during the upscaling process. Along with these concerns, stem cell aging might be an additional drawback that has to be overcome, particularly when ASCs are used in the elderly. Finally, it might be conceivable to stimulate the body’s regenerative capacity rather than performing stem-cell transplantation.

Acknowledgments

We like to thank Dr. Luc Koster for editing the manuscript.

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