Tissue Engineering, Stem Cells, and Cloning
Authors: Chester J. Koh, Anthony Atala
Journal: Journal of the American Society of Nephrology (2004)

Introduction to Tissue Engineering As one of the major components of regenerative medicine, tissue engineering follows the principles of cell transplantation, materials science, and engineering toward the development of biologic substitutes that can restore and maintain normal function. Tissue engineering strategies generally fall into two categories: acellular matrices, where matrices are used alone and depend on the body's natural ability to regenerate for proper orientation and direction of new tissue growth, and matrices with cells. Acellular tissue matrices are usually prepared by removing cellular components from tissues via mechanical and chemical manipulation to produce collagen-rich matrices (1–4). These matrices tend to slowly degrade on implantation and are generally replaced by the ECM proteins that are secreted by the ingrowing cells. When cells are used for tissue engineering, a small piece of donor tissue is dissociated into individual cells. These cells are either implanted directly into the host or are expanded in culture, attached to a support matrix, and then reimplanted into the host after expansion. The source of donor tissue can be heterologous (such as bovine), allogeneic (same species, different individual), or autologous. Ideally, both structural and functional tissue replacements will occur with minimal complications. The most preferred cells to use are autologous cells, where a biopsy of tissue is obtained from the host, the cells are dissociated and expanded in culture, and the expanded cells are implanted into the same host (4–21). The use of autologous cells avoids rejection, and thus the deleterious side effects of immunosuppressive medications can be avoided. One of the limitations of applying cell-based regenerative medicine techniques toward organ replacement has been the inherent difficulty of growing specific cell types in large quantities. Even when some organs, such as the liver, have a high regenerative capacity in vivo, cell growth and expansion in vitro may be difficult. By studying the privileged sites for committed precursor cells in specific organs, as well as exploring the conditions that promote differentiation, one may be able to overcome the obstacles that could lead to cell expansion in vitro. For example, urothelial cells could be grown in the laboratory setting in the past, but only with limited expansion. Several protocols were developed over the last two decades that identified the undifferentiated cells, and kept them undifferentiated during their growth phase (12,22–24). By use of these methods of cell culture, it is now possible to expand a urothelial strain from a single specimen that initially covers a surface area of 1 cm2 to one covering a surface area of 4202 m2 (the equivalent area of one football field) within 8 wk (12). These studies indicated that it should be possible to collect autologous bladder cells from human patients, expand them in culture, and return them to the human donor in sufficient quantities for reconstructive purposes (12,13,23–32). Major advances have been achieved within the last decade on the possible expansion of a variety of primary human cells, with specific techniques that make the use of autologous cells possible for clinical application. For cell-based tissue engineering, the expanded cells are seeded onto a scaffold synthesized with the appropriate biomaterial. In tissue engineering, biomaterials replicate the biologic and mechanical function of the native ECM found in tissues in the body by serving as an artificial ECM. As a result, biomaterials provide a three-dimensional space for the cells to form into new tissues with appropriate structure and function, and also can allow for the delivery of cells and appropriate bioactive factors (e.g., cell adhesion peptides, growth factors), to desired sites in the body (33). Because the majority of mammalian cell types are anchorage dependent and will die if no cell-adhesion substrate is available, biomaterials provide a cell-adhesion substrate that can deliver cells to specific sites in the body with high loading efficiency. Biomaterials can also provide mechanical support against in vivo forces such that the predefined three-dimensional structure is maintained during tissue development. Furthermore, bioactive signals, such as cell-adhesion peptides and growth factors, can be loaded along with cells to help regulate cellular function. The ideal biomaterial should be biocompatible in that it is biodegradable and bioresorbable to support the replacement of normal tissue without inflammation. Incompatible materials are destined for an inflammatory or foreign-body response that eventually leads to rejection and/or necrosis. In addition, the degradation products, if produced, should be removed from the body via metabolic pathways at an adequate rate that keeps the concentration of these degradation products in the tissues at a tolerable level (34). Furthermore, the biomaterial should provide an environment in which appropriate regulation of cell behavior (e.g., adhesion, proliferation, migration, and differentiation) can occur such that functional tissue can form. Cell behavior in the newly formed tissue has been shown to be regulated by multiple interactions of the cells with their microenvironment, including interactions with cell-adhesion ligands (35) and with soluble growth factors (36). In addition, biomaterials provide temporary mechanical support that allows the tissue to grow in three dimensions while the cells undergo spatial tissue reorganization. The properly chosen biomaterial should allow the engineered tissue to maintain sufficient mechanical integrity to support itself in early development, whereas in late development, the properly chosen biomaterial should have begun degradation such that it does not hinder further tissue growth (33). Generally, three classes of biomaterials have been used for engineering tissues: naturally derived materials (e.g., collagen and alginate), acellular tissue matrices (e.g., bladder submucosa and small intestinal submucosa), and synthetic polymers (e.g., polyglycolic acid (PGA), polylactic acid (PLA), and poly(lactic-co-glycolic acid) (PLGA). These classes of biomaterials have been tested in respect to their biocompatibility (37,38). Naturally derived materials and acellular tissue matrices have the potential advantage of biologic recognition. However, synthetic polymers can be produced reproducibly on a large scale with controlled properties of their strength, degradation rate, and microstructure. Collagen is the most abundant and ubiquitous structural protein in the body, and may be readily purified from both animal and human tissues with an enzyme treatment and salt/acid extraction (39). Collagen implants degrade through a sequential attack by lysosomal enzymes. The in vivo resorption rate can be regulated by controlling the density of the implant and the extent of intermolecular cross-linking. The lower the density, the greater the interstitial space and generally the larger the pores for cell infiltration, leading to a higher rate of implant degradation. Collagen contains cell-adhesion domain sequences (e.g., RGD) that exhibit specific cellular interactions. This may assist to retain the phenotype and activity of many types of cells, including fibroblasts (40) and chondrocytes (41). Alginate, a polysaccharide isolated from seaweed, has been used as an injectable cell delivery vehicle (42) and a cell immobilization matrix (43) because of its gentle gelling properties in the presence of divalent ions such as calcium. Alginate is relatively biocompatible and approved by the US Food and Drug Administration (FDA) for human use as wound dressing material. Alginate is a family of copolymers of D-mannuronate and L-guluronate. The physical and mechanical properties of alginate gel are strongly correlated with the proportion and length of polyguluronate block in the alginate chains (42). Acellular tissue matrices are collagen-rich matrices prepared by removing cellular components from tissues. The matrices are often prepared by mechanical and chemical manipulation of a segment of tissue (1–4). The matrices slowly degrade upon implantation, and are replaced and remodeled by ECM proteins synthesized and secreted by transplanted or ingrowing cells. Polyesters of naturally occurring α-hydroxy acids, including PGA, PLA, and PLGA, are widely used in tissue engineering. These polymers have gained FDA approval for human use in a variety of applications, including sutures (44). The ester bonds in these polymers are hydrolytically labile, and these polymers degrade by nonenzymatic hydrolysis. The degradation products of PGA, PLA, and PLGA are nontoxic, natural metabolites and are eventually eliminated from the body in the form of carbon dioxide and water (44). The degradation rate of these polymers can be tailored from several weeks to several years by altering crystallinity, initial molecular weight, and the copolymer ratio of lactic to glycolic acid. Because these polymers are thermoplastics, they can be easily formed into a three-dimensional scaffold with a desired microstructure, gross shape, and dimension by various techniques, including molding, extrusion (45), solvent casting (46), phase separation techniques, and gas foaming techniques (47). Many applications in tissue engineering often require a scaffold with high porosity and ratio of surface area to volume. Other biodegradable synthetic polymers, including poly(anhydrides) and poly(ortho-esters), can also be used to fabricate scaffolds for tissue engineering with controlled properties (48). Stem Cells Most current strategies for tissue engineering depend upon a sample of autologous cells from the diseased organ of the host. However, for many patients with extensive end-stage organ failure, a tissue biopsy may not yield enough normal cells for expansion and transplantation. In other instances, primary autologous human cells can not be expanded from a particular organ, such as the pancreas. In these situations, pluripotent human embryonic stem cells are envisioned as a viable source of cells because they can serve as an alternative source of cells from which the desired tissue can be derived. Combining the techniques learned in tissue engineering over the past few decades with this potentially endless source of versatile cells could lead to novel sources of replacement organs. Embryonic stem cells exhibit two remarkable properties: the ability to proliferate in an undifferentiated but pluripotent state (self-renew), and the ability to differentiate into many specialized cell types (49). They can be isolated by immunosurgery from the inner cell mass of the embryo during the blastocyst stage (5 d after fertilization), and are usually grown on feeder layers consisting of mouse embryonic fibroblasts or human feeder cells (50). More recent reports have shown that these cells can be grown without the use of a feeder layer (51), and thus avoid the exposure of these human cells to mouse viruses and proteins. These cells have demonstrated longevity in culture by maintaining their undifferentiated state for at least 80 passages when grown using current published protocols (52,53). Human embryonic stem cells have been shown to differentiate into cells from all three embryonic germ layers in vitro. Skin and neurons have been formed, indicating ectodermal differentiation (54–57). Blood, cardiac cells, cartilage, endothelial cells, and muscle have been formed, indicating mesodermal differentiation (58–60). And pancreatic cells have been formed, indicating endodermal differentiation (61). In addition, as further evidence of their pluripotency, embryonic stem cells can form embryoid bodies, which are cell aggregations that contain all three embryonic germ layers, while in culture, and can form teratomas in vivo (62). Therapeutic Cloning Nuclear cloning, which has also been called nuclear transplantation and nuclear transfer, involves the introduction of a nucleus from a donor cell into an enucleated oocyte to generate an embryo with a genetic makeup identical to that of the donor. Although there has been tremendous interest in the field of nuclear cloning since the birth of Dolly in 1997, the first successful nuclear transfer was reported over 50 yr ago by Briggs and King (63). Cloned frogs, which were the first vertebrates derived from nuclear transfer, were subsequently reported by Gurdon in 1962 (64), but the nuclei were derived from nonadult sources. In the past 6 yr, tremendous advances in nuclear cloning technology have been reported, indicating the relative immaturity of the field. Dolly was not the first cloned mammal to be produced from adult cells; in fact, live lambs were produced in 1996 using nuclear transfer and differentiated epithelial cells derived from embryonic discs (65). The significance of Dolly was that she was the first mammal to be derived from an adult somatic cell using nuclear transfer (66). Since then, animals from several species have been grown using nuclear transfer technology, including cattle (67), goats (68,69), mice (70), and pigs (71–74). Two types of nuclear cloning, reproductive cloning and therapeutic cloning, have been described, and a better understanding of the differences between the two types may help to alleviate some of the controversy that surrounds these revolutionary technologies (75,76). Banned in most countries for human applications, reproductive cloning is used to generate an embryo that has the identical genetic material as its cell source. This embryo can then be implanted into the uterus of a female to give rise to an infant that is a clone of the donor. On the other hand, therapeutic cloning is used to generate early stage embryos that are explanted in culture to produce embryonic stem cell lines whose genetic material is identical to that of its source. These autologous stem cells have the potential to become almost any type of cell in the adult body, and thus would be useful in tissue and organ replacement applications (77). Some useful applications would be in the treatment of diseases, such as end-stage kidney disease, neurodegenerative diseases, and diabetes, for which there is limited availability of immunocompatible tissue transplants. Therefore, therapeutic cloning, which has also been called somatic cell nuclear transfer, provides an alternative source of transplantable cells that theoretically may be limitless. Figure 1 shows the strategy of combining therapeutic cloning with tissue engineering to develop tissues and organs. According to data from the Centers for Disease Control and Prevention, it has been estimated that approximately 3000 Americans die every day of diseases that could have been treated with embryonic stem cell–derived tissues (78). With current allogeneic tissue transplantation protocols, rejection is a frequent complication because of immunologic incompatibility, and immunosuppressive drugs are usually administered to treat and hopefully prevent graft-versus-host disease (77). The use of transplantable tissue and organs derived from therapeutic cloning may lead to the avoidance of immune responses that typically are associated with transplantation of nonautologous tissues. As a result, with therapeutic cloning, the variety of serious and potentially life-threatening complications associated with immunosuppressive treatments may be avoided (79). Figure 1. : Strategy for therapeutic cloning and tissue engineering.Current Limitations of Cloning Technology Although promising, somatic cell nuclear transfer technology has certain limitations that require further improvements before therapeutic cloning can be applied widely in replacement therapy. Currently, the efficiency of the overall cloning process is low. The majority of embryos derived from animal cloning do not survive after implantation (80–82). In practical terms, multiple nuclear transfers must be performed to produce one live offspring for animal cloning applications. The potential for cloned embryos to grow into live offspring is between 0.5% to 18% for sheep, cattle, pigs, and mice (83). However, greater success (80%) has been reported in cattle (84), which may be in part due to the availability of advanced bovine supporting technologies, such as in vitro embryo production and embryo transfer, which have been developed for this species for agricultural purposes. To improve cloning efficiencies, further improvements are required in the multiple complex steps of nuclear transfer, such as enucleation and reconstruction, activation of oocytes, and cell cycle synchronization between donor cells and recipient oocytes (85), that will more readily produce viable sources of cells. Furthermore, common abnormalities have been found in newborn clones if they survive to birth, including enlarged size with an enlarged placenta (large offspring syndrome) (86), respiratory distress and defects of the kidney, liver, heart, and brain (87), obesity (88), and premature death (89). These may be related to the epigenetics of the cloned cells, which involve the reversible modifications of the DNA or chromatin, while the original DNA (genetic) sequences remain intact. Faulty epigenetic reprogramming in clones, where the DNA methylation patterns, histone modifications, and the overall chromatin structure of the somatic nuclei are not being reprogrammed to an embryonic pattern of expression, may explain the above abnormalities (77). Reactivation of key embryonic genes at the blastocyst stage is usually not present in embryos cloned from somatic cells, but embryos cloned from embryos consistently express early embryonic genes (90,91). Proper epigenetic reprogramming to an embryonic state may help to improve the cloning efficiency and reduce the incidence of abnormal cloned cells. Tissue Engineering of Specific Structures Investigators around the world, including our laboratory, have been working toward the development of several cell types and tissues and organs for clinical application. Urethra Various biomaterials without cells, such as PGA and acellular collagen-based matrices from small intestine and bladder, have been used experimentally (in animal models) for the regeneration of urethral tissue (1,92–96). Some of these biomaterials, like acellular collagen matrices derived from bladder submucosa, have also been seeded with autologous cells for urethral reconstruction. Our laboratory has been able to replace tubularized urethral segments with cell-seeded collagen matrices. Acellular collagen matrices derived from bladder submucosa by our laboratory have been used experimentally and clinically. In animal studies, segments of the urethra were resected and replaced with acellular matrix grafts in an onlay fashion. Histologic examination showed complete epithelialization and progressive vessel and muscle infiltration, and the animals were able to void through the neourethras (1). These results were confirmed in a clinical study of patients with hypospadias and urethral stricture disease (6,97). Decellularized cadaveric bladder submucosa was used as an onlay matrix for urethral repair in patients with stricture disease and hypospadias (Figure 2). Patent, functional neourethras were noted in these patients with up to a 7-yr follow-up. The use of an off-the-shelf matrix appears to be beneficial for patients with abnormal urethral conditions and obviates the need for obtaining autologous grafts, thus decreasing operative time and eliminating donor site morbidity. Figure 2. : Tissue engineering of the urethra using a collagen matrix. (A) Representative case of a patient with a bulbar stricture. (B) Urethral repair. Strictured tissue is excised, preserving the urethral plate on the left side, and matrix is anastomosed to the urethral plate in an onlay fashion on the right. (C) Urethrogram 6 mo after repair. (D) Cystoscopic view of urethra before surgery on the left side and 4 mo after repair on the right side.Unfortunately, the above techniques are not applicable for tubularized urethral repairs. The collagen matrices are able to replace urethral segments only when used in an onlay fashion. However, if a tubularized repair is needed, the collagen matrices should be seeded with autologous cells to avoid the risk of stricture formation and poor tissue development (98,99). Therefore, tubularized collagen matrices seeded with autologous cells can be used successfully for total penile urethra replacement. Bladder Currently, gastrointestinal segments are commonly used as tissues for bladder replacement or repair. However, gastrointestinal tissues are designed to absorb specific solutes, whereas bladder tissue is designed for the excretion of solutes. Because of the problems encountered with the use of gastrointestinal segments, numerous investigators have attempted alternative materials and tissues for bladder replacement or repair. The success of the use of cell transplantation strategies for bladder reconstruction depends on the ability to use donor tissue efficiently and to provide the right conditions for long term survival, differentiation and growth. Urothelial and muscle cells can be expanded in vitro, seeded onto polymer scaffolds, and allowed to attach and form sheets of cells (7). These principles were applied toward the creation of tissue engineered bladders in an animal model that required a subtotal cystectomy with subsequent replacement with a tissue engineered organ in beagle dogs (18). Urothelial and muscle cells were separately expanded from an autologous bladder biopsy sample and seeded onto a bladder-shaped biodegradable polymer scaffold. The results from this study showed that it is possible to tissue-engineer bladders that are anatomically and functionally normal. Male Genital Tissues Reconstructive surgery is required for a wide variety of pathologic penile conditions, such as penile carcinoma, trauma, severe erectile dysfunction, and congenital conditions such as ambiguous genitalia, hypospadias, and epispadias. One of the major limitations of phallic reconstructive surgery is the scarcity of sufficient autologous tissue. Phallic reconstruction using autologous tissue, derived from the patient's own cells, may be preferable in selected cases. The major components of the phallus are corporal smooth muscle and endothelial cells. The creation of autologous functional and structural corporal tissue de novo would be beneficial. Autologous cavernosal smooth muscle and endothelial cells were harvested, expanded and seeded on acellular collagen matrices and implanted in a rabbit model (100,101). Histologic examination confirmed the appropriate organization of penile tissue phenotypes, and structural and functional studies, including cavernosonography, cavernosometry, and mating studies, demonstrated that it is possible to engineer autologous functional penile tissue. Our laboratory is currently working on increasing the size of the engineered constructs. Female Genital Tissues Congenital malformations of the uterus may have profound implications clinically. Patients with cloacal exstrophy and intersex disorders may not have sufficient uterine tissue present for future reproduction. We investigated the possibility of engineering functional uterine tissue using autologous cells (102). Autologous rabbit uterine smooth muscle and epithelial cells were harvested, then grown and expanded in culture. These cells were seeded onto preconfigured uterine-shaped biodegradable polymer scaffolds, which were then used for subtotal uterine tissue replacement in the corresponding autologous animals. Upon retrieval 6 mo after implantation, histologic, immunocytochemical, and Western blot analyses confirmed the presence of normal uterine tissue components. Biomechanical analyses and organ bath studies showed that the functional characteristics of these tissues were similar to those of normal uterine tissue. Breeding studies that use these engineered uteri are currently being performed. Similarly, several pathologic conditions, including congenital malformations and malignancy, can adversely affect normal vaginal development or anatomy. Vaginal reconstruction has traditionally been challenging because of the paucity of available native tissue. The feasibility of engineering vaginal tissue in vivo was investigated (103). Vaginal epithelial and smooth muscle cells of female rabbits were harvested, grown, and expanded in culture. These cells were seeded onto biodegradable polymer scaffolds, and the cell-seeded constructs were then implanted into nude mice for up to 6 wk. Immunocytochemical, histologic, and Western blot analyses confirmed the presence of vaginal tissue phenotypes. Electrical field stimulation studies in the tissue-engineered constructs showed similar functional properties to those of normal vaginal tissue. When these constructs were used for autologous total vaginal replacement, patent vaginal structures were noted in the tissue-engineered specimens, whereas the non–cell-seeded structures were noted to be stenotic (104). Kidney We applied the principles of both tissue engineering and therapeutic cloning in an effort to produce genetically identical renal tissue in a large animal model, cattle (Bos taurus) (105). Bovine skin fibroblasts from adult Holstein steers were obtained by ear notch, and single donor cells were isolated and microinjected into the perivitelline space of donor enucleated oocytes (nuclear transfer). The resulting blastocysts were implanted into progastrin-synchronized recipients to allow for further in vivo growth. After 12 wk, cloned renal cells were harvested, expanded in vitro, and seeded onto biodegradable scaffolds. The constructs, which consisted of the cells and the scaffolds, were then implanted into the subcutaneous space of the same steer from which the cells were cloned to allow for tissue growth. The kidney is a complex organ with multiple cell types and a complex functional anatomy that renders it one of the most difficult to reconstruct (5,106). Previous efforts in tissue engineering of the kidney have been directed toward the development of extracorporeal renal support systems made of biologic and synthetic components (107–113), and ex vivo renal replacement devices are known to be life-sustaining. However, there would be obvious benefits for patients with end-stage kidney disease if these devices could be implanted long term without the need for an extracorporeal perfusion circuit or immunosuppressive drugs. Cloned renal cells were seeded on scaffolds consisting of three collagen-coated cylindrical polycarbonate membranes (Figure 3A). The ends of the three membranes of each scaffold were connected to catheters that terminated into a collecting reservoir. This created a renal neo-organ with a mechanism for collecting the excreted urinary fluid (Figure 3B). These scaffolds with the collecting devices were transplanted subcutaneously into the same steer from which the genetic material originated and retrieved 12 wk after implantation. Figure 3. : Combining therapeutic cloning and tissue engineering to produce kidney tissue. (A) Illustration of the tissue-engineered renal unit. (B) Renal unit seeded with cloned cells, 3 mo after implantation, showing the accumulation of urinelike fluid. (C) There was a clear unidirectional continuity between the mature glomeruli, their tubules, and the polycarbonate membrane. (D) ELISPOT analyses of the frequencies of T cells that secrete IFN-γ after primary and secondary stimulation with allogeneic renal cells, cloned renal cells, or nuclear donor fibroblasts.Chemical analysis of the collected urinelike fluid, including urea nitrogen and creatinine levels, electrolyte levels, specific gravity, and glucose concentration, revealed that the implanted renal cells possessed filtration, reabsorption, and secretory capabilities. Histologic examination of the retrieved implants revealed extensive vascularization and self-organization of the cells into glomeruli- and tubulelike structures. A clear continuity between the glomeruli, the tubules, and the polycarbonate membrane was noted that allowed the passage of urine into the collecting reservoir (Figure 3C). Immunohistochemical analysis with renal-specific antibodies revealed the presence of renal proteins, RT-PCR analysis confirmed the transcription of renal specific RNA in the cloned specimens, and Western blot analysis confirmed the presence of elevated renal-specific protein levels. Because previous studies have shown that bovine clones harbor the oocyte mtDNA (114–116), the donor egg's mtDNA was thought to be a potential source of immunologic incompatibility. Differences in mtDNA-encoded proteins expressed by cloned cells could stimulate a T cell response specific for mtDNA-encoded minor histocompatibility antigens when the cloned cells are implanted back into the original nuclear donor (117). We used nucleotide sequencing of the mtDNA genomes of the clone and fibroblast nuclear donor to identify potential antigens in the muscle constructs. Only two amino acid substitutions were noted to distinguish the clone and the nuclear donor, and as a result, a maximum of two minor histocompatibility antigens could be defined. Given the lack of knowledge regarding peptide-binding motifs for bovine MHC class I molecules, there is no reliable method to predict the effect of these amino acid substitutions on bovine histocompatibility. Oocyte-derived mtDNA was also thought to be a potential source of immunologic incompatibility in the cloned renal cells. Maternally transmitted minor histocompatibility antigens in mice have been shown to stimulate both skin allograft rejection in vivo and cytotoxic T lymphocytes expansi