Chapter 8 – Regenerative Medicine

178 Regeneration refers to the restoration of normal tissue and organ architecture and function after injury or disease. Although numerous complex organisms retain impressive capacity to regenerate limbs and organs throughout adult life, humans have sacrificed regenerative ability for speed and strength of repair. This has allowed us to enjoy remarkable evolutionary success, but it also leads to significant scarring that causes significant loss of function and aesthetic consequences. It may be possible to improve on the normal recovery from injury and illness by promoting true tissue regeneration instead of repair through fibrosis and scarring. Surgeons have understood these dynamics for decades, but comprehensive tissue and organ regeneration have remained elusive in clinical practice. The field of regenera- tive medicine is largely focused on stem cells, which are power- ful undifferentiated cells that have the ability to self-renew and give rise to one or more different cell types. As basic scientific research has uncovered the biology of stem cells, translational opportunities for stem cell–based therapies have become increas- ingly plausible. In addition to stem cell biology, the field of regenerative medicine includes the disciplines of tissue engineer- ing and biomaterials, which aim to create molecular and struc- tural niches to deliver regenerative therapies. This chapter provides an overview of the current status of stem cell biology and tissue engineering research and outlines the future steps required for regenerative medicine to become clinically useful. STEM CELL SOURCES Stem cells are defined by their capacity to self-renew and dif- ferentiate into multiple functional cell types (Table 8-1). Tradi- tionally, they have been divided into two main groups based on their potential to differentiate (Fig. 8-1). Pluripotent stem cells (embryonic) can differentiate into every cell of the body, whereas multipotent stem cells (adult) can differentiate into multiple, but not all, cell lineages. In addition to the traditional stem cell classification, a new class of stem cells has recently been described—induced pluripotent stem (iPS) cells—which are derived from genetically reprogrammed adult cells. These diverse cell populations hold much promise to provide researchers and clinicians with an expanded armamentarium to treat diseased and dysfunctional organs. Embryonic Stem Cells During development, two distinct lineages emerge during the transition from morula to blastocyst, the trophoectoderm and the inner cell mass. Embryonic stem cells (ESCs) are immortal cell lines derived from the inner cell mass of the blastocyst. The two hallmark characteristics of ESCs are their unlimited in vitro self-renewal capacity and their ability to differentiate into all somatic cell types.1 A number of transcription factors, most prominently Oct4, Sox2, and Nanog, are essential regulators that ensure the maintenance of pluripotency while suppressing differentiation.2 The two glycolipid antigens SSEA3 and SSEA4 are operational cell surface markers used to identify human ESCs.3 Since the successful isolation of mouse and human ESCs, their potential for cell replacement therapy and regenera- tive medicine has been widely acknowledged.4 Both mouse and human ESCs have demonstrated an in vitro capacity to form cardiomyocytes, hematopoietic progenitors, neurons, skel- etal myocytes, adipocytes, osteocytes, chondrocytes, and pancre- atic islet cells when cultured under specific growth factor conditions.5,6 However, a number of limitations currently exist regarding the use of human ESCs in regenerative medicine. Although pluripotentiality and unlimited ability for self-renewal make ESCs attractive for cell replacement therapy, these same charac- teristics simultaneously translate into unregulated differentiation and formation of teratomas and teratocarcinomas. These tumors contain differentiated cells that contain all three primary germ layers, as well as undifferentiated pluripotent stem cells. This tendency to form tumors has been observed when ESCs are transplanted into mice, raising the concern that human ESC– based therapy may also lead to unwanted tumor formation.1 Without the elimination of this possibility, the clinical use of ESC-derived tissue will remain limited. In addition, any cell-based therapy must be free of animal contaminants that might contain pathogens or elicit an immune reaction after transfer to a host. Both mouse cell and human ESC lines are generally grown on a mouse-derived feeder layer of fibroblasts that provides additional factors that promote ESC proliferation as well as inhibit their differentiation. One example of possible animal product contamination is the demonstration that human ESCs grown on mouse feeder cells express a nonhu- man sialic acid that could elicit a host’s immune response.7 stem cell sources bioengineering for regenerative medicine clinical applications of stem cells CHAPTER 8 REGENERATIVE MEDICINE Jason P. Glotzbach, Sae Hee Ko, Geoffrey C. Gurtner, and Michael T. Longaker RegeneRative Medicine  Chapter 8  179 SECTIO N I SU Rg ic aL B aSic PR in c iPLeS Concerns have also been raised over the possible transfer of murine viruses from feeder layers to human ESCs. Many labo- ratories are working to solve this problem, with some studies demonstrating the ability to culture human ESCs under serum- free defined medium conditions on human cell-derived feeders or under feeder-free conditions.8 Furthermore, there are significant political and ethical hurdles that hinder further investigations of human ESCs. At this time, the limited number of ESC lines available and the restrictions placed on their use have precluded major progress in ESC-based applications. Although President Obama in recent months has largely reversed the restrictions put in place by President Bush, alternative solutions are needed to advance cell- based regenerative strategies. FIGURE 8-1 Schematic of stem cell organization. eScs, derived from the inner cell mass of the blastocyst, have the highest stem cell capacity (pluripotent) and are the least committed to any tissue lineage. adult stem cells such as HScs and MScs are multipotent and are limited to certain tissue lineages, although they remain in a relatively undifferentiated state at rest. tissue-specific stem cells, such as skin follicular bulge cells, are limited to producing a single cell and tissue type (unipotent), although they retain considerable proliferative capacity to regenerate their specific tissue. Mature lineage cells, such as mature epithelium, do not have regenerative potential. iPS cells are mature lineage cells or adult stem cells that have been reprogrammed to a state of relative pluripotency and have much of the same regenerative potential as eScs. ESC St em c el l c ap ac ity N on e Un ip ot en t M ul tip ot en t P lu rip ot en t iPS cells HSC MSC/ASC Tissue-specific stem cells Differentiation Mature lineage cells Embryonic Adult R E P R O G R A M M I N G table 8-1  Definitions of Stem Cell-related terms terM DeFINItION totipotent ability to form all cell types and lineages of organism (e.g., fertilized egg) Pluripotent ability to form all lineages of the body (e.g., embryonic stem cells) Multipotent ability of adult stem cells to form multiple cell types of one lineage (e.g., mesenchymal stem cells) Unipotent cells form one cell type (e.g., follicular bulge skin stem cells) Reprogramming dedifferentiation into an embryonic state; can be induced by nuclear transfer, genetic manipulation, viral transduction, and related methods 180  SeCtION I SURgicaL BaSic PRinciPLeS genes had more than a fivefold difference in expression.16 Fur- thermore, chimeras and progeny mice derived from iPS cells had higher than normal rates of tumor formation than those derived from ESCs, which in some cases may have been caused by reac- tivation of the transfected c-Myc oncogene.17 These key differ- ences need to be elucidated further to define the safety of iPS cell use in regenerative medicine. Another potential complication with the generation of iPS cells is the use of retroviral and lentiviral vectors to activate the necessary reprogramming transcription factors. Specifically, the viral genome could be inserted near endogenous genes, resulting in gene activation or silencing. This risk of insertional mutagen- esis could lead to uncontrolled modification of the genome, with potential development of cancer. Much progress has been made in generating integration-free murine iPS cells, and various recent studies using adenoviral, plasmid-based, and recombinant protein-based strategies have reported that viral integration is not required for the reprogramming process.18,19 Even without viral integration, the safety of iPS cells needs to be rigorously tested, because all essential reprogramming factors are oncogenes and their overexpression has been linked with cancers.20 The characterization of iPS cells will be enhanced by ongoing improvements in the high-resolution analysis of genomic integ- rity via DNA sequencing technology to identify even minor deletions, inversions, or loss of individual alleles readily. The generation of iPS cells is likely to create a major impact on regenerative medicine. These iPS cells can be generated from human adipose-derived stem cells (ASCs) in a feeder-free condi- tion with a faster speed and higher efficiency than comparable strategies targeting adult human fibroblasts.21 Given the ease of isolating a large quantity of ASCs from lipoaspirates, ASCs could be an ideal autologous source of cells for generating individual-specific iPS cells. The therapeutic potential of iPS cells has been demon- strated in several preclinical models. For example, Wernig and colleagues have demonstrated that neurons derived from repro- grammed fibroblasts could alleviate the disease phenotype in a rat model of Parkinson’s disease.22 Using a humanized sickle cell anemia mouse model, Hanna and associates23 have shown that the genetic defect could be corrected using transplantation of hematopoietic stem cells (HSCs) derived from iPS cells (derived from fibroblasts of those mice) that had homologous recombina- tion of an intact wild-type β-globin gene. Although these early preclinical studies are very promising, iPS cell technology will require further refinement before clinical applications can be feasible. Fetal Stem Cells Although less prominently discussed, fetal stem cells represent another source for a regenerative building block with clinical potential. Fetal stem cells can be derived from fetal blood, liver, bone marrow, amniotic fluid, and placenta, and are rich in a population of stem cells that proliferate more rapidly and exhibit greater multipotentiality than adult stem cells.24,25 Fetal stem cells have been found to expand in culture for at least 20 passages, and their capacity for adipogenic, osteo- genic, and chondrogenic differentiation has been demon- strated under appropriate culture conditions.26 In addition, transplantation into a xenogeneic sheep model has shown the ability of these cells to engraft and undergo site-specific tissue differentiation. Somatic Cell Nuclear Transfer Somatic cell nuclear transfer (SCNT), also referred to as repro- ductive cloning, involves the transfer of nuclei from postnatal somatic cells into an enucleated ovum. Mitotic divisions of this cell in culture lead to the generation of a blastocyst capable of yielding a whole new organism. Major advances in this field came in 1997 with the production of a normal sheep (Dolly),9 and this procedure has been reproduced in other mammals, including mice, cattle, pigs, cats, and dogs.10 These experimental studies suggest that a similar approach using SCNT might work in humans for therapeutic cloning, whereby human ESCs produced by this approach could be subsequently differentiated into therapeutically useful cells and transplanted back into patients with degenerative diseases. A recent report on primate ESC lines, which were derived from rhesus macaque SCNT blastocysts using adult male skin fibro- blasts as nuclear donors, is an important step in this direction.11 However, similar to human ESCs, SCNT is embroiled in an ethically complex debate about the moral status of created embryo and concerns about obtaining human unfertilized eggs. The technical limitations of this procedure have also dampened early enthusiasm, because several studies have reported less than 10% efficiency in the derivation of SCNT-generated ESCs.12 Despite the controversy, SCNT and therapeutic cloning may still be a promising means to generate genetically matched stem cell lines. Long-lasting cell lines from patients with diseases created via SCNT can be used to screen potentially useful drugs or other treatments and may provide replacement cells for damaged organs. Induced Pluripotent Stem Cells Given the complex logistical and ethical considerations sur- rounding donated oocytes for SCNT, alternatives that recapitu- late the reprogramming process in vitro while avoiding the need for oocytes altogether are ultimately preferable. A groundbreak- ing study in 2006 by Takahashi and Yamanaka13 defined a spe- cific set of transcription factors, Oct4, Sox2, Klf4, and cMyc, that were sufficient to reprogram adult mouse fibroblasts back into a pluripotent state, thus creating ESC-like induced plu- ripotent stem (iPS) cells. Takahashi and coworkers14 quickly demonstrated that the same combination of transcription factors is sufficient for the pluripotent induction of human cells as well. The ease and reproducibility of generating iPS cells compared with SCNT has raised the hope that iPS cells might fulfill much of the promise of human ESCs in regenerative medicine. It is widely accepted that mouse and human iPS cells closely resemble molecular and developmental features of blastocyst-derived ESCs.13,15 A number of research groups have shown that iPS cells injected into immunodeficient mice give rise to teratomas comprising all three embryonic germ layers, similar to ESCs. In addition, when injected into blastocysts, iPS cells generated viable high-contribution chimeras (mice that show major tissue contributions of the injected iPS cells in the host mouse) and contributed to the germline.13,15 Furthermore, using reverse transcription polymerase chain reaction (RT-PCR) assays and immunocytochemistry, studies have shown that iPS cells express key markers of ESCs. However, recent evidence has demonstrated that iPS cells are not identical to ESCs. Global gene expression analysis com- paring iPS cells with human ESCs using microarrays has dem- onstrated that approximately 4% of the over 32,000 analyzed RegeneRative Medicine  Chapter 8  181 SECTIO N I SU Rg ic aL B aSic PR in c iPLeS In the heart, resident stem cells have limited regenerative poten- tial and have not been shown to engraft when administered exogenously after myocardial injury.36 More experimental work is needed before these populations of tissue-specific resident stem cells can be effectively exploited for regenerative medicine applications. Adult Multipotent Stem Cells Multipotent cells exist in several reservoirs in the adult and retain the ability to form many different cellular lineages (Fig. 8-2). Although the differentiation potential of these cells is not as complete as ESCs or induced pluripotent stem cells, their rela- tive abundance and ease of isolation from adult patients estab- lishes adult stem cells as a highly relevant cell type for regenerative medicine applications. Accordingly, adult multipo- tent stem cells have been a focus of intensive research efforts over the past several decades. Hematopoietic Stem Cells HSCs have been the most studied and best characterized adult multipotent stem cell type after being definitively isolated in mice several decades ago.37 These blood-forming cells reside in specialized niches within adult bone marrow and function to maintain homeostasis of all lineages of hematopoietic cells. HSCs have become the paradigm for the experimental investiga- tion of adult stem cell biology. They form the basis of the most successful clinical application of stem cell–based therapy—bone marrow transplantation for hematologic malignancies and other disorders, through which HSCs repopulate all lineages of the hematopoietic system after bone marrow ablation.28 Despite the enormous ability of HSCs to regenerate the hematopoietic system, the preponderance of evidence does not support the concept that HSCs can transdifferentiate into other tissue lineages, thus limiting their usefulness in cell-based therapeutic interventions outside of the hematopoietic system.38 In addition, HSCs cannot readily be grown in cell or tissue culture conditions in vitro, further limiting their usefulness for regen- erative medicine applications. Although direct transplantation of HSCs is not likely to be used for regenerative medicine (outside of hematopoietic deficiencies and malignancies), stem cell biologists have been investigating a possible role for HSCs in the induction of tolerance in preparation for organ transplantation.39 Mesenchymal Stem Cells The stromal fraction of adult bone marrow contains of hetero- geneous population of cells that were originally described as supportive cells for hematopoietic cells and later termed mesen- chymal stem cells (MSCs). This group of multipotent cells is derived from embryonic mesenchyme and can differentiate into mesenchymal-derived structures, such as bone, fat, cartilage, and muscle.40 MSCs are rare in the bone marrow, because they only make up approximately 1 of 10,000 total bone marrow cells. They have traditionally been isolated in vitro through their ability to adhere to polystyrene tissue culture plastic; however, it is increasingly recognized that this isolation method produces a heterogeneous mix of cells, which has made comparison of experimental protocols and standardization of results difficult. Reports of human MSC surface antigen expression profiles vary widely; there is no one agreed on group of surface markers that can be used for prospective isolation protocols. A comprehensive Despite these promising findings, however, significant debate has been raised over the issue of using cells from fetuses and the attendant risks associated with intrauterine procedures. Nonetheless, fetal stem cells may still provide a novel means whereby future autogenous in utero cellular and genetic thera- pies can be devised. Adult Stem Cells Once embryonic development has completed, humans and other complex organisms lose their cache of embryonic stem cells. During adult life, the regenerative capacity of tissues and organs is maintained by adult stem cells, which reside in mature tissues and in general repositories throughout bone marrow and adipose tissue. Unlike embryonic stem cells and induced plu- ripotent stem cells, adult stem cells are multipotent; they can differentiate into some but not all tissue lineages and are typi- cally confined to a certain tissue type and microenvironment, usually termed a stem cell niche.27 The most studied and best characterized adult stem cell types is the hematopoietic stem cell, which has served as the experimental paradigm for basic studies into the biology of adult stem cell biology.28 Recently, much insight has been gained into the organization and function of mesenchymal stem cells and adipose strom